ALBERT

Notes: Abstract The mutarotation rates of glucose in aqueous mixtures of tetrahydrofuran andtert-butanol in the mole fraction (xi) range 0〈xi〈0.2 have been measured at 5° intervals in the range 5–35°C. The kinetic deuterium isotope effects have been determined for the same solvent compositions at 25 and 35°C. A statistical analysis of the Arrhenius plots indicates that the experimental errors, although small, are too large for the establishment of any compensation behavior between ΔH

Notes: Abstract Tetramethylurea was hydrolyzed in aqueous hydrochloric acid at temperatures of 80, 90, and 100°C. All reactions were carried out in an excess of both tetramethylurea and water, with first-order dependence on acid concentration being observed. As the concentration of water was increased, the rate of hydrolysis decreased until equimolar amounts of water and tetramethylurea were present. Subsequent addition of water had no effect on the observed rate.

Notes: Summary The egg-shell of Japanese quail was studied by several techniques. Semithin sections (1μm thick) of non-decalcified shell were observed by normal and polarized light microscopy. Thin sections of non-decalcified shell, examined by transmission electron microscopy, permitted us to observe the forms and dimensions of crystals of calcite within different layers of the shell: mammilary layer, layer of cones, palissade layer and surface crystal layer. There appears to be two distinct zones in the layer of cones as well as in the superficial crystal layer. Electron microdiffraction revealed the orientation of calcite crystals in the columns. Some crystal defects (twins?) were described and the possibility of their artefactual formation during ultramicrotomy is discussed. Localization of Ca, Mg, P and S were made by X-ray microanalysis of semithin sections. This technique shows that shell membranes, and chiefly the true cuticle, are also mineralized but, in these layers, minerals are not crystallized. Otherwise the distribution of Mg is not uniform throughout the shell thickness; it is less concentrated in the external zone of the layer of cones. These results together with observation of developing shells by scanning electron microscopy allowed us to propose a scheme for shell organization of the quail egg. This organization was related with decalcification which occurs during hatching.

Notes: Summary Electron microscopical observations of the size and shape of bone mineral crystallites have not been in complete agreement with X-ray diffraction findings. The two prevalent viewpoints consider bone mineral crystals to be either rod, or plate like in habit. There appears to be agreement that the smallest dimension of the crystals is about 5 nm, but there is discrepancy in the reported c-axial lengths. The method of dark field imaging is used to obtain a quantitative measurement of the c-axial length distribution in rabbit, ox and human bone: mean c-axial lengths 32.6 nm, 36.2 nm and 32.4 nm, respectively, show no significant difference at the 5% level to the mean c-axial length measured by X-ray line broadening. Both bright and dark field images strongly suggest that bone mineral has a plate like form. Reasons for past discrepancies are discussed.

Notes: Summary Newly formed osteodentin obtained from the anterior extremities of fetal or young rat incisors was observed by means of electron microscopy and electron probe X-ray microanalysis. Cells related to osteodentin formation frequently showed membrane bound intracellular bodies containing varying amounts of fine, needle-shaped crystals, which were identified as apatite. The intracellular clusters of apatite crystals were extruded from the cells through membrane fusion or cellular degeneration. These extracellular clusters seemed to be gradually incorporated into the mineralizing collagenous matrix, which developed around them. Frequent occurrence of dense, dotshaped or filamentous profiles suggested that the dense bodies seen in the perinuclear regions or in the Golgi area were the sites of crystal formation. Energy dispersive X-ray point analysis showed that the intracellular or extracellular apatite clusters contained sulfur in a concentration higher than was present in the mineralizing collagenous matrix. Furthermore, wave dispersive X-ray line analysis showed that the concentration of sulfur was higher in the osteodentin matrix than in the dentin matrix. The sulfur detected is presumed to be contained in acid mucopolysaccharides, which were distributed more heavily in the osteodentin matrix than in the dentin matrix. On the basis of these data, it was concluded that the unique chemical and structural characteristics of the osteodentin result primarily from the incorporation of apatite clusters of intracellular origin and associated acid mucopolysaccharides.

Notes: Summary The comparative ultrastructure of fish bone osteogenesis and resorption induced by scale removal was described in the osteocytic (cellular-boned)Carassius auratus and the anosteocytic (acellular-boned)Tilapia macrocephala. Osteocytes, present in osteocytic bone, were lacking in anosteocytic bone. In osteocytic bone the osteoblast secreted a collagenous preosseous matrix in which it became enmeshed and then was termed a preosteocyte. When the preosseous matrix mineralized, the preosteocyte was termed an osteocyte and was completely surrounded by bone. In anosteocytic bone the osteoblasts receded from the mineralizing front and never became trapped as osteocytes. During resorption, types A and B resorptive cells, present in both bone types, invaded the matrix and demineralized the osseous zone. These cells were characterized by large amounts of granular endoplasmic reticulum and intracellular inclusions containing crystal-like material. Although functionally similar to mammalian osteoclasts, these cells lacked a characteristic ruffled border and were not multinucleated. The osteocytes of cellular bone did not appear to be involved during demineralization.

Notes: Summary The effects of parathyroid hormone (PTH) on the size of the osteoclasts, nuclei, ruffled borders, and clear zones in long bones of thyroparathyroidectomized (TPTX) rats were quantitated as a function of time. These data were compared with the number of osteoclasts in the bone and with plasma calcium levels. A significant increase in the average size of the ruffled borders was demonstrated 30 min after injection of 50 U of purified bovine PTH, and of the clear zones 30–90 min after PTH. This was followed at 90 min by an increase in the average size of the cells. The sizes of ruffled borders and clear zones dropped sharply to control levels after 6 h, whereas the size of the cells remained elevated up to 12 h and returned to control values at 24 h. Plasma calcium levels were increased, but not significantly, between 30 min and 6 h. An increase in the number of osteoclasts was significant after 12 h. Removal of the parathyroid glands did not diminish the normal activity of osteoclasts. In animals with intact glands injection of 50 U of PTH did not cause a significant change in cell size or resorbing apparatus. It is concluded that PTH acts to rapidly stimulate the bone resorptive activity of osteoclasts and to cause a delayed increase in their number.

Notes: Summary The nucleotide sequence ofDrosophila melanogaster 5S RNA has been determined and appears to be homogeneous both in the KC cell line and in the insect at different developmental stages. Experimental evidence on the conformation of this molecule is in agreement with a general class of 5S RNA models.

Notes: Abstract The ultrastructural organization of the spores of the sporocarp of Endogone flammicorona was studied. Two types of organization are described. Initially the spore possessed a vacuolate protoplasm and was bound by two cell wall layers. The spore was surrounded by a hyphal mantle formed of a sheet of vacuolized hyphae with uniformly thin walls. Secondly, although the ultrastructural features of the spore appeared the same, it was now surrounded by a hyphal mantle with unevenly thickened walls (i. e., the so-called flaming crown) due to the gradual and irregular deposition of granules and lamellae. This crown gives the spore its most commonly observed morphological feature and is the preminent character employed taxonomically to speciate Endogone flammicorona Trappe & Gerdemann.

Notes: Abstract An ultrastructural investigation of six different species of Prototheca showed that all of them contained starch grains enclosed in double-membrane-bounded structures recognized as plastids. It is concluded that these unicellular species of Prototheca must be considered as non-photosynthetic algae.

Notes: Abstract Four thiocyanatopyrazole derivatives were synthesized and their fungistatic activity was demonstrated in vitro against a number of dermatophytic fungi. In Trichophyton mentagrophytes, the most active compound induced an unusual increase of the plasma membrane with production of intra and extracytoplasmic complexes, a deterioration of nuclear and mitochondrial membranes and a formation of autophagic-like vacuoles. Plasmolysis, accompanied by an almost complete disorganization of cytoplasmic structures, seemed to be the final event. A possible mechanism of action of the compounds was discussed.

Notes: Abstract Point defect agglomerates in dislocation-free silicon crystals, usually called “swirls”, have been investigated by means of high-voltage electron microscopy. It was found that a single swirl defect consists of a dislocation loop or a cluster of dislocation loops. By contrast experiments it could be shown that these loops are formed by agglomeration of self-interstitial atoms. Generally the loops have a/2〈110〉 Burgers vectors, but in specimens with high concentrations of carbon (∼1017 cm−3) and oxygen (∼1016 cm−3) also dislocation loops including a stacking fault were observed. In crystals grown at growth rates higher thanv=4 mm/min no swirls are observed; lower growth rates do not markedly affect the size and shape of the dislocation loops. With decreasing impurity content (particulary of oxygen and carbon) the swirl density decreases, whereas the dislocation loop clusters become larger and more complex. A model is presented which describes the formation of swirls in terms of agglomeration of silicon self-interstitials and impurity atoms.

Notes: Summary Injection of α-ecdysone into the larval haemolymph of late third instar larvae ofD. virilis induces both the extrusion of secretory proteins and the inactivation of the enzyme glutamine-fructose-6-phosphate-aminotransferase (E.C. 2.6.16) in the salivary glands. In the presence of actinomycin D or cycloheximide the hormone is ineffective. If before adding these inhibitors RNA synthesis is allowed to proceed for 1.5h, or protein synthesis for 2h after ecdysone injection, however, the protein extrusion and the enzyme inactivation do occur. It is proposed that ecdysone controls these two cytoplasmic events at the transcriptional level by the activation of specific Correlations with puff activities are discussed.

Notes: Summary 1. The developmental potentials of dissociated cells of the different regions of the male foreleg disc ofDrosophila melanogaster were analysed. To this end, various amounts of foreleg disc material were dissociated together with an excess of heavily irradiated wing discs (“feeding layer”), and the reaggregates were cultured for 10 days in the abdomens of adult hosts prior to metamorphosis. 2. The foreleg disc cells were in most cases unable to regenerate missing structures in a circular direction within the leg segments. Instead they strongly tended to adopt the specifications of more distal leg segments (distal transformation), irrespective of the region of origin of the ancestor cells within the disc. 3. The distal transformation occurred mainly, if not exclusively, during an early phase (“initial phase”) in the reaggregates. 4. The extent of distal transformation was most pronounced in those series in which the foreleg cells were initially least diluted by the “feeding layer” cells. 5. Cells of the lower lateral quadrant were very poor both in proliferative activity and in the extent of distal transformation, compared to cells of the three remaining quadrants. In the experiments with a low initial dilution of the foreleg cells, cells of the lower medial quadrant underwent distal transformation much more distinctly than cells of the upper medial and the upper lateral quadrants. 6. Allotypic structures occurred exclusively in reaggregates of the upper medial and upper lateral quadrants. In these implants, however, the frequency of transdetermination was extremely high. 7. Two alternative mechanisms are discussed which could have led to the general occurrence of distal transformation. They differ in the basic assumption of whether or not the “feeding layer” cells were able to interact with the leg cells to influence their regulative behaviour. In addition, interactions among the leg cells themselves seemed to stimulate proliferation to varying degrees and may account for the observed differences in the degree of distal transformation.

Notes: Summary Three-hundred and twenty fertile,pal-induced Y-chromosome mosaic males and females were obtained. Fractional analysis of the sons of 55 somatically mosaic flies that were also germinally mosaic tentatively suggests that the number of functional primordial germ cells inDrosophila melanogaster is variable and that it is seldom greater than 24. From the observed 0.17 frequency of germinal mosaicism it was estimated that the average number of pole cells at the end of blastoderm formation is 45. At present, the germ cells afford the only opportunity to compare genetic estimates of the number of blastoderm or primordial cells with available histological counts. The good agreement between them suggests that both the fractional and the mosaic frequency methods for estimating primordial or blastoderm cell numbers of various larval and imaginal anatomical structures provide reasonably close approximations of the actual values.

