ALBERT

Notes: Summary The elaboration of bone matrix glycoprotein by osteoblasts of alveolar bone was investigated by radioautography after the intravenous injection of3H-fucose into young rats. At selected times after injection, animals were sacrificed by intracardiac perfusion and demineralized specimens were prepared for light and electron microscope radioautography. At 5 and 10 min after injection, when the blood fucose level was high, silver grains were restricted to the spheroidal and cylindrical saccules of the Golgi apparatus. At 20 min membrane-limited secretory granules were also labeled. By 35 min, the blood fucose level had dropped and silver grains were detected over the apical cortical cytoplasm, in association with secretory granules located therein. Some grains were present over osteoblast processes and the adjacent prebone matrix. By 4 h most of the silver grains had left the cell. At that time they were observed over prebone, adjacent to osteoblast processes, as well as over the prebone-bone junction where a distinct band of label was noted. In demineralized preparations an electron-dense granular material was present at the prebone-bone junction in association with collagen fibrils. These findings provide evidence that osteoblasts in alveolar bone synthesize fucose-containing glycoprotein and indicate that the addition of3H-fucose occurs in the Golgi apparatus. The glycoprotein passes to the apical cortical cytoplasm by way of membrane-limited secretory granules, is exteriorized, and accumulates at the site where prebone transforms into bone (the prebone-bone junction). Since this is also the site of the calcification front, the deposition of labeled glycoprotein may be related to the deposition of bone mineral.

Notes: Summary Recently published bone fluoride values from Iowa are very high compared to earlier reports, suggesting an increase in fluoride intake. Reanalysis of the Iowa specimens shows levels one-fourth those reported by the Iowa laboratory indicating an error in the original report. Seventeen bone specimens, collected from long-term residents of Rochester, New York, drinking 1 ppm F− water, had a mean value of 2085±270 ppm F− on an ashed-weight basis. This value is not significantly different from that predicted by the data of Zipkin et al. in 1958. These data, therefore, do not support the contention that there has been an increase in fluoride intake.

Notes: Summary A conventional spectrophotometer was used to do radiographic photodensitometry of bone mass. An experiment to produce osteoporosis in guinea pigs required quantification of changes in bone mass. Lack of a densitometer necessitated finding another method for quantification. The method developed involved using industrial film in a closed X-ray system. Optical density of X-rays was then read in a spectrophotometer at nine femur locations. Highly significant correlations between optical density and femur calcium content and femur dry weight per millimeter were obtained. Histological sections from an animal with low optical density measurements confirmed bone loss by showing decreased cortical width and loss of trabecular bone. In addition to using readily available equipment, this method offers the advantage of being able to detect bone loss in specific areas of the bones. In addition, bones are left intact and can be used for other purposes.

Notes: Summary Carbonate-containing hydroxyapatite, enamel, and bone were irradiated by an X-ray and investigated between 77° and 350°K by means of electron spin resonance (ESR) spectroscopy. The ESR spectrum of enamel irradiated at 77°K in vacuum and observed at the same temperature was almost the same as that of the carbonate-containing hydroxyapatite. The temperature dependence of signal intensities confirms a spin-energy exchange between the mineral and organic constituents in bone, but in enamel no or very little spinenergy exchange between the mineral and organic constituents. Considerable similarity among the ESR spectra of enamel, bone, and carbonate-containing apatite was obtained after X-ray irradiation in air at 300°K with both an X-band and a Q-band ESR spectrometer. The Q-band spectrum can be interpreted in terms of two paramagnetic species. One is identified as a CO 3 3− anion radical which has an axial symmetry withg factors of 2.0029 and 1.9972. The other species is found to be centered atg=2.0019.

Notes: Abstract Photovoltaic effect (infrared and visible light) is observed in bone and its two major components, collagen and apatite, at room temperature. A dimunition in the magnitude of photovoltage is observed after exposure to ultraviolet light in all the cases. The drift mobility of the charge carriers is obtained by measuring I versus V relationships in sandwich samples and relating them to the permitivity of the medium. Lifetime of the injected carriers is measured in the usual way. The data are consistent with the hypothesis that the effects are due to protonic conduction phenomena.

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 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 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 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 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 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 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 study describes the ultrastructure of the mineralized portion of chick tibiae from 10 days in ovo to 2 days post-hatch. At 10 days a single mineralized cylinder surrounds the diaphysis. On its outer surface columnar trabeculae join to form ridges parallel to the long axis of the bone. These ridges are covered by another cylinder and form the haversian canals. At 11 days vascular invasion of the marrow cavity occurs and resorption of the endosteal surface begins. This type of periosteal deposition and endosteal resorption is repeated during and subsequent to embryonic development. The mineralized portion of 10-day chick tibiae cultured for 2 days in modified BGJ medium was compared with 10-, 11-, and 12-day tibiae in ovo. Cultured tibiae were similar in length and calcium content to 11-day tibiae in ovo. The form of mineral deposited in ovo and in culture was the same, namely, aggregates of spherical mineral clusters. Differences in culture included the following: (a) few concentric cylinders were deposited as compared with tibiae in ovo; (b) trabeculae were not arranged in rows and ridges in culture; (c) osteocytic lacunae were restricted to bases of trabeculae rather than uniformly distributed as in ovo; and (d) the endosteal surface of tibiae in culture appeared etched.

Notes: Summary Osteolathyrism has been used as an experimental model for the study of calcification nodules during the mineralization process. Periosteal exostoses developing in osteolathyrism characteristically have spherical basophilic structures (calcification nodules) in the vicinity of developing bone spicules. In thin sections, the nodules were seen scattered between collagen fibers in the intercellular matrix. Collagen fibers did not appear to be present within the nodules but sometimes were packed just outside them. Matrix vesicles were also present in areas of early mineralization. After EDTA decalcification, the majority of the nodules consisted of a fine granular material surrounded by an electron-dense peripheral zone. The peripheral dense zone was occasionally incomplete in small nodules in areas of early mineralization. An electron-dense central area could be observed in the center of the nodules. Evidence has been presented indicating that the calcification nodules arise from smaller mineralization foci, presumably matrix vesicles. The calcification nodules enlarge to approximately 1.0 µm in size, at which point development is slowed or halted allowing the formation of the peripheral dense zone. Although coalescence of nodules was observed, this was more a random event. The further mineralization of the trabeculae was achieved by the calcification of the collagen fibers. The mineralized trabeculae reflected this pattern of nodular and collagenous calcification. It is suggested that this pattern of calcification is characteristic of rapidly developing woven bone.

Notes: Summary In order to ascertain whether the intrinsic strength of human bone changes with age or not, we have determined the ultimate tensile strength and density of strips of femoral cortical bone. These femora were collected from cadavers varying in age from 13 to 97 years. The results show that both density and intrinsic strength of bone increase up to about the fourth decade of life and then decrease with age. However, the rate of decrease of strength is greater than that of density. This indicates that the density of bone is not the sole determining factor of its strength, and that some other factors play an important part.