Notes: Summary 1. Histological analyses were made of imaginal discs and histoblasts during the larval development ofDrosophila melanogaster to determine the number of cells, the patterns of cell division and the growth dynamics in these adult primordia. Histological studies were also made of the imaginal rings which are the primordia of the adult salivary gland, fore-and hindgut, the anlage cells of the midgut and several larval and embryonic tissues. 2. In the newly-hatched larva, the immature eye-antenna, wing, haltere, leg and genital discs contain about 70, 38, 20, 36–45 and 64 cells respectively. These numbers include cells destined to form cuticular elements as well as peripodial, tracheal and nerve cells and probably the progenitors of adepithelial cells. The number of cells counted in the various imaginal disc anlagen is 1.5 to 4 times higher than the numbers deduced from genetic mosaic analyses by other investigators and reasons for these differences are given. 3. About 12 h after fertilization, mitosis ceases in all tissues of the embryo except the nervous system. After the larva hatches, mitosis resumes in most of the imaginal anlagen and in some larval tissues. The time of resumption of mitosis in the imaginal anlagen was determined after treating the larvae with colchicine for 2 h. 4. Among the imaginal discs, the eye disc is the first to begin cell division, at about 13–15 h after the hatching of the larva (first instar) followed by the wing (15–17 h), the haltere (18–20 h), the antenna, leg, and genitalia (24–26 h, early second instar), and finally the labial and dorsal prothoracic discs (52–54 h, early third instar). The cell doubling time for various discs was calculated from cell counts and the times agree closely with the doubling times deduced from clonal analyses by other workers: e.g., 7.5 h for the cells of the wing disc. 5. The imaginal ring of the hindgut first shows cell division early in the second instar. The imaginal rings of the foregut and salivary glands, the anlage cells of the midgut and the cells of the segmental lateral tracheal branches begin to divide early in the third instar. 6. The histoblasts which are the anlagen of the integument of the adult abdomen do not increase in number from the time of larval hatching until about 5 h after pupation when they begin to divide. Their behaviour contrasts with that of the histoblasts of the other dipterans such asCalliphora, Musca andDacus, which begin to divide during the second instar. 7. The histoblasts are an integral part of the larval abdominal epidermis and, unlike imaginal disc cells, secrete cuticle during larval life. Each hemisegment consists of an anterior dorsal, a posterior dorsal, and a ventral histoblast nest containing about 13, 6 and 12 cells respectively. The 62 histoblasts in each larval segment represent about 7–8% of the total number of cells that form the integument of that segment. 8. The number of cells in a particular type of histoblast nest was constant for both male and female larvae and among the different abdominal segments, except that the anterior dorsal group of the first and the seventh segments contains fewer cells than those of the other segments. Although the male and female adultDrosophila lack the first abdominal sternite and the male lacks the seventh abdominal tergite and sternite, the ventral histoblast nests of the first and the dorsal and ventral nests of the seventh abdominal segments are present in the larval stages as well as in the prepupa and have the same morphology and cell number as similar nests in the rest of the abdominal segments. 9. The cells of the imaginal discs increase in volume about six-fold and their nuclei increase in volume three-fold between the time of hatching and the initiation of mitosis. The histoblasts increase in volume about 60-fold and their nuclei increase in volume about 25-fold between larval hatching and pupariation. 10. Prior to each cell division, the nuclei of the columnar cells of the disc epithelium and of the histoblasts appear to migrate toward the apical surface of the epithelium. The cells round up and shift toward the apical region where mitosis occurs. After cytokinesis, the daughter cells move back to deeper positions in the epithelium. Because the nuclei of the non-dividing cells continue to lie deep in the epithelium, this intermitotic migration of nuclei gives these epithelia a pseudostratified appearance. 11. Analyses of the growth of larval cells and of organs confirmed the observations of earlier investigators that cell division occurs only in a few larval tissues, whereas growth in the rest of the larval tissues is by cell enlargement and polyteny. During larval life, cell division was detected only in the central nervous system, gonads, prothoracic glands, lymph glands and haemocytes. Each tissue began mitosis at a characteristic stage in larval life. The larval cells that did not divide, grew enormously, e.g., epidermal cells increased in volume 150-fold and their nuclei increased in volume 80-fold. 12. The adepithelial cells, which give rise to some of the imaginal muscles, were first identified between the thick side of the imaginal dise epithelium and the basement membrane at the beginning of the third larval instar (50–52 h). The origin of these precursors of mesodermal structures was analysed and evidence is presented that the adepithelial cells come from the disc epithelium. The question of the origin of the mesoderm of cyclorrhaphan Diptera is reviewed and it is suggested that the imaginal disc ectoderm may become segregated from the rest of the embryo before gastrulation has occurred, that is before the mesoderm has been established.

Notes: Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis. The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos. Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development. All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae. Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.

Notes: Summary The lineages of cells on the second-leg basitarsus ofDrosophila melanogaster were analyzed by examining gynandromorphs andMinute mosaics. Bracts lie proximal to bristles on the adult basitarsus, yet bract precursor cells were found to originate lateral to bristle precursor cells. In 6 of the 8 longitudinal rows of bristles on this segment, the bract cells arise ventral to the bristle cells; in the others they arise dorsally. The lateral cell origins are interpreted as reflecting a pattern of lateral cell movements associated with evagination of the leg disc. An unusual discrepancy was observed in the relative frequencies of male vs. female bracts and bristles in gynandromorphs. The discrepancy suggests that there is a cell-autonomous sexual difference in either the time at which cells begin moving during evagination or the speed with which they move. On the basis of the results, it is reasoned that the bristle pattern of the basitarsus does not originate in its final form. Prior to evagination, the bristle cells of each row are apparently closer together than in the final pattern, and the rows are farther apart. Evidence is presented which suggests that the bristle cells of each row may originally be arranged in a jagged line which is later straightened by cell movements. The two locations where the anterior/posterior compartment boundary of the second leg passes through the basitarsus were found to vary relative to the bristle pattern. If this boundary is assumed to be a fixed line of positional values, then the extent of the observed variability — which is estimated to be ± 1 or 2 cell diameters — provides a measure of the precision of patterning around the circumference.

Notes: Summary The differentiation of muscles in primary cultures of cells fromDrosophila melanogaster embryos was investigated. In early cultures, and in the absence of exogenous ecdysone, two main classes of muscle were found. Comparison, by light and electron microscopy, of one of these classes (the “myotube” class) with muscles from third instar larvae shows that this class corresponds to the muscles of the body wall of the larva. When α- or β-ecdysone is added to the cultures, these undergo a number of metamorphic changes. Most of the larval muscles disappear, and two new types of muscle form. Ultrastructural and light microscopic examination of these two types indicates that they correspond to the two classes of skeletal muscle (fibrillar and tubular) found in adult flies.

Notes: Summary Cartilaginous cells of aged newts (Triturus cristatus) were studied during hind limb regeneration. The electron microscope was used to study the structure and distribution of chromatin in the cell nuclei, while the DNA content of the chromatin was measured by means of a scanning cytophotometer. Changes in the ultrastructure of the cytoplasm during regeneration were also studied. It was observed that the structure and distribution of chromatin in the activated cell is greatly modified. In the non-activated cell of the aged newt, the chromatin is found highly condensed and distributed peripherally close to the nuclear membrane. In contrast, in the activated cells, the chromatin is much less condensed and is distributed throughout the nucleus. Moreover, cytoplasmic vacuoles, found only in the non-activated aged cells, disappear and an increase in the mitochondria and rough endoplasmic reticulum is also observed. Changes in the nuclear structure are observed prior to the cytoplasmic modifications. It is interesting to note that the process of activation induces structural changes in the aged cells which make these cells appear to be structurally identical to the young cells. This process of rejuvenation takes 3–5 days in the newt. We suggest that these structural changes of the chromatin and cytoplasm in the aged cells are necessary to increase the metabolic activity which precedes cell division. It may also explain why regeneration takes a longer time in the aged animals than in the young ones.

Notes: Summary The pathway of adult sensory nerves has been analysed in three experimental situations: (i) in flies with grossly abnormal thoracic morphology resulting from X-irradiation early during development, (ii) in flies which had been subjected to surgical operations late in the larval period, (iii) in homoeotic mutants. The results provide experimental support for a simple mechanism in which developing adult axons join the nearest larval nerve and are guided by it up to the central nervous system. In particular, experimental interference with normal development can result in nerves from different segments, or from dorsal and ventral appendages, joining each other and entering the central nervous system together.

Notes: Summary The mature labial disc, when implanted into a larva of the same age, undergoes metamorphosis along with the host and produces one lateral half of the medi- and distiproboscis. On the basis of results obtained from transplanted disc halves (including the separate peripodial membrane) a tentative fate map of the labial disc was constructed, which shows most of the presumptive mediproboscis to be located in the dorsal, and most of the presumptive distiproboscis in the ventral part of the disc. The distal protion of the peripodial membrane also contains imaginal anlagen, viz. part of the mediproboscis, prementum, and labellar cap anlagen. The involvement of this part of the peripodial membrane was checked by a careful histological analysis of labial disc development during the first ten hours after prepupation. The results were compared with the situation described forCalliphora imaginal discs. In addition, a detailed morphological analysis was made of the proboscis of the homoeotic mutantproboscipedia (pb). At 27°C,pb changes the distiproboscis into a “telopodite” (leg segments distal to the coxa); the (unchanged) prementum may therefore correspond to the coxa. At 15° C, the tarsus of this homoeotic “telopodite” is replaced to a greater or lesser extent by an arista. The present analysis thus confirms (a) the fundamental morphological correspondence of the medi- and distiproboscis with the labium of other insects, and (b) the fundamental developmental correspondence of the labial, antennal, and leg discs.

Notes: Summary A temperature-sensitive period during early embryogenesis for three stocks carrying thetuh-3 gene suggests that it is a homoeotic mutation involved in the initial determination of the eye-antennal disc rather in maintenance of the determination.

Notes: Summary The development of the adult abdomen ofDrosophila melanogaster was analyzed by histology, microcautery, and genetic strategies. Eight nests of diploid histoblasts were identified in the newly hatched larva among the polytene epidermal cells of each abdominal segment: pairs of anterior dorsal, posterior dorsal, and ventral histoblast nests and a pair of spiracular anlagen. The histoblasts do not divide during larval life but begin dividing rapidly 3 h after pupariation, doubling every 3.6 h. Initially they remain confined to their original area, but 15 h after pupariation the nests enlarge, and histoblasts replace adjacent epidermis cell by cell. The histoblasts cover half the abdomen by 28 h after pupariation and the rest by 36 h. Polytene epidermal cells of the intersegmental margin are replaced last. Cautery of the anterior dorsal nest caused deletion of the whole corresponding hemitergite, whereas cautery of the posterior dorsal nest caused the deletion of the macrochaetae of the posterior of the hemitergite. Cautery of the ventral nest deleted the hemisternite and the pleura, whereas cautery of the spiracular anlagen deleted the spiracle. Results of cautery also revealed that no macrochaetae formed on the tergite in the absence of adjacent microchaetae. Clonal analysis revealed that there were no clonal restrictions within a hemitergite at pupariation. Cautery of polytene epidermal cells other than those of the intersegmental margin failed to affect tergite development. However, cautery of polytene epidermal cells of the intersegmental margin adjacent to either dorsal histoblast nest caused mirror-image duplications of the anterior or posterior of the hemitergite in 10% of the hemitergites. Forty percent of the damaged presumptive hemitergites formed complete hemitergites, indicating extensive pattern regulation and regeneration. Pattern duplication and regeneration were accounted for in terms of intercalation and a model of epimorphic pattern regulation (French et al., 1976). Histoblasts in adjacent segments normally develop independently, but if they are enabled to interact by deleting the polytene epidermal cells of the intersegmental margin, they undergo intercalation which results in duplication or regeneration. The possible role of the intersegmental margin cells of insects in development was analyzed.

Notes: Summary The development of the rhabdomeric pattern in the compound eye ofDrosophila has been studied using combined transplantation and electron microscope techniques. In a first series of experiments eye imaginal discs of increasing age were implanted into larvae ready to pupate, thus losing variable amounts of the normal time for development. A sequence of differentiative abilities was found in the metamorphosed test pieces. As far as the photoreceptor cells are concerned, the most prominent steps of this sequence are: ability to form groups with other similar elements, anatomical polarization of microvilli, establishment of the rhabdomeric pattern and formation of an equator line. The stability of determination of the equator line was tested in a second experimental series. Fragment of different topographical origin within the mature eye anlage were brought to metamorphosis by implantation into larvae ready to pupate. It was found that an equator line differentiates only in those pieces which according to the published anlage maps contain the prospective equator region prior to metamorphosis. The mitotic abilities of implanted eye imaginal discs were investigated by means of “in vitro”3H-thymidine pulse-labelling and light microscope autoradiography of the differentiated test pieces. During the third larval stage the eye anlage is traversed by two consecutive mitotic waves, each one of them producing different categories of receptor cells. The first, anterior wave predominantly produces cells oriented toward the poles of the eye within the ommatidia, while the second, posterior wave gives rise to elements exclusively in an equatorial position. The dynamics of this proliferation are discussed in relation to the findings in the implantation experiments. Silver-grain counts support the possibility that at least two successive cell divisions occur in the eye anlage between labeling with tritiated thymidine and beginning of morphological differentiation. The relevance of this finding for the understanding of the concept of acquisition of competence is discussed.

Notes: Summary 1. Eggs of the midgeSmittia were investigated by light microscopy and transmission electron microscopy. This paper describes elements and architecture of periplasm and yolk endoplasm before the formation of pole cells. 2. The periplasm is coated externally by the oolemma and a multilayered egg shell. The periplasm consists of a cytoplasmic matrix rich in ribosomes; it contains mitochondria and ER cisternae, some annulate lamellae and an occasional Golgi complex. Microtubuli were demonstrated only rarely. Accumulations of a dense granulated substance resembling in its structure the oosome material were frequently observed. 3. The yolk endoplasm is a cytoplasmic network embodying proteid yolk particles, lipid droplets and accumulations of glycogen. The endoplasm is continuous with the periplasm and shows the same cell constituents. It may form between 3 and 7 cytoplasmic islands free of yolk particles. Rosette-shaped membranous structures in the yolk endoplasm are interpreted as nuclear envelope organizing centres. 4. Three carefully analysed eggs contained 2 nuclei each. both nuclei were situated in the posterior egg half. 5. Periplasm and yolk endoplasm are characterized by random distribution of cell elements. No zonation or special accumulations could be recognized. 6. The spatial distribution of the egg components studied did not indicate that any of these components could function as a determinant in embryonic pattern formation.

Notes: Summary 1. The egg ofPollicipes polymerus, the common intertidal gooseneck barnacle, has been studied by electron microscopy. Constriction rings, similar to the contractile rings of cleaving cells and polar lobes, move unidirectionally from the animal to the vegetal pole of newly fertilized eggs. This is referred to as peristaltic constriction. The present paper describes the fine structure of the egg during first polar body formation and peristalsis. 2. During formation of the polar body, dense bodies are produced by the Golgi and extracellular plaques are observed. Thin microfilaments (40–60 Å) are in the egg adjacent to the polar body. 3. In eggs undergoing peristalsis, the appearance of extracellular spheres, flocculent material and filaments is observed. Intracellularly large numbers of multivesiculate bodies, glycogen granules, mitochondria and protein-carbohydrate and lipid yolk bodies are seen at the level of constriction. 4. Thin microfilaments are found in the cortical area of newly-fertilized eggs exclusively in peristaltic constriction rings. Filaments are oriented primarily in a meshwork, although circumferentially-oriented filaments are also found in rings near the vegetal pole. Microvilli extend into the space created between a constriction and the elevated egg membrane. 5. A model is proposed to explain the peristalsis in this species. It is suggested that information from a pacemaker region activates peristalsis by affecting filament polymerization and orientation. One function of peristalsis may be elongation of the egg from a sphere to an ovoid, although other possibilities such as elevation of the egg membrane, segregation of the lipid yolk to the vegetal pole and predetermination of the first cleavage plane are also discussed.

Notes: Summary The derivatives of 110 mosaic genital discs of gynandromorphs have been analysed microscopically. It has been found that theanalia of both sexes are homologous and derive from a single primordium (see Fig. 1a). Whether male or female anal plates are formed depends on the genetic constitution of the cells. This is analogous to the development of male sex combs versus female transversal rows on the forelegs of gynandromorphs. In contrast, the data for thegenitalia (see Fig. 1 b) are best explained if it is assumed that there are two genital primordia in everyDrosophila embryo: a male primordium that will only develop into genitalia if populated by XY (or XO) nuclei, and a female primordium that will only do so if populated by XX nuclei. This model, as depicted in Figure 2, is compatible with all our gynandromorph data and also with observations onMusca andCalliphora where in fact two separate genital primordia are found.

Notes: Summary Five regions of the compound eye have been found to be preferential boundaries for clones of labelledMinute + cells, and to act restrictively on the growth of cell clones after a given developmental stage. One of these regions is topographically related to the line of pattern inversion existing at the level of the equator. The results of experiments showing independency of origin of restriction lines and line of pattern inversion are reported.

Notes: Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.

Notes: Summary sev LY3,the only existing allele at thesev locus (1–33,2±0,2), behaves as strongly hypomorph or even as amorph. Ommatidia in asev compound eye have only seven receptor cells, the position of the R7 pattern element being vacant. Various criteria showing that the missing cell is R7 have been verified. These include (i) anatomical characteristics ofsev ommatidia; (ii) behaviour of central R cells insev rdgB double mutants; (iii) medullary projection of central R cell axons; and (iv) mitotic pattern ofsev imaginal discs. The analysis of morphogeneticsev-sev + mosaics has shown thatsev is expressed autonomously by R7 cells, indicating that thesev phenotype is not due to asev genotype of ommatidial pattern elements other than R7. The study of third instarsev imaginal discs has not brought any direct evidence for death of clustered presumptive R7 cells; however, clonal analysis of the developingsev compound eye has given evidence of developmental parameters comparable to those ofsev +, therefore favouring the hypothesis that R7 cells die insev mutants. On the other hand,sev + seems to be required for the determination of the R7 cells, since thesev phenotype cannot be uncovered during the last mitoses of heterozygous mutant cells.

Notes: Summary These experiments examined whether inDrosophila immature imaginal disc tissue and tissues from embryonic stages can influence pattern regulation in a disc fragment in the same way as can mature imaginal discs. Immature imaginal discs, or the cells of whole embryos, were mixed with a test fragment (presumptive notum) from a mature wing disc. The immature tissues in each mixture were genetically marked and had been heavily irradiated (25 Kr gamma) prior to mixing to prevent growth and maturation during subsequent culture in vivo. Alteration of the regulative behavior of the test fragment (that is, regeneration of wing) thus provided an assay for the communication of positional information by the immature tissues. The results suggest that this capacity arises well before competence to metamorphose, as early as the 16th hour of embryonic development, whereas prior to 16 h, essentially no stimulation of regeneration occurred. It is suggested that the imaginal disc (or presumptive disc) cells of the embryo may have been responsible for this early stimulatory capacity.

Notes: Summary The ontogeny of allozyme patterns has been studied in embryos ofDrosophilamelanogaster, which are doubly heterozygous for alleles specifying the slow and fast forms of alcohol dehydrogenase (ADH) and α-glycerophosphate dehydrogenase (GPDH). The ontogeny of esterase-2 was studied in embryos and young larvae of the flour mothEphestia kühniella, which are heterozygous for two of the three existing esterase-2 alleles. In freshly laidDrosophila eggs only the maternal enzyme forms are present and during the first 15 hours of development the staining of these forms becomes progressively fainter. After 16 and 17 h, the paternal and hybrid bands of ADH and GPDH respectively become obvious. Before hatching, the intensity distribution in the three-banded pattern of reciprocal hybrids is asymmetric in favour of the persisting maternal enzyme form. InEphestia embryos, however, there is no persistence of the maternal esterases. In all reciprocal heterozygotes a three-banded pattern suddenly appears 96 h after egg deposition, indicating synchronous activation of both parental alleles. The relative intensity distribution in the hybrid patterns approaches that of the mature larvae stepwise and in an allele-specific manner. This result and the fact that the various heterozygous types exhibit unequal total activities suggest that the Esterase-2 alleles have different activities, which are fixed late in embryogenesis.

Notes: Summary The females produced in the crossesD. melanogaster×D. simulans andD. melanogaster×D. mauritiana are sterile and have reduced ovaries. Normal and fertile ovaries were produced when genetically marked pole cells ofD. melanogaster were transplanted into eggs which gave rise to the hybrid females. These results eliminate the possibility that the sterility of these hybrids is due to the somatic component, i.e. the follicular cells of the ovaries, or to other physiological causes. The results also suggest that the control of gonadal morphogenesis is dependent mainly on the germ line.

Notes: Summary Gynandromorphs with female XX-and male XO-areas result from the loss of an unstable ring-X-chromosome in the early cleavage mitoses of ring/rod-X-chromosome heterozygotes. The phenotypes of the recessive alleles on the rod-X-chromosome are expressed in the XO-areas. 377 larval gynandromorphs of the genotypeR(1)2, In(1)w vC /y w sn3Iz50e mal were examined and scored for the phenotypes of 13 paired and 10 unpaired structures (Table 2, Fig. 2). This was possible mainly by the cell-autonomous expression of aldehyde oxidase activity in soft tissues and by the comparison of the distribution of enzyme activity in wildtype and gynander larvae. The distances between pairs of structures were calculated in sturt-units (Tables 3 and 4). A morphogenetic fate map with the presumptive areas of larval structures was constructed (Fig. 3). The relative positions of the structures agree well with Poulson's fate map (Fig. 4). In addition, the distribution of phenotypes was scored in 380 adult gynandromorphs Table (5). The fate map (Fig. 5) which was constructed from these data is very similar to the fate map of larval structures. This similarity becomes even more pronounced if fate maps are constructed which contain only structures analogous in larva and imago (Table 6, Fig. 6). Therefore an attempt was made to set up an integrated morphogenetic fate map containing the presumptive areas of both larval and imaginal structures (Fig. 7). The possibilities of further blastoderm mapping are discussed.

Notes: Summary Females homozygous for a newly isolated mutation induced by ethyl methane sulphonate,fs(1)K10, lay abnormally shaped eggs in which the dorsal appendages of the chorion are enlarged and fused ventrally. The eggs are usually not fertilized and development is never normal beyond the blastoderm stage. The mutant was mapped to the tip of the X-chromosome with a meiotic position of 1–0.5 and a cytological location between 2B17 and 3A3. Using germ line mosaics constructed by transplantation of pole cells, it was shown that the abnormal morphology and the sterility are obtained only when the germ line is homozygous for the mutant.

Notes: Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.

Notes: Summary The bristle pattern of the second-leg basitarsus inDrosophila melanogaster was studied as a function of the number and size of the cells on this segment in well-fed and starved wild-type flies, in triploid flies, and in two mutants (dachs andfour-jointed) that have abnormally short basitarsi. The second-leg basitarsi of well-fed, wild-type flies from 22 otherDrosophila species were studied in a similar manner. There are typically 8 longitudinal rows of evenly-spaced bristles on the second-leg basitarsus, and in each row the number of bristles was consistently found to vary in proportion to the estimated number of cells along the segment, and the interval between bristles was found to vary in proportion to the average cell diameter on the segment. These correlations are interpreted to mean that the spacing of the bristles within each row is controlled developmentally, whereas the number of bristles is not. The interval between bristles is evidently measured either as a fixed number of cells or as a distance which indirectly depends upon cell diameter.