Notes: Summary The perilacunar areas of low mineral density in microradiographs from cortical bone of patients with hypophosphatemic (vitamin D-resistant) rickets are not evenly distributed throughout the bone tissue. Their frequency and distribution were determined in bone from 9 patients with this disease. It was found that the lesion was more frequent in haversian bone than in interstitial bone, and along the inner circumference of growing haversian systems as compared with the outer circumference. These findings indicate that the lesion is the result of retarded mineralization, and that mineralization slowly proceeds in these areas as the bone becomes older. A relatively high frequency of the lesion was also found in osteons with an elliptical cross section along the long axis of the ellipse. The cause of the abundance of the lesion at these sites is not clear, but it is possible to explain the uneven distribution in elliptical osteons by assuming an unequal rate of bone formation in these structures.

Notes: Summary The present study was carried out to determine the influence of carbon dioxide and oxygen tension on the respiratory activity of bone cells in mouse calvaria in vitro. Five-day-old mouse calvaria were removed aseptically and incubated individually for 1 h at 37° C in a closed reaction chamber containing 1.5 ml of tissue culture medium made up of 60% horse serum in Gey's solution containing 100 unit/ml penicillin and 100µg/ml streptomycin. Before the calvaria were added, the medium in the incubation chamber was equilibrated with 10%, 20%, 30%, or 50% oxygen balanced with nitrogen. The effect of CO2 on oxygen utilization by the calvaria was determined by incubating the calvaria in a medium previously equilibrated with either 50% O2 balanced with N2 or 50% O2 and 5% CO2 balanced with N2. At each oxygen tension, the rate of oxygen utilization by the calvaria was measured polarographically by a Clark oxygen electrode. The results showed that the rate of oxygen uptake of bone increased as the oxygen tension increased and carbon dioxide stimulated significantly the rate of oxygen utilization by the bone cells. In view of the previous reports that both carbon dioxide and oxygen tension are implicated in the process of bone resorption, it is suggested that these two factors may affect bone resorption by influencing the oxygen utilization by bone cells and ultimately controlling their energy metabolism.

Notes: Summary Pyrolyzed scales, fin spines, and bone from the ray-finned bony fishPolypterus (Actinopterygii) showed two mineral phases on X-ray diffraction: hydroxyapatite (HA), Ca5(PO4)3OH, and whitlockite, Ca3(PO4)2. The ratio of HA/whitlockite varied with the structure (scale, spine, bone) within each individual fish. The relative proportions of HA to whitlockite in pyrolyzed samples reflected the Ca/P ratio of the sample. Whitlockite appears after pyrolysis when the Ca/P is lower than 1.67. Among the five fish investigated, for each structure a general trend was noted. The proportion of HA relative to whitlockite increased with size (age) of the fish. Thus the smallest fish, a juvenile, exhibited a low Ca/P mineral in its calcified tissues, whereas the larger fish had progressively more HA and less whitlockite.

Notes: Summary Vitamin D and mineral metabolism status was examined in five children maintained chronically on combined ketogenic diet-anticonvulsant drug therapy (KG), and the results compared to those obtained in 18 patients treated with anticonvulsant drugs alone (AD) and 15 normal controls. KG patients exhibited biochemical findings of vitamin D deficiency osteomalacia: decreased serum 25-hydroxyvitamin D (25OHD) and calcium concentrations, elevated serum alkaline phosphatase and parathyroid hormone concentrations, decreased urinary calcium and increased urinary hydroxyproline excretion, and decreased bone mass. Although the KG and AD groups demonstrated similar reductions in serum 25OHD concentration, the KG patients exhibited a significantly greater reduction in bone mass. In response to vitamin D supplementation (5000 IU/day), mean bone mass in the KG group increased by 8.1±0.9% (P〈0.001) over a 12-month period. These results suggest that ketogenic diet and anticonvulsant drug therapy have additive deleterious effects on bone mass and that these effects are partially reversible by vitamin D treatment.

Notes: Summary Osteoclast activating factor (OAF) is a lymphokine which may participate in the pathologic destruction of bone observed in a number of disorders. In the current studies, we investigated the action of OAF on cAMP accumulation by bones and isolated bone cells in culture. OAF was shown to stimulate accumulation of cAMP in mouse cranial bones at doses between 1 and 1000 ng/ml. Stimulation of bone resorption was observed in bones treated with the same doses of OAF. In order to investigate the cell types responsible for cAMP responses to OAF, we isolated bone cells and grew them in monolayer culture. The cells were isolated by a variety of techniques which separate bone cells into two types of parathyroid hormone (PTH)-responsive populations: (a) cells derived from the periosteal regions of the bone, which also respond to calcitonin with increases in cAMP; and (b) cells derived from the matrix, which do not respond to calcitonin. OAF caused elevation of cAMP levels in both the periosteum-derived cells and the matrix-derived cells. The magnitudes and time courses of OAF effects in these populations resembled the effects previously reported for PTH in the same populations. OAF stimulated adenyl cyclase in both types of cell populations, but did not produce significant changes in cAMP phosphodiesterase activity. OAF differed from PTH in that its effects on cAMP accumulation decreased sharply at supramaximal doses in both bone and isolated cells, especially in the matrix-derived populations. Bone resorption did not decrease as markedly as did cAMP accumulation at high doses of OAF, suggesting that cAMP accumulation and resorption could be dissociated under some conditions. These results indicate that OAF has effects on cAMP production in the same cell populations as PTH, and suggest that OAF could modify not only resorption but also formation of bone in vivo. OAF may exert its effects on bone by means of cAMP-dependent mechanisms, but more data will be necessary to establish this unequivocally. The observed differences between OAF and PTH may be of relevance in the mechanism and treatment of pathologic bone destruction in vivo.

Notes: Summary In paraplegia, osteoporosis below the neurological lesion occurs early after the spinal cord affection. The serum levels of parathyroid hormone (PTH) and calcitonin (CT), using a radioimmunoassay for the measurement of immunoreactivity, were studied in 12 paraplegic patients for 9 months following onset. Serum Ca and P levels, urinary hydroxyproline excretion, and the kinetic metabolic clearance of45Ca have also been measured. P and immunoreactive (i) CT levels were found the highest at the beginning of the observation and progressively decreased with time. Ca and iPTH serum levels varied inversely with time, the highest level of Ca and the lowest level of iPTH being recorded at the third month following the paraplegia. Mean values of Ca, iPTH, and iCT were in the normal range throughout the study. P levels were increased during the first 3 months. Hydroxyprolinuria was also high and45Ca kinetics showed increased values of Vt, Vo+, and Vu. These parameters indicate a high degree of bone turnover. The results were consistent with the assumption that PTH is not responsible for the increased resorption of bone in paraplegia. Likewise, a deficiency of CT does not seem to be responsible for this bone resorption. These endocrine modifications could be secondary to an increase in the calcium flux from bone to blood and resulting from bone destruction as attested by the increase of urinary calcium and urinary hydroxyproline excretion.