Notes: Summary Electron microscopic analysis of primary cultures derived from larvalXenopus liver has shown that these cells, although they form only two-dimensional aggregates, retain and presumably also develop structural characteristics typical of liver parenchyma cells, such as bile canaliculi with microvilli and epithelial junctional complexes. As judged from structural criteria, primary cultures contain 80–90% hepatocytes. In contrast to the intact tissue, primary cultures showed excessive development of microfilaments, however. Incorporation of labeled amino acids has revealed further that the capacity for protein synthesis is maintained in culture and that synthesis of liverspecific protein albumin is maintained in vitro, even in liver cultures derived from thyrostatic tadpoles. This latter result suggests that initiation of albumin synthesis in the larval liver is probably not dependent upon thyroid hormones but rather reflects the protodifferentiated state of this tissue.

Notes: Summary Salivary gland cells of members of theDrosophila melanogaster group (from four different subgroups) were examined electron microscopically and histochemically during the late larval period of development. The secretory product, which is supposed to be utilized as ‘glue’ at the time of puparium formation, appears, by analogy to Palade and Jamieson's results, to be synthesized partially in the rough endoplasmic reticulum (RER) and partially in the Golgi complex. The latter is also the usual site of the packaging of the product into secretory granules, except in the case of one of the secretory granule components ofD. lucipennis. The phylogenetic relationships among the subgroups, implied by the morphological appearance of the secretory granules, fit well with the existing phylogenetic relationships within the group. The secretory granules of each species have their own morphological features; granules of species of the same subgroup share some of these features. Secretion occurs from the cells via exocytosis during which the morphology of the secretory granules changes. Light microscope examination of PAS (Periodic Acid-Schiff reaction) stained glands shows a strong positive reaction in most species, with the exception of the species of thesuzukii subgroup which show a weak, or a negative reaction (D. rajasekari). Electron histochemical localization of polysaccharides in the secretory granules was possible inD. melanogaster and the species of theananassae subgroup.

Notes: Summary The generalogical relationships of photoreceptor cells within the compound eye ofDrosophila have been studied using cell labelling, with either3H-thymidine or recessive mutations, during the third larval stage. It has been found that photoreceptor and secondary pigment cells arise from different precursor cells. Under the present experimental conditions, precursors of receptor cells give rise to about 8 elements which differentiate as R cells of two different groups. One of the cells differentiates as R7 and the remaining as any one of the R1 to R6. The last cells behave initially as equivalent, and can differentiate within the same or within different, but neighbouring, ommatidia. The class of R1 to R6 cell in which each one of these elements differentiates, seems to depend on the time of its origin. The implications of these findings for the formation of the ommatidial pattern are discussed.

Notes: Summary The morphogenetic differentiation of synapses of the optic tectum of the rainbow trout was investigated at different stages of development (from hatching to adult) and compared with the improvement in visual discrimination (minimum separable). (1) The main phase of synaptogenesis (increase in number of synapses, length of contact zone and number of vesicles) begins about one week after hatching and continues up to the age of one month, when the larvae start swimming freely. (2) Myelination begins 26 days after hatching and induces the end of the synaptogenesis period. (3) The visual discrimination (minimum separable) of trout larvae improves from 30 degrees of arc on the 10th day after hatching to 1 degree on day 30, then to about 14 to 18 min of arc in the adult. The results are discussed with special reference to previous biochemical investigations on changes in the ganglioside composition of the trout brain during comparable periods of development.

Notes: Summary We report on the size distribution of clones marked by mitotic recombination induced by several different doses of X-rays applied to 72 h oldDrosophila larvae. The results indicate that the radiation significantly reduces the number of cells which undergo normal proliferation in the imaginal wing disc. We estimate that 1000 r reduces by 40–60% the number of cells capable of making a normal contribution to the development of the adult wing. Part of this reduction is due to severe curtailment in the proliferative ability of cells which nevertheless remain capable of adult differentiation; this effect is possibly due to radiation-induced aneuploidy. Cytological evidence suggests that immediate cell death also occurs as a result of radiation doses as low as 100 r. The surviving cells are stimulated to undergo additional proliferation in response to the X-ray damage so that the result is the differentiation of a normal wing.

Notes: Summary Immature ovaries ofDrosophila mercatorum were injected into young larvae and into adult males ofD. mercatorum, D. melanogaster, D. hydei, D. virilis, andZaprionius vittiger. These homo- and heteroplastic transplantations allow normal vitellogenesis to occur in the donor ovary. By SDS gel electrophoresis, we identified the major species-specific yolk proteins of mature eggs (stage 14) which were exclusively of donor-specific origin. Other experiments withD. hydei andZ. vittiger showed that, when females were used as hosts, the host-specific yolk proteins became incorporated into the donor eggs. When two immature ovaries, one ofD. mercatorum and one ofD. hydei, were co-cultured in males, again only the donor-specific yolk proteins were found in the mature eggs implying that these yolk proteins were not released into the host hemolymph. A parthenogenetic strain ofD. mercatorum was used to demonstrate the ability of transplanted immature ovaries to produce viable eggs which can give rise to fertile adults. The role of the species-specific yolk proteins is discussed with respect to the dual origin of these proteins during normal vitellogenesis, i.e., an autonomous synthesis within the ovary itself in addition to the well-known production by the fat body. Further experiments with pupae as hosts indicate that even in the absence of juvenile hormone and in the presence of high doses of ecdysone, vitellogenesis can proceed within the donor ovary. Based on these experiments, a new hyopthesis on the hormonal control of vitellogenesis inDrosophila is presented. We propose that yolk proteins derived from the fat body are controlled by juvenile hormone, whereas the independent and autonomous vitellogenesis within the ovary itself is controlled by endogenously synthesized ecdysone.

Notes: Summary We estimate the number of blastoderm cells which generate the thoracic imaginal discs ofDrosophila. At hatching the wing disc is twice the size of the haltere disc, but the results suggest that both discs develop from a similar number of blastoderm cells. Two homeotic mutations, which transform the haltere into wing, affect embryonic growth but not the primordial number. All the segmental primordia may be of similar size and each may be similarly subdivided into a larger anterior, and a smaller posterior polyclone.

Notes: Summary The embryonic organization of the sexually dimorphic genital disc was studied in genetic mosaics resulting (a) from early loss of a chromosome or (b) from mitotic recombination. (a) Early Loss of a Chromosome. Three types of mosaics were produced — purely female mosaics, purely male mosaics, and gynandromorphs. They show that the genital disc arises from a group of cells in the ventral region of the embryo somewhat larger than that giving rise to a single foreleg (Table 2). Within this group of cells three regions can be distinguished that are present in both sexes: an anterior, a medial, and a posterior one, with distances of only 3–4 sturts between adjacent regions. The anterior region gives rise to the female genitalia, the medial region to the male genitalia, and the posterior region forms the analia of both sexes and the parovaria of the female (Figs. 2 and 3). The relative positions of the three regions were deduced from sturt distances (Tables 1 and 5), and from frequencies of mosaicism (Table 2). (b) Mitotic recombination was induced at the blastoderm stage in order to produce twin spots in the external genitalia and analia of purely male and female flies. Clone sizes indicate that these structures arise from a small number of precursor cells (Table 4). Clones overlapped right and left sides, but no clones were found extending over analia and genitalia. However, within either the analia or the genitalia of each sex, no clonal restrictions could be observed, and the clones comprised structures that were up to 12 sturts apart. A comparison of clone sizes and sturt distances in the foreleg and in the genital disc indicates that equal gynandromorph distances involve equal numbers of cells in different regions on the ellipsoid egg (Fig. 5). The results obtained from all mosaics provide a consistent picture of the embryonic organization of the genital disc. This becomes apparent in the summarized fate maps (Fig. 4), where the map derived from normal gynandromorphs can be produced by a simple superposition of the male and the female maps. The data are also discussed with respect to mechanisms of sexual differentiation in the genital disc.

Notes: Summary Mutations of the bithorax complex result in segmental transformations in the thorax and abdomen ofDrosophila. The haltere discs from larvae homozygous forbx 3 orpbx are transformed so that the discs contain cells that will produce wing cuticle as well as cells that produce haltere cuticle. The pattern regulation behavior of these discs has been examined. The fate maps of the two discs were established, and then the regulative behavior of a number of fragments from both types of mutant discs was established by culturing the fragments in vivo prior to metamorphosis. The most important conclusion from this work is that the cells producing, haltere cuticle and wing cuticle within the same disc share the same positional information and that they communicate during pattern regulation.

Notes: Summary The ultrastructural changes in the wing bud afterapical ectodermal ridge (A.E.R.) removal was studied to re-examine the issue of distal mesenchymal cell death. The A.E.R. of the right wing bud was removed microsurgically from chick embryos of stages 18 to 22 (HH 1951). The wing buds were examined at three hour intervals up to twelve hours after the operation with light, transmission and scanning electron microscopy. The main findings were: (1) Immediate and temporary shrinkage of the mesenchymal extracellular space 100 to 150 μm and chromatin condensation in the cells 50 to 75 μm from the wound. (2) Death of ectodermal and mesenchymal cells in the immediate vicinity of the wound. (3) Formation of a single squamous-like layer of mesenchymal cells to cover the wound. (4) Occasional evidence of cell death in the distal mesenchyme at later times after the operation. The pattern of cell death observed suggests only a traumatic etiology, and gives little evidence for the postulated developmental significance of cell death following A.E.R. removal.

Notes: Summary Pairs of eye-antennal discs, attached to the cephalic ganglia, were cultured in vitro with a concentration of β-ecdysone optimal for imaginal differentiation. The eye-antennal discs fused to form a vesicle inside which the antennae were partially everted, and on the inner surface of which imaginal structures differentiated. The epithelium of the discs was continuous, and an integrated pattern of bristles and hairs differentiated in vitro. In particular, the median ocellus, a unified structure derived partially from each disc, differentiated normally.

Notes: Summary l(1)su(f)mad-ts (mad) is a new temperature-sensitive (ts) lethal mutant ofDrosophila melanogaster which produces duplicated legs after temperature pulse treatment during larval development. The ts-lethality was studied in temperature experiments and genetic mosaics. Temperature pulses given during two distinct TSPs of larval development result in two different types of leg pattern duplication. “Total” differ from “partial” duplications with respect to the affected leg compartments and the orientation of the planes of symmetry which are perpendicular to the dorso-ventral and the proximo-distal leg axes in total and partial duplications, respectively. Genetic mosaic studies indicate (i) disc autonomy of leg pattern duplication, (ii) clonal separation of the anlagen of the two pattern copies, and (iii) clonal restriction along the antero-posterior compartment border in the two pattern copies of totally duplicated legs. The results suggest thatmad leg pattern duplication is caused by a change in positional information rather than by cell death and subsequent regeneration. Our data are compatible with the assumption that during normal development the leg disc cells acquire information about their position within the disc with respect to the different leg axes independently and at different times.

Notes: Summary Two possible mechanisms are considered for the occurrence of experimentally or genetically induced duplications of bristles: extra cell division of a bristle mother cell versus determination of more than one mother cell. From a clonal analysis it appears that duplications induced by actinomycin-D arise by the latter mechanism, whereas those found in the mutantspl seem to arise by the former mechanism.

Notes: Summary The calcium distribution in cartilage and bone cells during beginning ossification of fetal mouse long bones was studied after fixation with 2% K-pyroantimonate in 1% osmium. In the developing periosteum, the future osteoblasts showed a sparse cation-antimonate precipitate over the cytoplasm. In young osteoblasts the precipitate was accumulated on the mitochondrial membranes and the plasmalemma. Both organelles were sharply outlined by precipitate in the mature osteoblasts at the onset of mineralization. X-Ray microprobe analysis of these organelles demonstrated the presence of both Sb and Ca. In the extracellular compartment, a collagen-associated precipitate with 50 to 60 nm periodicity appeared during osteoblastic differentiation. During the initial phase of matrix mineralization, a random gross precipitate appeared in the matrix and seemed to be accumulated by osmiophilic matrix vesicles while the collagen fibrils lost their precipitate. Subsequently, during the confluent phase of matrix mineralization, the precipitate rapidly disappeared from the cells, leaving them devoid of precipitate once they were surrounded by mineralized matrix. Similar changes were found in the chondrocytes of the growth plate, but cartilage collagen, unlike osteoid collagen, did not bind precipitate. The results indicate that both osteoblasts and calcifying cartilage cells bind calcium prior to matrix mineralization. Bone collagen has strong pyroantimonate binding capacity, but it is not directly involved with initial stages of matrix mineralization, which starts in close association with matrix vesicles.

Notes: Summary The mantle edge of the freshwater pulmonate snailsLymnaea stagnalis andBiomphalaria pfeifferi was investigated with histochemical and ultrastructural methods. The mantle edge gland, which is involved in shell formation, consists of the periostracal groove and the belt. This belt appears to be composed of various regions. In the area of the periostracal groove a number of subepithelial gland cell types occur; these release their products into the groove. Between the groove cells ciliated free nerve endings terminate; the corresponding perikarya occur in the subepidermal connective tissue. Also in the posterior belt region free nerve endings were observed between the epithelial cells; in addition, a particular type of subepithelial gland cell was found in this area. The epithelial cells of this part of the belt have the ultrastructural characteristics of ion and water transporting cells; they are probably involved in calcium deposition and resorption. The possible role of the free nerve endings and of the subepithelial gland cells is discussed.

Notes: Summary The fine structure of the extracellular phase of avian medullary bone and embryonic chick femur was examined in thin sections prepared by ultracryotomy and ultramicroincineration. Since contact with solutions was completely avoided, little or no loss or dislocation of mineral constituents could occur. Amorphous bone mineral (ABM) was present in two forms: as 15–30 nm spheres and as a structure-free haze. Removal of all organic material by low temperature ashing left the ABM intact. Crystals were usually associated with the ABM. In newly ossifying regions clusters or nodules of randomly oriented crystals and ABM appeared to coalesce when they reached approximately 1 μm in diameter. In highly calcified regions crystals appeared to be oriented along collagen fibers. ABM did not appear to be associated with collagen. Unmineralized collagen was visible in osteoid after staining with dry OsO4 vapor and it appeared to be diverted around nodules. Structures which resembled matrix vesicles were present. Selected area electron diffraction patterns indicated the presence of hydroxyapatite.

Notes: Summary The ultrastructure of calcifying cartilage and bone has been examined under the electron microscope after using three different methods of decalcification. The first was carried out before embedding (by soaking specimens in EDTA or formic acid), the second after embedding (by floating ultrathin sections on formic acid), and the third after embedding (by soaking embedded specimens in EDTA or formic acid), and with later re-embedding. The first procedure invariably induces drastic changes in the fine structure of the cells and calcified matrix, probably as a results of the extraction of organic material along with extraction of mineral. The second and third procedures make it possible to preserve ultrastructural details perfectly in both cells and calcified matrix. Of the two, the third procedure is preferable because of its greater simplicity. In areas that are still calcifying, these post-embedding decalcification techniques reveal the presence of crystal-associated, filamentous organic structures which are not recognizable in specimens decalcified before embedding. These structures, which could have a key role in inducing and regulating crystal formation and growth, are less evident in fully calcified areas (but not at their borders). This may partly be due to the loss of glycan components in the matrix during calcification. The most important determinant, however, seems to be the fact that during calcification the components of the matrix, including collagen fibrils, are involved in an aggregation process which reduces the amounts of free chemical groups available for reaction with the stain solution. Because post-embedding decalcification does not disturb this state of aggregation, the stainability of the matrix and the electron microscopic evidence of its components remain very low.

Notes: Summary An ultrastructural study of teeth with amelogenesis imperfecta revelaed various aspects of microcavities in the enamel surface, which ranged from isolated imprints of ameloblasts corresponding to the mildest lesions at the end of amelogenesis, to pits caused by the death of 20 to 30 ameloblasts at the beginning of amelogenesis. Abnormalities in the shape of the prisms can be observed. Further, crystals are distributed randomly within a prism or at the junction of 2 contiguous prisms while intercrystalline spaces are widened, indicating in various places the lack of a preferred orientation of the crystals. In amelogenesis imperfecta, two different crystalline periods are found: 1 of about 250 Å, the other of about 500 Å and over. The fact that amorphous areas are found among the crystals of enamel may be related to different stages of crystallization. However, it was not possible to find any lattice defect.

Notes: Summary Examination of microradiographs from the deciduous teeth of pigs revealed large lacunae or radiolucent zones close to the cemento-dentinal junction. Electron microscopic studies of the ground sections showed areas or irregularly shaped zones devoid of mineral and filled with collagen fibers. In the wide unmineralized zones, spherical clusters of crystallites were noted. Several cementum lacunae bordered by a broad rim of unmineralized collagen fibers were noted and some lacunae also contained zones of a moderately electron dense material. This material did not yield a diffraction pattern, while the mineralized part of the cementum gave the diffraction pattern typical of hydroxyapatite.

Notes: Summary The aragonite crystals in the molluscan bivalve hinge ligament are surrounded by an organic sheath which is distinct from the remainder of the ligament matrix. Methods have been developed to isolate these sheathed crystals from the ligaments ofSpisula solidissima andMercenaria mercenaria employing a papain digestion of the matrix protein. The sheathed crystals fromSpisula have a CaCO3/protein ratio of 11.1 and those fromMercenaria a ratio of 29.6. The sheathed crystals and the empty crystal sheaths have been examined by electron microscopy for structural integrity. The sheath proteins exhibit much smaller proportions of the amino acids glycine and methionine than the hinge ligaments. These are characteristic amino acids of high concentration in the hinge ligaments of both species. The concentrations of acidic and basic amino acids are increased about two fold in the sheaths over those of the ligaments. Otherwise there is little similarity in the amino acid composition of the sheaths in the two species. However, SDS electrophoresis shows the sheaths of both to contain a major protein component with a molecular weight of about 25,000. The sheath protein from theMercenaria ligament contains about 5% carbohydrate and that ofSpisula sheaths less than 1% carbohydrate.

Notes: Summary The X-ray diffraction method based on pole figures has been applied to single osteon samples in order to obtain information about the texture of the inorganic bone fraction and the way it changes during calcification. The osteon samples were cylindrically shaped, with axes corresponding to those of the haversian canals. Selection was carried out according to the degree of calcification and the orientation of collagen bundles and inorganic particles. Osteons at both the initial and final stages of calcification were chosen. Arrangements of fiber bundles and inorganic particles in successive lamellae characteristic of three types of osteons were selected: longitudinal, alternate, and transversal. The results indicate that in all three types of osteons, the long axis of the sample is apparently the only direction of orientation because the transversally oriented crystallites give an isotropic diffuse scattering as would be expected if all the inorganic particles were irregularly oriented around the osteon axis. The number of longitudinally oriented crystallites increases progressively from transversally oriented osteons to alternately and longitudinally oriented ones. The crystallite orientation in an axial direction increases in fully calcified osteons. This last result is in agreement with the electron microscopic finding that the long needle-shaped crystallites covering much more than a major collagen period and measuring 40–45 Å in width increase in number as calcification proceeds.

Notes: Summary To obtain information on the changes in the inorganic bone fraction during calcification, low- and wide-angle X-ray diffraction techniques and electron microscopy have been applied to single osteon samples. The samples were cylindrically shaped and their axes corresponded to the axes of the Haversian canals. The selection was made according to the degree of calcification and the orientation of collagen bundles and inorganic particles. Osteons at both the initial and final stages of calcification were chosen. Arrangements of fiber bundles and inorganic particles in successive lamellae characteristic of three types of osteon were selected, that is, longitudinally structured osteons, transversely structured osteons, and alternately structured osteons. The results indicate that in osteonic lamellar bone there are two types of inorganic particles: (1) granules arranged in linear or needle-shaped entities with maximum width 40–45 Å, which are regularly distributed at the level of the main band of the collagen fibrils where their maximum length reaches the length of the main band itself; that is, about 400 Å; and (2) very long crystallites, with a diameter of 40–45 Å, which grow with their crystallographicc-axis parallel to the collagen fibrils and cover much more than a major collagen period.

Notes: Abstract Tillers of Festuca arundinacea Schreb. were subjected to-8°C in a bath of methylated spirits for three-quarters of an hour. They were thawed at room temperature and some material taken from the shoot apical meristem and leaf blade for electron microscopy. Similar material was taken from control plants for electron microscopy. Nine tillers subjected to-8°C and thawed subsequently failed to regrow. Nine control tillers regrew. All the treated meristem cells and about half the treated leaf mesophyll cells were extensively altered. Their nuclei were contracted, organelles were swollen or partly disrupted, plasmalemma and nuclear membranes were broken or absent and vacuoles were sometimes disrupted. Strongly osmiophilic material accumulated in the vicinity of membranes. About half the leaf mesophyll cells differed from the control mesophyll cells only in having more spherosomes and narrower thylakoids. Parallels with other ultrastructural studies of stress damage and the indications the results give of possible primary damaging events are discussed.

Notes: Abstract Embryogeny in the nasturtium is characterized by the development of a large, tripartite suspensor and storing cotyledons. A light and electron microscopic study revealed an early diversification of the plastids in the various regions of the suspensor and the embryo proper. Amyloplasts are found in the developing cotyledons of the heart-like embryo, while chloroplasts occur within the meristematic part of the embryo and the adjacent portion of the suspensor. The cells between the meristem and the storing cotyledons display undifferentiated leukoplasts, whereas leukoplasts with an electron-dense matrix occur in the basal cell mass of the embryo-suspensor. Etioplasts develop in several cells of the placental haustorium of the suspensor. The carpel haustorium shows rather undifferentiated leukoplasts, which are transformed into electron-dense plastids during autolysis of the suspensor. This early plastidal differentiation in discussed with respect to its control and functional significance.

Notes: Summary The pharmacokinetics of the anti-epileptic drug di-n-propylacetate (DepakineR) have been studied in 7 patients, in whom plasma concentrations were determined during and following subchronic treatment. Elimination of the drug appeared to follow a monophasic exponential course; biological half lives were 8 to 15 hours. The data supported the assumption that an open one-compartment model can be used to describe the kinetics of dipropylacetate in man. The drug appeared to have a relatively restricted distribution: calculated relative distribution volumes ranged from 0.15 to 0.40 1/kg. There were large interindividual differences in clearance rate. The therapeutic range was considered to be between 50 and 100 mg/1 plasma. Plasma levels of phenobarbital were markedly raised during treatment with dipropylacetate for an unknown reason. Determination of the plasma concentrations of drugs at accurately fixed times appears to be a reliable method for pharmacotherapeutic monitoring of epileptic patients.