Notes: Summary Vitamin D and phosphate deficiency were produced in rats in order (a) to evaluate the degree of bone mineral and matrix maturation using a bromoform/toluene density gradient technique; and (b) to compare the aforementioned bone maturational changes due to vitamin D and phosphate deprivation to those produced with superimposed severe acidosis. Rats were fed a diet deficient in vitamin D and phosphorus (0.2%) from 3 weeks through 7 weeks of age. To examine the additional contribution of dietary calcium, we gave one-half of the animals either a low (0.06%) or high (1.3%) calcium diet. Following the 4 weeks of vitamin D deficiency, one-half of each group was given 1.8% NH4Cl in the drinking water for 4 succeeding days to induce an acute, severe acidosis. The degree of bone maturation was quantitated via bromoformtoulene density gradient fractionation; total mineral and hydroxyproline (collagen) levels were quantitated as well. The vitamin D-deficient rats deprived of adequate dietary phosphate responded by conserving phosphorus, and as a consequence total bone phosphorus levels were maintained within that level for control rats. This conservation was independent of calcium intake but was extremely sensitive to acute acid loading, where a significant reduction in total bone phosphorus was noted. The bone maturational profile obtained from the vitamin D-phosphate deficient rats, however, revealed a significant accumulation of less mature or dense bone collagen and mineral with a corresponding decrease in the most mature or dense moieties. In contrast to the reduction of the total bone phosphorus content by acute acidosis, the skeletal collagen-mineral maturational profile was not significantly affected by the short-term systemic acidosis. The observed retardations in the bone collagen and mineral maturation of the vitamin D-deficient, phosphate-deprived state provide an additional observation which may well relate to the progressive osteopenia documented in states of chronic, mild acidosis.

Notes: Summary Proline biosynthetic and degradative enzymes are unevenly distributed in differentiated mammalian tissues. Activities of the synthetic enzymes are relatively high in collagenous tissues, whereas activities of the degradative enzymes are high in noncollagenous tissues. In order to further characterize tissue-specific proline biosynthesis and degradation, we have determined proline enzyme activities during cartilage and bone formation induced by demineralized bone matrix. We can thus follow temporal changes in enzyme activity in a single tissue as different cell types develop. Ornithine aminotransferase and pyrroline-5-carboxylate reductase have peaks of activity which correlate with maximal type II collagen synthesis by chondrocytes. Both enzymes also are active during bone formation. In contrast, proline oxidase and pyrroline-5-carboxylate dehydrogenase are present at low levels and do not change as new cell types appear. Arginase activity peaks during the first 3 days and then rapidly decreases by the time cartilage and bone formation begin. These observations further substantiate the importance of proline biosynthesis in collagenous tissues. The close correlation between ornithine aminotransferase activity and type II collagen synthesis suggests that the pathway from ornithine to proline may be especially important during formation of type II collagen.

Notes: Summary The in vivo bending rigidity and bone mineral content of monkey ulnae and tibiae were measured. Bending rigidity in the anteroposterior plane was measured by an impedance probe technique. Forced vibrations of the bones were induced with an electromechanical shaker, and force and velocity at the driving point were determined. The responses over the range of 100–250 Hz were utilized to compute the bending rigidity. Bone mineral content in the cross section was determined by a photon absorption technique. Seventeen male monkeys (Macaca nemestrina) weighing 6–14 kg were evaluated. Repeatability of the rigidity measures was 4%. Bone mineral content was measured with a precision of 3.5%. Bending rigidity was correlated with the mineral content of the cross section,r=0.899. Two monkeys were evaluated during prolonged hypodynamic restraint. Restraint produced regional losses of bone most obviously in the proximal tibia. Local bone mineral content declines 17 to 24% and the average bending rigidity declines 12 to 22%. Changes in bones leading to a reduction in mineral content and stiffness are discussed.

Notes: Summary Parameters of mineral metabolism were examined in 6 patients with moderately severe anticonvulsant drug-induced osteomalacia. Compared to 15 matched controls, the patients exhibited significantly reduced serum calcium, inorganic phosphate, and 25-hydroxyvitamin D concentration, elevated serum alkaline phosphatase and immunoreactive parathyroid hormone (iPTH) concentration, reduced intestinal47Ca absorption, reduced urinary calcium and increased urinary hydroxyproline excretion, and reduced forearm bone mass. Intestinal absorption of vitamin D3 was normal. Following 4 months of treatment with vitamin D3 (4000 units/day), serum 25-OHD concentration was increased to 3 times mean normal values and all parameters except serum iPTH, urinary calcium excretion, and forearm bone mass were returned to levels not significantly different from normal. Serum iPTH concentration was reduced by 39% (P〈0.05); 24-h urinary calcium excretion rose by 98% (P〈0.001), and forearm bone mass increased by 5.6% (P〈0.05). It is concluded that moderate-dose vitamin D3 supplementation is effective in normalizing parameters of mineral metabolism in this disorder, despite evidence of resistance to the biologic effects of vitamin D.

Notes: Summary We measured mineral and acid balances, serum iPTH, urinary cAMP/creatinine, and plasma concentrations of 25OHD and 1,25(OH)2D in 7 healthy adults during control conditions and during increased fixed acid production achieved either by the administration of NH4Cl (N=3) or by increased dietary protein intake (N=4). When acid production was increased, the subjects were in positive acid balance and negative Ca balance because of increased urinary Ca excretion. Serum iPTH fell slightly but urinary cAMP and the plasma levels of vitamin D metabolites did not change. We conclude that the accelerated skeletal and urinary losses of Ca that occur when fixed acid production is increased are not contributed to nor compensated for by the parathyroid-vitamin D endocrine systems.

Notes: Summary Compact bone tissue chondroitin sulphate, previously considered to be pure chondroitin-4-sulphate, was isolated from three adult human femoral diaphyses and digested with chondroitinase ABC. Assaying these digests by means of high performance liquid chromatography we could demonstrate that 12 to 14% of the disaccharide residues were 6-sulphated. The 4/6-ratios were also studied in chondroitin sulphate fractions of different molecular size. Slightly increasing amounts of 6-sulphated disaccharides were found with increasing size of the chondroitin sulphates.

Notes: Summary We have examined the effects of H+, CO2, and HCO3 − concentrations during metabolic and respiratory acidosis and alkalosis on bone resorption in vitro. Rat fetal bones prelabeled with45CaCl2 were cultured at 2%, 5%, and 10% CO2 for up to 120 h, and the release of45Ca was measured in devitalized bones (non-cell-mediated45Ca release) and in live bones (cell-mediated45Ca release) cultured with or without PTH and 1,25(OH)2D3. Non-cell-mediated mineral loss was linearly related to H+ concentration but not to CO2 or HCO3 − concentration. This effect was observed on both labeled and stable calcium. Over a wide pH range (6.9–7.5) H+, CO2, or HCO3 − concentrations did not influence cell-mediated bone resorption in control or in PTH-and 1,25(OH)2D3-stimulated cultures. However, inhibition of cell-mediated bone resorption was observed at higher or lower pH irrespective of CO2 or HCO3 − concentrations. These observations demonstrate that the bone mineral mobilizing effect of acidosis in vitro is mainly due to the effect of changing H+ concentration on devitalized bone. Effects on cell-mediated bone resorption and hormonal response were observed only at extremes of pH. The effects of H+ were independent of changes in CO2 or HCO3 − concentration and could be responsible for the negative calcium balance and increased urinary loss observed in metabolic acidosis in vivo, but do not explain the reported differences in effects on calcium metabolism between respiratory and metabolic acidosis.

Notes: Abstract Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed “enzyme A” (MW 82,000) and “enzyme B” (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl2, are required for activity; optimal activity occurs at pH 7.4 and 30 C; the K m for dihydroneopterin triphosphate is 10 µm; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.

Notes: Abstract Four glycolytic enzymes in Drosophila melanogaster have been genetically and/or cytogenetically mapped. The structural gene for aldolase (Ald) has been genetically mapped to 3-91.5 and cytogenetically localized to 97A-B. Tpi, the structural gene for triosephosphate isomerase, has been genetically mapped to 3-101.3 and cytogenetically localized to 99B-E. Utilizing closer-flanking markers than the previous mapping, Pgk, the structural gene for 3-phosphoglycerate kinase, has been mapped to 2-5.9; cytogenetically it was found to lie in the interval between 22D and 23E3. The cytogenetic location of Pgm, the structural gene for phosphoglucomutase which has been located genetically at 3-43.4, was determined to be in 72D1-5.

Notes: Abstract NADP-malic enzyme (NADP-ME) (E.C. 1.1.1.40) is situated in the cytosol of Drosophila melanogaster. Both the tissue activity and CRM level of NADP-ME parallel changes in the dosage of a gene, Men +, located in region 87C2-3 to 87D1-2 of the third chromosome. The tissue activity of NADP-ME is very high in early third instar larvae, providing about 33% of the NADPH at this life stage. The tissue activity declines during pupal development but increases as the adult ages. The concentration of NADP-ME CRM and tissue activity are coordinately increased in third instar larvae by dietary carbohydrate and decreased by dietary lipid.

Notes: Abstract Isoelectrofocusing of abdominal extracts of Drosophila melanogaster revealed the existence of two forms of sucrase (E.C. 3.2.1.26). One form exhibited an isoelectric point of 4.63±0.02 while the other form exhibited an isoelectric point of 4.83±0.02. The localization of the structural gene for sucrase is proposed on the basis of enzyme determinations in a series of duplication- and deletion-bearing aneuploids. We suggest that the sucrase structural gene lies between 31CD and 31EF on the left arm of chromosome 2 and that the two forms of abdominal sucrase derive from a common protein coded for by a single sucrase gene designated Sucr +.

Notes: Abstract Genetic and cytogenetic locations of the structural genes for the NAD-dependent malate dehydrogenases have been studied. The mitochondrial form (mMDH) is coded for by a gene (Mdh) found at 62.6 on the third chromosome and included in Df(3R)P14, which includes 90C2–91A3 in the salivary gland chromosomes. Based on its inclusion within several J (Jammed; 2–41.0) deficiencies, the structural gene (cMdh) for the cytoplasmic form (cMDH) was determined to lie in region 31B-E, confirming the earlier finding of Grell. Flies lacking any cMDH activity (cMdhn-γ10069/ Df(2L)J-der-27) were both viable and fertile.

Notes: Abstract When adult Drosophila are placed on medium containing 0.5% acetone, their level of alcohol dehydrogenase activity drops rapidly. At the same time, the proportion of activity in the various electrophoretic forms of the enzyme shifts; most of the activity becomes localized in what is ordinarily a minor form of the enzyme. Moreover, the loss of enzyme activity occurs in vivo as well, as shown by sensitivity to ethanol poisoning, insensitivity to pentenol treatment, and inability to utilize ethanol as an energy source. These observations are discussed in light of a model advanced for the origin of the multiple forms of alcohol dehydrogenase in Drosophila.

Notes: Abstract Several in vitro properties of partially purified form II RNA polymerase from Drosophila melanogaster embryo nuclei are described. The enzyme preparation is free from contaminating RNase, protein kinase, and polyphosphate kinase activities and can be used to study the incorporation of γ-32P-labeled nucleoside triphosphates. The enzyme exhibits a biphasic heat inactivation pattern which is probably related to differential lability of its two subforms. However, a considerable protection against heat inactivation is provided by the nucleoside triphosphates present in the in vitro reaction system such that the enzyme catalyzes RNA synthesis in a nearly linear mode for over 2 hr at 30 C. Two initiation inhibitors, rifamycin AF/013 and polyriboinosinic acid (poly[I]), were tested against this enzyme. Rifamycin AF/013 was found unsuitable for critical studies because of the high concentrations necessary for total inhibition (200 µg/ml) and particularly because of the obligate use of solvents which secondarily have a destabilizing effect on native DNA. Poly[I] was found to effectively block initiation at very low concentrations (1 µg/ml). The enzyme rapidly forms poly[I]-resistant preinitiation complexes on both double- and single-stranded DNA. These complexes decay with a half-life of 2.5–3 min. RNA synthesis from poly[I]-resistant complexes amounts to 10% of the total potential synthesis on both double- and single-stranded DNA. Enzyme-DNA saturation experiments indicate that the form II enzyme discriminates two types of sites on Drosophila DNA, tight binding and weak binding, from which RNA synthesis proceeds slowly and rapidly, respectively. The tight-binding sites appear to be analogous to those sites with which the enzyme is able to form poly[I]-resistant complexes.

Notes: Abstract Allozyme frequency data from natural populations of Drosophila buzzatii were analyzed for genotype-environment relationships. Allele frequency and heterozygosity at six loci polymorphic throughout eastern Australia and a number of environmental factors (both means and variabilities) were examined by a variety of multivariate techniques. Significant genotype-environment associations were found for five of the six loci, and after correcting for geographic location significant associations remained for Est-2 and Adh-1 gene frequencies and heterozygosities and for Pgm gene frequencies. The results are discussed in relation to selection and gene flow and provide the basis for laboratory studies to disentangle confounded effects of (1) environmental means and environmental variabilities and (2) allele frequency and heterozygosity, and thus to further test for and determine the nature of any natural selection at particular allozyme loci.