Notes: Summary An oral dose of 5 mg of14C-isosorbide dinitrate was rapidly absorbed, biotransformed and excreted by human subjects. Peak whole blood concentrations of radioactivity were reached after 1.5 to 2 hours and declined relatively slowly. The radioactivity in whole blood mainly represented metabolites, isosorbide mononitrates. The peak concentrations found were 4.5, 11.7 and 34.3 ng/ml of isosorbide dinitrate, isosorbide 2-mononitrate and isosorbide 5-mononitrate, respectively, in the blood of one subject and 5.9, 15 and 61.3 ng/ml, respectively, in the blood of another subject. However, concentrations of the metabolites declined relatively slowly during 6 h after the oral dose. Up to 99% of an oral dose of isosorbide dinitrate was excreted during 5 days, mainly in the urine of the first day (ca. 78%). The results showed that isosorbide mononitrates were available to contribute to the pharmacological action.

Notes: Summary In 11 healthy untrained volunteers the increase in plasma dopamine-β-hydroxylase (DBH) activity during graded physical exercise has been examined as a true measure of increased activity of the sympathetic nervous system. The correlation between DBH activity, catecholamine concentration (CA) in plasma and heart rate was studied. When work on an electrically braked bicycle ergometer was gradually increased from 12.5 to 100, 200 and 300 watts there was a linear increase in DBH activity and heart rate; the increase in CA concentrations followed an exponential function. The peak values for DBH activity and CA concentration in plasma after the 300 watt work load (as percentages of the resting levels) were 130±3% and 820±71%, respectively; the adrenaline concentration in plasma increased only to 150±19% (p〉0.05). There were significant correlations between heart rate and work load, DBH and work load and log CA and work load. The data imply direct correlations between heart rate and DBH, heart rate and log CA and DBH and log CA. The exponential increase in noradrenaline concentration in plasma might be due either to a greater net “overflow” from sympathetic nerve endings, and/or to increased secretion by the adrenal medulla. In the latter case, the release of noradrenaline would not be accompanied by secretion either of adrenaline or DBH. After work ceased there were sharp falls in heart rate and CA concentration, which indicate an immediate drop in sympathetic activity. DBH activity in plasma returned to normal very slowly; it reached half maximum values after 20 – 22 min. It is concluded that increased sympathetic activity in man can be estimated in vivo as changes in DBH and/or CA concentration in plasma. In contrast, a rapid decrease in sympathetic activity is directly reflected only by a rapid fall in the plasma concentrations of CA.

Notes: Summary The diminished sympathomimetic pressor activity of monohydroxylated phenylalkylamines after oral administration has been attributed to incomplete enteric absorption. Therefore, urinary excretion of the unchanged drug and its metabolites has been compared after intravenous and oral administration of3H-m-octopamine to eight patients. Identical amounts of3H-activity (80% of the dose) were excreted after the two routes of dosing, so enteric absorption has been assumed to be complete. Significant differences were found in the fraction of free urinarym-octopamine, which amounted to 10.5% of the dose after infusion and 0.58% after oral administration. The only metabolic pathways form-octopamine are deamination and conjugation. Following oral administration the percentage of conjugates was considerably higher than after intravenous infusion. This metabolic pattern appears typical of all phenylalkylamines with a hydroxyl group in themeta position. Ring hydroxylation to catecholamines was not observed. The enzymes mainly responsible for conjugation after oral administration are located in the gut wall. The resulting “first pass effect”, i.e. metabolism prior to the access to the central compartment, can account for the diminished pharmacodynamic effect after dosing by this route.

Notes: Summary After oral administration of oxyfedrine to healthy volunteers, norephedrine was identified in the urine by thin layer chromatography and gas liquid chromatography and mass spectrography. 30 hours after single oral doses of 8, 16 or 24 mg of oxyfedrine, about 4, 8 and 9 mg, respectively, of norephedrine were found in the urine, i.e. on a molar base 75–100% of the dose was excreted as norephedrine. The peak of excretion occurred within 2–4 hours after administration of the drug. No accumulation of oxyfedrine and/or its metabolite was observed after administration of 16 mg of oxyfedrine t.i.d. for three days. It could not be decided whether oxyfedrine was metabolized to norephedrine by liver enzymes, as in rats, or was spontaneously degraded to norephedrine, e.g. in duodenal fluid before absorption. 30–150 min after oral oxyfedrine (24 mg) norephedrine was demonstrable in duodenal fluid. Thus, in addition to the directβ-sympathomimetic effects of oxyfedrine, it may also have indirect sympathomimetic effects because of the noradrenaline-releasing properties of its metabolite norephedrine.

Notes: Summary It is apparent from studying recent articles on pharmacokinetics that a number of misunder-standings exist, both about the design of experiments and the analysis of results. The purpose of this paper is to outline many of the common pitfalls associated with the design of experiments and also the limitations upon the analysis of results. The paper describes mathematical, laboratory and clinical aspects which must be examined in designing a protocol for pharmacokinetic experiments. Simulated data is presented to demonstrate the dangers of using standard computer programs for parameter estimation. Even when convergence is obtained the answers may be dependent on the method employed. A mathematical model is of little use unless a reasonable amount of good, accurate data is obtained.

Notes: Summary Clonazepam (CNP) and its principal metabolite in plasma, 7-amino-CNP (ACNP), have been investigated in a prospective study of 27 newly diagnosed epileptics and correlated with specified side-effects. At a daily dose of 6 mg, the average plasma levels of both substances were about 50ng/ml, and individual values ranged from 30 to about 80ng/ml. There was a linear correlation between changes in dose and the resulting plasma levels, which indicates first order elimination kinetics. Side-effects were frequent, but neither their severity nor their occurrence could be related to plasma levels or to the rate of increase in plasma concentration of the drug. Three out of five patients who developed serious dysphoria had significantly high CNP levels. The concentration of ACNP was considerably increased in four patients who subsequently suffered from withdrawal symptoms. Drug interaction with diphenylhydantoin, i.e. decreased CNP level, was observed in all five patients who received both compounds. In general it is not yet possible to define an upper limit for the plasma levels of CNP and ACNP at which toxicity occurs. In patients treated with conventional doses of CNP, measurement of plasma concentration is not required, except in special circumstances, because of the lack of correlation between plasma level and side-effects.

Notes: Summary After administration of a single loading dose (80 mg p.o.) of fluorophenindione, the prothrombin level decreased to 37 % in 24 h, and the effect lasted for 48 h. Accordingly, fluorophenindione can be classified as an anticoagulant with an “intermediate” effect. Its elimination half-life was 31 h, which is longer than that of phenindione, because of the greater stability of the fluorinated derivate.

Notes: Summary Plasma concentrations of nortriptyline have been assayed in four subjects after intravenous infusion of 57 mg nortriptyline hydrochloride. The data were evaluated according to a two compartment open model. The calculated best-fitting curves were in good agreement with the experimental data, better than could be expected from a simpler model. This justifies the assumption that the kinetics of nortriptyline in man may be described by this model with an appropriate input function. The data permitted estimation of all the parameters of the model. The meaning of the parameters is discussed, particularly in relation to individual variation.

Notes: Summary A 55-year old man with lithium intoxication showed increasing serum concentrations in spite of forced diuresis and dialysis. A high lithium content was found in gastric juice three days after the compound had been taken. The serum lithium level began to fall after gastric lavage.

Notes: Summary A quantitative thin layer chromatographic (TLC) method has been developed for determination of the antiarrhythmic quaternary ammonium compound N, N-bis (phenylcarbamoylmethyl) dimethylammonium chloride (QX-572) in biological materials. Prior to chromatography QX-572 was transferred into chloroform as perchlorate by ion pair extraction. Tritium-labelled QX-572 was used as the internal standard and a TLC scanning spectrophotometer equipped with a linear detector system afforded the required accuracy, specificity and simplicity. The method was used to determine QX-572 in plasma from 11 patients with various cardiac diseases who received QX-572 8 mg/kg body wt. as an intravenous infusion over 30 min. There was a rapid initial decay of the plasma levels from 11.0±1.1 µg/ml (mean ± SE) at the end of infusion to 3.5±0.5 µg/ml after 30 min. 240 min after commencement of the infusion the plasma level was 0.7±0.1 µg/ml. In these patients 22±2% (mean±SE) of the total administered dose of QX-572 was excreted unchanged in urine during the 24 hours following infusion of the drug. A second group of 28 patients with acute myocardial infarction also received QX-572 8 mg/kg body wt. Their plasma levels did not differ significantly from those found in the first group of patients. There was a poor correlation between the amount of QX-572 administered and plasma level at the end of the infusion. The study has provided some preliminary data about the pharmacokinetics of QX-572, but before a detailed analysis can be done data from longer periods of observation is required. The present results suggest that in future QX-572 can be administered in a standardized dosage, what would be advantageous in practice.

Notes: Summary The plasma elimination of carbamazepine (Tegretol®) was studied in five newborns who had got the drug transplacentally from their epileptic mothers. The half-lives ranged from 8.2 – 27.7 hours which is comparable or even shorter than those found in adults after a single oral dose, but in the same range as those found in adults after multiple oral doses. This suggests that the newborns' drug metabolizing capacity has been induced during fetal life.

Notes: Summary The importance of individualizing the dosage of potent drugs in order to maximize their therapeutic effectiveness and safety is generally accepted. Whenever possible the dosage of a drug should be “titrated” directly in each patient against the intensity of its therapeutic or toxic actions. Unfortunately, for many drugs convenient clinical yardsticks of the intensity of their pharmacologic effects are lacking. Determination of the serum concentration of such compounds can help to guide adjustment of dosage during their therapeutic use. By measuring the serum level of drugs one bypasses the largest source of individual differences in doseeffect relationships — the pharmacokinetic variation between subjects. However, the relationship between the serum concentration of a drug and the intensity of its pharmacodynamic action is influenced by many other factors, which must always be considered in interpretation of serum levels. Therapeutic decisions should never be based solely on the serum concentration of a compound, nor can such measurements ever substitute for careful medical observation and judgement.

Notes: Summary 35S-furosemide was administered orally (n=7) or i.v. (n=2) to healthy subjects. The average gastrointestinal uptake estimated by comparison of the urinary recovery of label and the areas under the plasma curves after the two routes of administration was 65%. The half life of radioactivity in the plasma after oral35S-furosemide was 90 ± 17 min (estimated on the slope between 2 and 6 h); the corresponding figure after35S-furosemide i.v. was 47–53 min (slope 0.5–4 h). There was probably a slower phase after 4–6 h. Fractionation of labelled material in urine from two subjects demonstrated that approximately two thirds of the label recovered at 24 h had the same chromatographic properties as furosemide. A major part of the metabolite(s) was probably furosemide glucuronide. There was no evidence that 4-chloro-5-sulfamoylanthranilic acid was formed in man. The total urinary recovery of label (5–7 d) after oral and intravenous administration was 55.1±3.2 (mean±SD) and 82–84%, respectively. After35S-furosemide i.v., 6–9% of the label was recovered in faeces, and it could not be accounted for solely by biliary excretion of furosemide.

Notes: Summary Twenty male volunteers received oral doses (2100, 1050, and 525 mg) of a pivampicillin-probenecid salt in a 1 to 1 molar ratio (MK-356) at 12 hour intervals. After each dose peak serum concentrations of probenecid were observed 2 hours later than peak concentrations of ampicillin. Following the first dose of MK-356 the apparent elimination rate of ampicillin was dose-dependent and did not follow first order kinetics, as it showed a longer apparent half life after a higher dose. An equal dose of MK-356 administered 12 hours later caused an increase in the peak serum ampicillin level greater than expected from the concentration of ampicillin after the preceding dose. In twelve male volunteers who received at random 525 mg of MK-356 or 350 mg of pivampicillin, each three times daily for 4 days, the areas under the ampicillin concentration curve were the same after the first or last dose of either drug. When 2100 or 1050 mg of MK-356 was taken as an initial dose, 30 to 40 per cent of the ampicillin was recovered from urine in the ensuing 12 hours. The results indicate that when at least 400 mg probenecid was coadministered twice daily with 700 mg pivampicillin (MK-356), the peak serum concentrations of ampicillin were increased and its elimination rate slowed following successive doses.