Notes: Abstract A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.

Notes: Summary As a test of the hypothesis that adult temperature stress is an important component of natural selection maintaining the α-gpdh polymorphism, we have looked for differential survival among genotypes subjected to (i) heat shock and (ii) cold shock. Factorial ANOVAR, taking account of genotype, sex and temperature-stress indicated that genotype did not contribute to the variance of survival proportion per vial. We have not therefore found evidence to support our hypothesis. Incidental to the above was a significant sex-temperature interaction. Thus, adult females showed higher survival than males under heat stress, while under cold stress, there was no indication of a survival difference between the sexes.

Notes: Abstract Comparative studies of oviposition site preference (OSP) inDrosophila suggest that choice of oviposition site is an important adaptive behavior which influences individual fitness and the potential of populations for speciation. OSP has been investigated under conditions which provided females with a choice of standard medium or medium containing ethanol for oviposition. OSP is an extremely labile behavior in the laboratory, but a technique has been developed which minimizes variation between replicates and allows the detection of OSP differences between semispecies of a single species. An analysis of the OSP of 14Drosophila species shows that this behavior is not correlated with phylogenetic relationships. OSP with respect to ethanol may be correlated with the presence of ethanol in the environment and the activity of alcohol dehydrogenase in the species tested.

Notes: Abstract An alternative explanation to the pheromonal control of mating through chemoreceptor saturation proposed by Averhoff and Richardson (1974) is offered for the apparent rise in heterogamic mating in their experiments, after several generations of full-sib mating. In a multiple-choice mating between two genotypic strains differing in their level of sexual vigor, there is a sequence from heterogamic to homogamic mating. It is proposed that, by reducing mating speed, inbreeding changes the rate of this sequence but not its pattern, so the apparent level of heterogamic mating will increase during inbreeding, for a fixed observation period. This hypothesis was tested using the Kence-Bryant model of mating success.

Notes: Abstract Two alcohol dehydrogenase genotypes (Adh F /Adh F andAdh S /Adh S ) exhibit different behavioral responses when presented with a choice between ethanol and nonethanol environments at the larval stage but not at the adult stage. The larval preferences are correlated with alcohol dehydrogenase activity, which also differs between genotypes. Since ethanol is important in the ecology of this species, the preference may be related to microhabitat selection in nature.

Notes: Abstract Selection for the wing vibration component of courtship in the Oregon-R stock ofD. melanogaster was practiced for 44 generations. Selection was successful, indicating that there is genetic variation for the trait in the Oregon-R stock. The mean realized heritability of the trait, based on the first 11 generations of selection, was 15%. Biometrical analysis showed that there is some additive genetic variance for the trait with the possibility of some ambidirectional dominance. No maternal effects for the trait were found.

Notes: Abstract It is shown that minority mating advantages, so commonly observed inDrosophila, would be selectively advantageous in a heterotic system. When an allele is below the equilibrium frequency maintained by heterosis, females mating with that homozygote produce offspring of highest mean fitness.

Notes: Abstract Using Hirsch-Hadler phototaxis mazes, selection for photopositive and photonegative behavior was carried out for 21 generations inDrosophila ananassae. The chromosomes that are important in influencing photomaze behavior inD. ananassae are different from what has been observed for other members of themelanogaster species group, and the differences cannot be entirely attributed to the chromosome rearrangements which have occurred during the evolution of these related species.

Notes: Abstract Experiments on frequency-dependent fitness often consist of forming pairwise mixtures of distinguishable types at several frequency combinations. These mixtures are allowed to undergo competition, after which the performance of each type is enumerated. A statistical method for analyzing such experiments is described in this article. This method, suggested previously for other purposes, is superior to the statistical procedures now commonly employed. It involves the maximum likelihood estimation of parameters for two logistic regression models: one which assumes that fitness is frequency-dependent, the other that fitness is constant over changing frequency. Estimators for both models can be calculated without difficulty using an iterative numerical algorithm implemented in a Fortran computer program available from the authors. Fitting both models allows for the construction of a likelihood ratio statistical test for whichever model is more appropriate. The method is illustrated by application to publishedDrosophila data from differential mating success experiments.

Notes: Summary Pinocytotic activity has been analyzed in Drosophila oocytes following either in vivo or in vitro exposure to horseradish peroxidase. The enzyme tracer gains access to the yolk spheres only when supplied to the oocyte in vivo. In oocytes cultured in vitro, peroxidase remains restricted to the residual coated vesicles and to the tubular profiles formed in excess in the cortical ooplasm. In an attempt to induce peroxidase uptake by oocytes cultured in vitro, various incubations were tested. Among these, hemolymph from both sexes is capable of promoting peroxidase uptake up to a level comparable to that detectable in vivo. On the other hand, fat body extracts fail to promote such cellular activity. Finally, the juvenile hormone analogue ZR-515 is shown to be the only factor required to promote pinocytotic activity under the experimental conditions tested. The observations are interpreted to indicate that vitellogenin has no inductive role on pinocytosis but simply acts by adhering to the forming coated vesicles which in turn are produced by the oolemma in response to the action of juvenile hormone.

Notes: Abstract Specimens of dry human cortical bone of a disc form were subjected to sinusoidal loading under controlled conditions. The results show a nonlinear increase of the measured peak potential with frequency which reaches a limiting value at about 200 Hz. The analysis, which is based on the circuit representation proposed previously by Shamos, indicates that such a behaviour can be attributed to the measuring circuit parameters. It was found that some relevant results can be interpreted qualitatively by similar analysis.

Notes: Summary Mineral particle cluster, corresponding to calcospherites or matrix vesicle-initiated clusters in calcifying cartilage and dentin and to collagen bundle-related mineralization in lamellar bone, have been isolated from NaOCl solutions used to dissolve the organic matrix in preparation of anorganic mineralizing fronts for scanning electron microscopy.

Notes: Summary Osteoclastic bone resorption involves the solubilization of the mineral salts and the degradation of noncollagen bone matrix and collagen fibrils. As no recognizable collagen fibrils have ever been reported within cytoplasmic vacuoles in osteoclasts, it is generally assumed that the collagen fibrils are digested extracellularly in the resorption zone. The extent to which lysis occurs extracellularly and whether or not the osteoclasts phagocytose the degradation products remain to be established. In the present communication, a hypothesis is presented suggesting the possibility that osteoclastic resorption of bone involves the participation of two different cell types. According to this hypothesis, osteoclastic bone resorption is initiated by osteoclasts that demineralize areas of bone and degrade noncollagen bone matrix. After the osteoclasts have moved away or become partially detached from the demineralized site, the exposed collagen fibrils are phagocytosed by mononuclear, fibroblast-like or monocyte-derived cells.