Notes: Summary The absorption, biotransformation and elimination of sulfinpyrazone, 1,2-diphenyl-3,5-dioxo-4-(2′-phenylsufinylethyl)-pyrazolidine, have been studied by administration of single 200 mg oral doses of a14C-labelled preparation to two male volunteers. Absorption from the gastro-intestinal tract was rapid and complete and the plasma concentration of unchanged drug reached maximum values of 22.67 and 13.04 µg/ml, respectively, after 1 – 2 hours. The elimination half-life in the two subjects, calculated from the decline between 3 and 8 hours, was 2.7 and 2.2 hours. The integrated concentration of unchanged sulfinpyrazone in plasma, estimated from the area under the concentration curves (AUC), was almost as high as that of total14C-substances, so the proportion of metabolized drug in plasma was low. In no case did the AUC of the three specifically determined metabolites, i.e. the sulphone G 31 442, the “para-hydroxy”-compound G 32 642 and the “4-hydroxy”-compound GP 52 097, exceed 4% of the sulfinpyrazone value. More than 95% of whole blood radioactivity was confined to plasma. The oral dose was rapidly and completely excreted, since within 4 days more than 95% was recovered, 85% from urine and 10% from faeces. A large proportion of the dose was excreted as unchanged drug in the two volunteers: 51 and 54% of total urinary radioactivity was present as sulfinpyrazone; 8.2 and 8.8% was present as “para-hydroxy”-metabolite, 2.7 and 3.0% as sulphone-metabolite, and 0.6 and 0.8% as “4-hydroxy”-metabolite. About 30% of urinary radioactivity consisted of highly polar metabolites. Spectroscopy of them showed that they were the C-β-glucuronides of sulfinpyrazone (28%) and the corresponding sulfone (2%). In these metabolites the C(4) of the pyrazolidine ring was directly attached to glucuronic acid, and thus they represent a new type of biosynthetic conjugate.

Notes: Summary Eight healthy volunteers were given single i.v. and oral doses of clonazepam (2 mg). The disposition curves after i.v. administration showed a biexponential decline and the data were applied to a two-compartment open model. The volume of distribution ((Vd)β) ranged between 1.5 and 4.4 l/kg and the plasma half-life (t1/2) between 19 and 60 hours. Absorption after oral administration was fast, with peak plasma concentrations within 4 hours in all subjects. Five of the subjects received repeated oral doses of clonazepam 0.5 mg bid for 15 days. The plasma level during steady state (estimated as Cmin within the dose interval) could be predicted from the constants A, B, α and β obtained in the single dose study with a coefficient of variation of 6%. The plasma half-lives after cessation of the subchronic dosing were of the same magnitude as after single doses.

Notes: Summary The metabolic fate of orally administered (−)-[14C]-ephedrine has been studied in 3 human subjects and the urinary excretion of metabolites determined quantitatively by solvent extraction, paper chromatography and reverse isotope dilution procedures. Following an oral dose of the drug (0.35 mg/kg, 1.6 µCi), 97% of the dose was excreted in the urine within 48 h, 88% in the first 24 h. Unchanged drug was the major urinary excretory product (53–74%), with N-demethylation occurring to a variable extent (8–20%) although there was little interindividual variation in urine pH. Oxidative deamination was also variable (4–13%); the main identified products of this were benzoic acid (free and conjugated) and 1,2-dihydroxy-1-phenylpropane (free and conjugated). No phenolic metabolites could be detected, and thus it would appear that these compounds cannot be implicated in the acquisition of tolerance to ephedrine which can occur on repeated dosage.

Notes: Summary Pharmacokinetic and metabolic studies with3H-etilefrine were performed to assess the importance of a first-pass effect on the pharmacodynamic action of this sympathomimetic amine. Identical amounts of3H-activity, ca. 80% of the dose, were excreted in the urine after intravenous or oral administration, which indicates complete enteral absorption of the drug. Comparison of the areas under the plasma curves of unchanged etilefrine after both routes of administration resulted in a bioavailability factor of 0.55, which can be explained by an extensive first-pass effect. The time curve of plasma levels of etilefrine was compatible with an open 2-compartment model characterized by a rather large volume of distribution (Vd, β) of 160 1, and a predominant half life of 2 hours. The pharmacodynamic action corresponded to the amount of drug in the central compartment. The major pathway of metabolism of etilefrine was conjugation to form the phenolic sulphate, and a very minor proportion of the drug was excreted as the corresponding hydroxymandelic acid. This metabolic pattern seems to confirm our hypothesis that phenylalkylamines with the hydroxyl group in the m-position of the benzene ring are predominantly conjugated in contrast to p-hydroxylated compounds which are mainly deaminated.

Notes: Summary A method has been developed for the quantitative determination of heptabarbital [5-(1-cyclohepten-1-yl)-5-ethylbarbituric acid] in human plasma after administration of single therapeutic doses of the drug. It involves a single extraction step followed by gas chromatography with alkali flame ionization detection, and the results were linear in the concentration range 0.125 – 5.0 µg/ml plasma. The pharmacokinetics and relative bioavailability of heptabarbital and heptabarbital sodium were studied in a crossover design in 7 healthy volunteers after oral administration of 20 tablets containing 200 mg heptabarbital and hard gelatine capsules containing an equivalent amount of its sodium salt. Heptabarbital concentrations in plasma were determined at regular intervals. The absorption of heptabarbital from the tablets was quite slow and peak level times varied from 1.5 to 4 h. The sodium salt was absorbed more rapidly and peak concentrations occurred between 1/3 and 2 h. In all cases the elimination of heptabarbital could be described by a single first-order process with an average half-life of 7.6 h (range 6.1 – 11.2 h). The half-life of the drug in each individual was about the same in the two trials. The relative bioavailability in each volunteer was estimated by comparing the areas under the plasma concentration curves. The sodium salt had an average bioavailability of 83% relative to the free acid. In some volunteers urinary excretion of unchanged heptabarbital was measured; cumulative excretion amounted to 0.16 – 0.30% of the administered dose. Four volunteers received one tablet each night for eight or ten days, but no accumulation was found. In three volunteers the half-life of the drug prior to and after these experiments did not change, whereas in the other volunteer the half-life decreased from 7.1 to 4.6 h. The possibility of enzyme induction should be considered when heptabarbital is taken regularly. It was concluded that heptabarbital was a suitable drug for the treatment of insomnia, since its half-life was rather short. Heptabarbital sodium may be used for induction of sleep, whereas Medomin® tablets, i.e. heptabarbital free acid, may be prescribed when the maintenance of sleep is the primary reason for treatment with a hypnotic drug.

Notes: Summary Five healthy volunteers took 1.2 g rifampicin daily for 8 days, and before and afterwards each received hexobarbital (7.32 mg/kg) and tolbutamide (20 mg/kg) by i.v. infusion on two consecutive days. The plasma concentrations of the two drugs were determined during and after infusion. The average elimination half-life of hexobarbital had decreased from 325 to 122 min and of tolbutamide from 418 to 183 min following rifampicin treatment. It was calculated that the metabolic clearance of hexobarbital had increased about three-fold and that of tolbutamide more than two-fold. Significant changes in the distribution kinetics of the two drugs were not observed. The results suggest that rifampicin is capable of inducing drug metabolism in man, which leads to an increased rate of elimination of drugs that undergo biotransformation in the liver.

Notes: Summary A clinical-pharmacological study was performed to determine the effect of acetylsalicylic acid upon the serum concentration of indomethacin. 14 rheumatic patients were given indomethacin orally (25 mg × 4 for 4 days) and concurrently acetylsalicylic acid 3.7 g orally (0.9 g × 3 and 1.0 g × 1 daily), and 21 rheumatic patients were given indomethacin rectally in the morning (100 mg × 1) and concurrently acetylsalicylic acid 3.7 g orally (0.9 g × 3 and 1.0 g × 1 daily). On comparison with treatment with oral or rectal indomethacin alone, it was found that peak serum concentrations of indomethacin were significantly reduced (1% level), the times of the peaks were not shifted, and the areas beneath the serum concentration curves of indomethacin were smaller, but significantly so only if compared with rectal administration. In 12 rheumatic patients given indomethacin by rectum in the evening (100 mg × 1) and concurrently acetylsalicylic acid 3.7 g (0.9 g × 3 and 1.0 g × 1 daily), the serum level of indomethacin on the following morning (after 11 h) did not differ from that found after rectal treatment. A statistically but not biologically significant difference was observed between the mean serum half-lives of indomethacin given orally and rectally. For unknown reasons, concurrent doses of acetylsalicylic acid and indomethacin made the mean serum half-life of indomethacin longer than after its oral administration, but shorter than when the same dose of indomethacin was given rectally. There was no difference between serum levels of salicylate after oral administration of acetylsalicylic acid alone or after a concurrent oral or rectal dose of indomethacin. The results have been related to those reported previously, with respect to the interaction between indomethacin and acetylsalicylic acid, the serum levels of indomethacin after oral and rectal dosing, and the serum half-life of indomethacin based upon a one- or two-compartment model. The clinical relevance of the study is discussed.

Notes: Summary The pharmacokinetics of quercetin, a flavonoid, have been studied in 6 volunteers after single intravenous (100 mg) and oral (4 g) doses. The data after iv administration were analyzed according to a two compartment open model with half lives of 8.8±1.2 min for the α phase and 2.4±0.2 h for the β phase (predominant half life), respectively. Protein binding was 〉98%. The apparent volume of distribution was small at 0.34±0.03 l/kg. Of the intravenous dose 7.4±1.2% was excreted in urine as a conjugated metabolite, and 0.65±0.1% was excreted unchanged. After oral administration no measurable plasma concentrations could be detected, nor was any quercetin found in urine, either unchanged or in a metabolized form. These results exclude absorption of more than 1% of unchanged drug. Recovery in faeces after the oral dose was 53±5%, which suggests extensive degradation by microorganisms in the gut. The data obtained show that oral administration of flavonoids may be of questionable value.

Notes: Summary Canrenone was administered in doses of 2×82 mg and 2×164 mg per day over a period of 10 days to diabetic patients without cardiovascular, liver or kidney involvement. Aldosterone excretion and plasma aldosterone increased only slightly during both regimes. There was a clear-cut increase in aldosterone excretion only after discontinuation of carenone. Excretion of sodium potassium and fluid was not significantly changed either during or after treatment. The lack of effect of canrenone on the kidney was in contrast to the significant decrease in serum sodium and increase in serum potassium, and the significant, dose-dependent rise in plasma renin activity following canrenone administration. The increased plasma renin activity persisted for some days after discontinuation of canrenone. It is suggested that canrenone primarily exerted its effect in the distal part of the large intestine where ionic movements are most affected by aldosterone. The disproportionately slight increase in plasma aldosterone concentration and aldosterone excretion, in spite of the greatly elevated plasma renin activity and serum potassium level, is considered to be due to a direct inhibitory effect of canrenone on aldosterone production in the adrenals.

Notes: Summary The pharmacokinetics of lofepramine, an imipramine analogue, have been studied by administering single oral doses to volunteers, determination of plasma levels of lofepramine and desmethylimipramine after ten days of oral administration to patients, and by relating plasma levels to the effect on uptake of noradrenaline by isolated rat irides and brain slices of plasma samples collected during treatment. The results indicate that lofepramine undergoes pronounced first pass elimination and that desmethylimipramine is a major metabolite of it. During steady-state conditions the plasma level of lofepramine fluctuates considerably between doses. A linear relation was found between inhibition of neuronal uptake of noradrenaline and the plasma concentration of desmethylimpramine. No effect was seen on the uptake of 5-hydroxytryptamine in brain slices incubated in patients' plasma which suggests that neither lofepramine nor its metabolites formedin vivo in man affect neuronal uptake of this amine. Lofepramine belongs to the group of tricyclic anti-depressants which preferentially inhibit noradrenaline uptake.