Notes: Summary Plasmaα 2HS-glycoprotein is specifically accumulated in calcified tissues. In the present studies this glycoprotein was isolated from plasma and after iodination with iodine-125 was injected intravenously into young rabbits. The tissue distribution and plasma disappearance rate of this radioactively labeled material were determined. Of the various tissues studied, bone showed the greatest retention of labeled glycoprotein expressed as percentage of the injected dose per gram tissue relative to the plasma content. The rate of loss of iodinatedα 2HS-glycoprotein from plasma was similar to that ofα 2HS-glycoprotein labeled endogenously by using14C-glucosamine or3H-glucosamine. The uptake of exogenously labeled3I-α 2HS-glycoprotein into bone tissue expressed as a percentage of the injected dose was similar to that of endogenously labeled14C-α 2HS-glycoprotein. These results suggest that the125I-labeled material can be used to study further the metabolism ofα 2HS-glycoprotein by bone tissue.

Notes: Summary Mithramycin suppresses bone resorption. Its effect on the synthesis and release of β-glucuronidase (a referent for lysosomal enzymes) in mouse calvarial explants was studied in an in vitro culture system. A newly described medium (designated as KT medium) was introduced in this specific study. Mithramycin initially inhibited the release of β-glucuronidase into the medium and resulted in an ultimate accumulation of this enzyme in the bone. These results suggest that inhibition of bone resorption by mithramycin may be attributed to interference in release of lysosomal enzymes from bone cells.

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 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 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 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 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 Exposure of bone mineral to X-rays generates free radicals. These are usually very labile, but can be stabilized at liquid nitrogen temperatures for study by electron spin resonance spectroscopy. The free radicals thus detected in the present study included one with resonances arising from an electron excess center and 2 species with electron-deficit centers: a phosphate anion radical and a radical associated with carbonate. Each of these radicals seemed to be located chiefly at the mineral surface and was sensitive to the surface environment. Presence of an organic phase, as in whole bone, modified free radical production in a manner that suggests interference with the formation of electron deficit centers. Comparison with other synthetic minerals suggests that precipitated carbonateapatites are good models for bone mineral.

Notes: Summary The purpose of this study was to measure in an in vitro system the movement of Ca and phosphate (Pi) out of bone when treated with parathyroid hormone (PTH). Tibiae from 13-day chick embryos were incubated for up to 8 h in a defined medium containing 1.8 mM Ca. Medium samples were collected every 2 h and were analyzed for Ca, Pi and lactate. Net effluxes from the bones were calculated. When bones were incubated with PTH in the medium (1 U/ml), net Ca efflux was increased 44, 60 and 100% at 4, 6 and 8 h, respectively. At no time was net Pi efflux affected by the hormone. The well known PTH-stimulated lactate production was not seen until 8 h. Lower doses of PTH (0.1 and 0.3 U/ml) were also effective. Comparing PTH (1 U/ml) responsiveness at higher (2.2 mM) and lower (0.9 mM) medium Ca concentrations, showed that with 2.2 mM Ca no increased Ca efflux was seen, while with 0.9 mM Ca significant elevation in medium Ca occurred 2 h sooner than in the experiments using 1.8 mM Ca. In another experiment, varying the medium Pi level from 1 to 2 mM had no effect on the Ca response to PTH. In neither experiment was Pi release affected by PTH. The results of this study have led to the following conclusions: (1) PTH acts on bone to cause an early dose related increase in net Ca efflux; (2) the effect is specific for Ca, since it is not accompanied by an increased Pi efflux, and may be saturated by raising the medium Ca level; and (3) PTH-stimulated Ca efflux in this system is not correlated with, and is probably not a result of increased lactate production.

Notes: Summary A fraction (brA), which causes resorption of fetal rat bones in vitro, has been concentrated from bovine serum albumin by anion exchange column chromatography on DEAE Sephadex. This active fraction has also been prepared using DEAE Sephadex A-50 by a batch method with a 0.09M NaCl, 0.1M TRIS buffer, pH 8.35. BrA was 10–30 times more potent than the original albumin. The retained material, which constitutes the bulk of the protein and has less activity than the original albumin, elutes with 0.45M NaCl. Similar treatment of serumα,β or γ globulins does not yield brA. Further enhancement of the bone resorbing activity of brA can be obtained with (NH4)2SO4 fractionation or extraction with CH3OH∶CHCl3. Heating at 55° C for 2 h or at 100° C for 10 min does not affect the activity; overnight incubation with protease destroys the bone resorbing effect. The bone resorbing activity is not removed by dialysis and does not correlate with the protease activity of the fraction. The action of brA is inhibited by 3 mM PO4, 1 μg/ml calcitonin or glucagon, 10−7 M dexamethasone or 0.02 μg/ml actinomycin D. The bone resorbing activity of brA is partially inhibited by 10−7–10−5 M indomethacin. PTH did not elicit bone resorption when added to cultures incubated in chemically defined medium supplemented with 0.1 mg/ml brA. However, brA did not inhibit PTH-induced resorption.

Notes: Summary Attempts were made to demonstrate the presence of specific oestrogen binding proteins (“receptors”) in bone cells. High speed cytosol preparations of bone were incubated with several concentrations of radioactive oestradiol alone and with radioactive oestradiol in the presence of a specific antioestrogen, Nafoxidine. Separation of bound and free oestradiol was carried out by dextran coated charcoal treatment and by sucrose gradient ultracentrifugation. Several types of bones likely to be oestrogen-sensitive were investigated: human femoral heads, human phalanx, rat and rabbit calvaria, humeri and femora of female rats. In all experiments we were unable to demonstrate the presence of specific oestrogen receptors in bone cell cytosol indicating that the direct effect of oestrogens on bone, if present, is not mediated by specific oestrogen receptors.

Notes: Summary A comparatively simple quantitative method for assessing bone morphology has been evolved. Microradiographs of thin sections of mandible have been scanned with a Joyce-Loebl double beam recording microdensitometer with a scanning autodensidater attachment, using a white beam. For each image the optical densities for all the pixels (picture elements) were divided into 10 groups. The limits of the division were fixed by the maximum and minimum densities occurring within the image. A computer generated map was produced which indicated the spatial distribution of the pixels within each group to which an arbitrarily chosen shading was attached. The number of pixels within each group is also shown on the map. The computer map was compared with the photomicrograph and, where necessary, the original section. The fractional area of hard tissue was then readily determined using the numerical values of each group of pixels.

Notes: Summary The surface areas of three different samples of hydroxyapatite, samples of deproteinized bone, and samples of whole and deproteinized enamel were determined by adsorption of an adduct (the diglycidyl ether of bisphenol A with N-phenylglycine) from methylene chloride solution. In all cases, the surface areas of these samples agree well with those obtained by the BET (N2) method.

Notes: Summary Although sex steroids are known to affect skeletal metabolism, their effects on bone collagen synthesis have not been studied. We have examined the direct effects of progesterone, 17β estradiol, testosterone, 5α dihydrotestosterone and dehydroepiandrosterone on bone collagen and noncollagen protein synthesis in cultures of calvaria obtained from 21-day fetal rats. Bones were incubated for 24 to 168 h and3H-proline was added for the last 2 h of culture. Incorporation of the label into collagenase-digestible protein (CDP)1 and noncollagen protein (NCP) was determined using repurified bacterial collagenase. Progesterone caused a dose dependent inhibition of the labeling of CDP at concentrations of 3×10−7 M to 10−5 M after 96 h of culture. A smaller inhibitory effect was observed on NCP. The inhibitory effect was slow in onset and persisted when bones were incubated for 48 h with progesterone and then transferred to control medium for 48 h. Progesterone also inhibited the incorporation of3H-thymidine and3H-uridine into fetal rat calvaria. After 24 h of culture, 17β estradiol and testosterone had no effects on the labeling of CDP and NCP. After 96 h, 17β estradiol had a small and inconsistent stimulatory effect on the labeling of CDP but testosterone had no effect. Neither hormone altered the inhibitory effects of parathyroid hormone, cortisol and progesterone. Dihydrotestosterone and dehydroepiandrosterone had no effects after 24 h and 96 h of culture. 17β estradiol, testosterone and dihydrotestosterone did not affect the incorporation of3H-uridine or3H-thymidine into fetal rat calvaria. Our studies indicate that progesterone is an inhibitor of bone collagen synthesis and estrogens and androgens are not major regulators of bone formation in vitro.

Notes: Summary 125I-labeled rat albumin injected intravenously into rats was taken up by growing bone. Some of this radioactive albumin could be removed from bone by washing with saline, the proportion so removed decreasing from 82.5% at 1 day to 7.4% at 8 days. Both the total radioactivity, and that remaining in bone after saline wash, were reduced when growth was slowed by alteration or restriction of the diet. Although the amount of125I albumin in rachitic bone was reduced, autoradiography showed that radioactivity was present in rachitic osteoid. Immunoprecipitation using anti-rat-albumin serum showed that about half of the radioactivity released from normal bone by EDTA was still attached to albumin. These results suggest that albumin plays some part in the growth of bone.

Notes: Abstract Three of the major protein species present in the hemolymph of Drosophila melanogaster larvae just prior to pupation are absent from second instar larvae but accumulate rapidly during the third instar. This article describes the purification and characterization of one of these, larval serum protein (LSP) 2, using an immunological assay. It is a homohexamer of molecular weight about 450,000, with a polypeptide molecular weight of 78,000–83,000. Fast and slow electrophoretic variants of this protein map between the markers vin and gs, at 36–37 on chromosome 3.

Notes: Abstract Only one molecular form of trehalase (E.C. 3.2.1.28) was detectable in adult Drosophila melanogaster by polyacrylamide gel electrophoresis and isoelectric focusing. An examination of duplication- and deletion-bearing aneuploids exhibiting do sage sensitivity indicated that the enzyme is encoded by a gene, Treh +, located between 55B and 55E of the second chromosome. The tissue-specific soluble and particulate forms of trehalase appear to be manifestations of a single protein encoded by a single gene.

Notes: Abstract Glutamine-dependent CPSase, ATCase, and DHOase from Drosophila, the first three enzymes in pyrimidine biosynthesis, show coordinate variation in activity throughout development. The three activities were highest in first instar larvae and decreased as development proceeded. The three activities cosediment in sucrose gradients as a single peak with a relative sedimentation coefficient of approximately 30S. CPSase, ATCase, and DHOase copurify during (NH4) 2SO4 fractionation and during DEAE-cellulose and hydroxylapatite chromatography.

Notes: Abstract It has been shown that crude extracts of Drosophila melanogaster adults contain three distinctly different enzymes which catalyze the oxidation of d-sorbitol into d-fructose. These include (1) a soluble NAD-dependent sorbitol dehydrogenase (NAD-SoDHs), (2) a mitochondrial NAD-dependent sorbitol dehydrogenase (NAD-SoDHm), and (3) a soluble NADP-dependent sorbitol dehydrogenase (NADP-SoDH). Developmental studies have shown that the activities of all three of these enzymes are lowest during the larval stages while highest levels are seen during or shortly prior to the adult period. With respect to NAD-SoDHs, studies of tissue distribution in adults have shown that highest activity is associated with thoracic musculature in both sexes and with organs of the male reproductive system. The developmental profile of this enzyme reveals a significant increase in activity at between 40 and 60 hr after hatching. This time interval corresponds closely to that during which the paternally derived NAD-SoDHs gene is expressed. An additional increase in activity is seen in male pupae at 160 hr and in female adults at 210 hr. The rapid increase in males takes place immediately following the developmental period during which the testes attach to their respective duct systems. NADP-SoDH activity is concentrated among organs of the thorax and abdomen in both sexes. Males show significantly higher levels of this enzyme during the late pupal and early adult periods. In contrast to the patterns of distribution seen for NAD-SoDHs and NADP-SoDH, 91–92% of the total NAD-SoDHm activity in adults is localized to the thoracic musculature. The developmental profile of this enzyme reveals a significant increase in activity during the late pupal and early adult periods, when flight muscle mitochondria are known to be proliferating and undergoing structural maturation.

Notes: Abstract Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55–65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.

Notes: Abstract It is shown that the gene controlling the synthesis of the organ-specific S-esterase of Drosophila virilis ejaculatory bulbs is located on the second chromosome (at approximate position 192.1±map units). The cells of the genital imaginal disks are determined for the synthesis of S-esterase 10–12 hr after the second molt. The organ-specific esterase can be detected after adult emergence only. It is preceded by an increase in RNA content and by enhancement of RNA synthesis in the cells of the ejaculatory bulbs. Interstock differences were found in the level of the activity of S-esterase, which is under the control of the X chromosome, as well as in the time of expression of enzyme activity, which is controlled by the fifth chromosome. It is suggested that the specific phenotypic expression of this enzyme depends on the system of genes with regulatory expression at both the transcriptional and posttranscriptional levels. The genetic control of the synthesis of the S-esterase described is a convenient model for studying mechanisms of gene activity regulation in eukaryotes.