Notes: Summary Single doses of β-methyl-digoxin 0.4 mg were given to groups of 17 – 18 healthy volunteers as an intravenous infusion lasting 2 hours, or orally as Lanitop Liquidum® or Lanitop® tablets. The serum glycoside concentration and urinary glycoside excretion were measured over 8 and 32 h. The absolute bioavailability from the oral preparations in comparison with the infusion was lower for the first 8 h than for the entire 32 h of the investigation; the relative bioavailability from tablets was the same as from the solution for both periods. For both periods the area under the serum concentration/time curve and the urinary glycoside excretion were significantly lower after administration of the tablets than after intravenous infusion. Taking the average of both parameters, the absolute bioavailability of β-methyl-digoxin was about 80% from the solution and about 70% from the tablets. In 18 patients undergoing intravenous or oral therapy with β-methyl-digoxin steady state glycoside concentrations were compared in a cross-over study of intravenous maintenance therapy with Lanitop® ampoules or oral treatment with Lanitop® tablets. For a standard daily dose of 0.2 mg β-methyl-digoxin the serum concentrations were 1.35±0.10 ng/ml during both intravenous and oral administration. The intra-individual variation in glycoside concentration after changing from intravenous to oral maintenance therapy, or vice versa, was about the same as during continued intravenous or oral administration. It is concluded that the rate of rise of serum concentration after a single dose may be a useful indicator of the rate of absorption, but that the area under the serum concentration/time curve and the urinary glycoside excretion up to 32 h are unsuitable for determining equivalent doses of different formulations or routes of administration of digitalis glycosides.

Notes: Summary A previously described GLC method has been modified and applied to measurement of antipyrine levels in plasma, blood and saliva of man following administration of a single oral dose (10 mg/kg). The levels in blood and saliva were comparable to those in plasma at every time studied. The half life of antipyrine determined in blood, plasma or saliva in any given individual was similar. The intersubject variation in half-life was about two-fold (n=5). Antipyrine levels in saliva were not affected by the rate of saliva flow when collections were made continuously for 20 minutes. This study has demonstrated that kinetic data about antipyrine comparable to that from plasma may also be obtained from readily accessible tissue fluids, such as saliva and capillary blood.

Notes: Summary A gas chromatographic method has been employed to determine chlorthalidone in plasma and whole blood after therapeutic doses. Radioactively labelled chlorthalidone was used for in vitro studies of the uptake of chlorthalidone from plasma by red blood cells. Chlorthalidone was markedly concentrated in red cells and as a compartment they would account for at least 30% of total drug in the body after multiple doses. The ratio between the plasma and red cell concentration of chlorthalidone varied between individuals. After a single oral dose of 50 mg in 6 healthy volunteers chlorthalidone was eliminated with a half-life of 51 to 89 hours. The apparent volume of distribution varied between 3 and 13 1/kg and the clearance between 53 and 145 ml/min. The mean steady-state plasma concentrations during treatment with a standard dose of 50 mg daily (n=10) varied 5-fold between individuals. During the steady state approximately 50% of the daily dose was excreted unchanged in the urine during 24 hrs. The plasma levels observed in patients were higher than those predicted from the single oral dose studies in healthy volunteers.

Notes: Summary The effects of sotalol after oral administration were measured on the tachycardia induced by strenuous exercise in normal subjects. Plasma sotalol levels were also determined. The oral administration of sotalol (50, 100, 200 and 400 mg) to 6 subjects produced a progressive reduction in the tachycardia induced by severe exercise. This was similar to the effects of 25, 50, 100, 200, 400 and 800 mg given to different subjects. Each increase in sotalol dose produced a successively greater reduction in exercise tachycardia. This did not appear to be maximum even with 800 mg. Oral sotalol was rapidly absorbed and produced peak blood levels in 2 – 3 hours. The plasma levels of sotalol measured 2 hours after the oral administration of 25 to 800 mg showed never more than a six-fold variation between different subjects. The half-life of sotalol in plasma was 12.7 ± SE 1.6 hours. There was a significant correlation between the logarithm of the plasma sotalol concentration and the percentage reduction of exercise heart rate. It is concluded that the oral administration of sotalol either once or twice daily (depending on dose level) will provide satisfactory 24-hour blockade of β-adrenoceptors.

Notes: Summary The effect of spironolactone on the metabolism of intravenously administered3H-digitoxin (80 µCi) was investigated in eight patients. In three of them the labelled glycoside was given on a second occasion after spironolactone treatment had been discontinued for at least 65 days. Of total urinary radioactivity 79 % was unaltered drug and 12 % consisted of water soluble compounds. No digitoxigenin or digoxigenin and only trace amounts (〈2 %) of digoxin and the bis- and monoglycosides of digoxigenin were found. After spironolactone total urinary radioactivity was unchanged but the fraction eliminated as unchanged digitoxin fell from 79 to 66 % and the water soluble compounds increased from 12 to 26 % (p〈0.05). In addition spironolactone caused a 20 % reduction in the half-life of serum radioactivity (p〈0.01) and a 16 % reduction in the volume of distribution (p〈0.05). Induction of hepatic enzymes by spironolactone is proposed to explain the alteration in the metabolism of digitoxin in man. Both the altered metabolic pattern and the reduction in the volume of distribution appear to contribute to the reduction in half-life.

Notes: Summary Serum zinc concentrations in peripheral venous blood were determined in 8 healthy volunteers at various times after oral administration of 50 mg Zn++. The same dose was given to 6 patients surgically treated for obesity by jejuno-ileostomy. In the healthy volunteers the mean serum zinc concentration before dosing was 0.89 µg/ml and a mean peak concentration of 2.39 µg/ml was found after 3 h. In the patients the starting level was lower, 0.67 µg/ml, and a mean peak concentration of 1.31 µg/ml was found 90 min after treatment. In the patients the areas under the serum concentration-time curve was approximately 1/3 of that in the healthy subjects. Zn++ 50 mg was also given to 3 patients undergoing transumbilical catheterization of the portal vein for diagnostic purposes and serum zinc concentrations were measured in portal and peripheral venous blood sampled simultaneously. No significant differences were found between the concentration of zinc in portal and peripheral venous blood during absorption, which suggests slow passage of zinc across the intestinal wall.

Notes: Summary The pharmacokinetics and relative bioavailability of cyclobarbital calcium have been studied after oral administration of Phanodorm, of tablets according to the Formularium Nederlandse Apothekers (1968; FNA), and an aqueous solution. Six healthy volunteers participated in the investigation on three occasions and each received the three preparations. The dose administered was 300 mg cyclobarbital calcium. Plasma concentrations of cyclobarbital were determined at regular intervals. Absorption from the three preparations was rapid and was faster from the solution. Peak concentrations were usually attained within 1 h. The elimination of cyclobarbital could be described by a single first-order process with an average half-life of 11.6 h (range 8 – 17 h). There was little intra-subject variation of the half-life. Relative bioavailability for each volunteer was estimated by comparing the areas under the plasma concentration curves. The FNA-tablets and Phanodorm exhibited similar bioavailability, whereas the average bioavailability of the solution was 78% of that of FNA-tablets; the reason for this unexpected finding is unknown. It was concluded that cyclobarbital cannot be regarded as a uniformly suitable drug for the treatment of insomnia. The long half-life that was apparent in some of the volunteers (15 – 17 h) creates a substantial risk of residual effects on the following morning. In principle, however, the calcium salt of cyclobarbital may be used for induction of sleep, because of its rapid absorption.

Notes: Summary Whole night EEG and polygraphic recordings were made in ten young, healthy, male volunteers after dixyrazine (12.5 mg, 25 mg, 50 mg), methaqualone (250 mg) and Isonox® (methaqualone 250 mg + etodroxizine 50 mg). A total of 156 recording nights (36 adaptation nights were not included in the analyses) were scored for different sleep stages according to accepted criteria. The smallest dose of dixyrazine (12.5 mg) had no significant effect upon sleep pattern: the larger doses (25 mg and 50 mg) caused significant decreases in REM-sleep during the first nights of administration. The decrease disappeared during the following two nights of treatment. No withdrawal effects were seen. Methaqualone also caused moderate depression of REM-sleep during the first night of treatment, and this effect, too, disappeared during prolonged administration. Isonox® (methaqualone + etodroxizine) had a somewhat stronger surpressive effect upon REM-sleep than methaqualone alone.

Notes: Summary Four subjects received 5 mg14C-glipizide orally, 3 subjects 1 mg intravenously and 2 subjects 5 mg14C-glibenclamide orally. Plasma levels of radioactivity, and urinary and faecal excretion were measured. For both drugs the disappearance of radioactivity from plasma followed complex kinetics and the apparent half-lives increased steadily with time. The two sulfonylureas were extensively metabolized and were excreted in the urine as hydroxylated or conjugated metabolites. The effects of both drugs on blood glucose and immunoreactive insulin were comparable. The findings are compared with other published results.

Notes: Summary Carbamazepine (2.7–3 mg/kg) was administered orally as an alcoholic solution (50% v/v) to eight healthy volunteers. Two of the subjects were also given 50 mg and 100 mg of carbamazepine in alcoholic solution and 200 mg as a tablet. Plasma concentrations, which were analysed by mass fragmentography, reached a maximum 1 – 7 hours after dosing, and then declined monoexponentially with half-lives ranging from 24 to 46 hours. The half-lives were independent of dose. The apparent distribution volume ranged from 0.79 to 1.40 l/kg. It was found that 72% of carbamazepine was bound to plasma proteins with little interindividual variation, and this was not influenced by the presence of diphenylhydantoin or phenobarbital in therapeutic concentrations. The pharmacokinetic parameters calculated from single oral doses were used to predict the steady-state plasma concentration expected after treatment with multiple doses of 200 mg three times daily. The predicted steady-state concentration was 2 – 3 times higher than that reported in patients undergoing chronic treatment with carbamazepine at this dose level, i.e. the pharmacokinetics of carbamazepine apparently change during multiple dosing.

Notes: Summary The haemodynamic effects of disopyramide have been studied in 11 patients with manifest or imminent heart failure. Disopyramide, 2 mg per kg body weight, was given intravenously during right and left heart catheterisation. The cardiac index decreased by an average of 28% (p〈0.01); mean maximal left and right ventricular end-diastolic pressures were increased by 5.0±0.9 mm Hg (〈0.01) and 5.0±0.6 mm Hg (p〈0.05), respectively; and left ventricular systolic pressure fell slightly but significantly (p〈0.05). No significant change in right ventricular systolic pressure was seen. Pulmonary wedge pressure rose on average by 2.7 mm Hg (p〈0.05). No significant change in heart rate was observed in 5 patients with sinus rhythm. In 6 patients with atrial fibrillation, there was a significant (p〈0.01) increase in heart rate; the average increase in heart rate for the entire group was 19,6 heats per minute. The maximum effect on all the parameters occurred 7–11 minutes after the injection, and it gradually subsided during the following 10 minutes. It was concluded that disopyramide had a potentially serious myocardial depressant effect.

Notes: Summary Co-trimoxazole, a mixture of one part trimethoprim (TMP) and five parts of sulphamethoxazole (SMX) in fixed ratio was given to 48 children aged between one and 48 months twice daily for up to seven days. Twenty were relatively healthy and 28 were very ill. Dosage was based on age. Plasma concentrations of both drugs were measured just before a dose was due and some three hours later. They were in the effective but not toxic range and serve to justify the simple regimen which generated them.