Notes: Abstract Starch and polyacrylamide gel electrophoresis were used to ascertain the substrate specificities of alcohol-oxidizing enzymes in 13 Drosophila species. The substrates used were a variety of long- and short-chain aliphatic alcohols, one aromatic alcohol, and benzaldehyde. Only one enzyme (product of a single-gene locus) showed significant NAD+-dependent alcohol dehydrogenase activity with short-chain aliphatic alcohols. The 13 species, belonging to four different Drosophila groups, all showed a similar complement of alcohol-oxidizing enzymes, although differences in electrophoretic mobility and in levels of activity existed from species to species. These findings are relevant to the adaptation of Drosophila to alcohol environments.

Notes: Abstract There is an association among resource utilization divergence, habitat selection, and taxonomic divergence in the genusDrosophila. Given permissive conditions of temperature, humidity, and light intensity, an enormous variety of resources is used in a diversity of habitats. These resources are considered in the cosmopolitan and endemic Australian fauna, providing evidence for habitat selection in the laboratory and field. Lek behavior in picture-winged species of subgenusHirtodrosophila, a case of parallel evolution with lek behavior in subgenusDrosophila in Hawaii, is discussed in detail. Other examples of habitat selection discussed concern behavioral reactions of larvae to alcohol and other metabolites and the avoidance by adults of extreme physical environments. Evolutionary strategies involved in habitat selection are considered at various taxonomic levels inDrosophila. These considerations show that it is essential to relate results from laboratory studies to natural environments in order to explore the genetics of habitat selection.

Notes: Summary Intercellular bridges have been detected in ovarian follicle cells of Drosophila melanogaster. These bridges occur widely between follicle cells of previtellogenic chambers, while, in vitellogenic chambers, they become restricted to the columnar follicle cells. Usually, only one bridge is detectable between adjacent follicle cells, but a single cell may form two cytoplasmic continuities. The fine structure of the intercellular bridges is similar to that previously described in the development of Drosophila. The bridge wall consists of two layers of which the more external is more electron dense and thinner than the inner one. The role played by the intercellular bridges in the determination of a synchronous differentiation of the linked follicle cells is discussed in relation to the known behaviour of these cells in the secretion of the egg covering precursors.

Notes: Abstract A method is presented for separating osteoclasts from the heterogeneous population of bone and marrow cells. Cell suspensions were prepared from femora of young rabbits by mechanical dispersion. The starting cell suspension typically contained only 1.0%±0.5 osteoclasts. Following an initial 45 min of unit gravity sedimentation in a lucite chamber osteoclasts were primarily distributed in fractions 2–5. A second 45-min sedimentation of these pooled fractions yielded cell suspensions containing greater than 30% osteoclasts (as much as a 50-fold increase over starting percentages). Linear scan analysis, however, revealed that osteoclasts accounted for 73.14%±0.58 of the cell colume. Subsequent in vitro experiments demonstrated linear incorporation of3H-leucine into TCA precipitable protein for cells comprising the osteoclast fraction. Concomitant radiautographs revealed radioactive label in isolated osteoclasts.

Notes: Summary Male rats weighing 100 g were assigned to groups and injected daily for 10 days with vehicle (control), 0.4, 2.0, 4.0, 10.0, or 20.0 mg/kg/day of ethane-1-hydroxy-1,1-diphosphonate (EHDP) or dichloromethylene diphosphonate (Cl2MDP). The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation parameters. From the data, information about hard tissue resorption was deduced. All doses of Cl2MDP and doses of 2.0 mg EHDP/kg/day and greater caused significant increases in percentage of hard tissues with Cl2MDP being more effective than similar doses of EHDP in decreasing bone resorption. Osteoclast population parameters were increased with all doses of both Cl2MDP and EHDP with Cl2MDP having a greater effect than similar doses of EHDP. Decreases in the proliferation of the osteoprogenitor pool parallel the decreases in osteoblasts and bone formation parameters. These decreases in osteoprogenitor pool proliferation do not account for the increases with diphosphonates in osteoclast population parameters.

Notes: Summary The stimulation of bone resorption, assessed by the release of45Ca from prelabeled bones, was associated with an increase in number of osteoclasts per bone section in parathyroid hormone (PTH)-treated bones, but not in lipopolysaccharide (LPS)-treated bones. By contrast the number of nuclei per osteoclast increased following LPS treatment, but was not affected by PTH. LPS-treated bones had more multinucleated cells, some having as many as 27 nuclei per osteoclast. More osteoclasts were adjacent to the bone collar in bones treated with LPS or PTH than in control bones. In LPS-treated bones this area also contained the largest osteoclasts, as determined by the greatest number of nuclei per osteoclast. The results suggest that LPS and PTH stimulate osteoclastic resorption by different mechanisms.

Notes: Summary The extractability of125I-labelled plasma albumin from bone pieces and from powdered bone has been compared after both in vivo and in vitro incorporation. The results show that albumin is more readily extracted from bone pieces than from bone powder which implies that tissue disruption exposes additional protein adsorption sites. It is suggested that incorporation of plasma albumin into calcified matrix during bone formation occurs mainly as a result of its strong interaction with bone mineral.

Notes: Summary Cortical bone remodeling along the femur diaphysis was determined in normal female rats (Sprague-Dawley) with the tetracycline technique. Three segments on the cortical bone circumference (the anterolateral, the medial, and the posterior) were found to be most suitable for the study of the remodeling process. Oxytetracycline was administered at age 60 and 75 days, and groups of animals were killed at age 75, 85, 95, and 105 days. The accumulated endosteal growth during age 60 to 75 days in the anterolateral segment was found to increase uniformly in the distal direction along the femur diaphysis. A method is described where this accumulated endosteal growth is used. This method eliminates the use of calipers to determine the section level and makes it possible to study comparable sections even after varying periods of time. The proximal part of the diaphysis showed periosteal apposition in all three segments. The periosteal apposition turned into resorption in the distal part of the diaphysis in the anterolateral and medial segments, whereas the periosteal appsition increased in the posterior segment. The endosteal growth increased in the distal direction in the anterolateral and medial segments. Irregular OTC bands made measurements of endosteal remodeling in the posterior segment impossible. The cortical width decreased in the distal direction along the femoral shaft. Comparison between the different age groups is described and also the relation between the accumulated endosteal growth and the diameter of the medullary cavity.

Notes: Summary A short review is given of the water in bone. Various analyses of bone water content are discussed, and its possible location is considered in relation to the behaviour of water in isolated components of bone. Some of the difficulties encountered in examining such microscopic phenomena as water structure in a heterogeneous system such as bone are also discussed.

Notes: Summary The present study is an investigation of the mechanism of hypercalcemia and hyperphosphatemia induced by the intravenous injection of lead acetate (Pb-Ac). A total of 118 male rats were injected with 30 mg/kg of Pb-Ac, or with 16.5 mg/kg of sodium acetate as the control. The levels of serum calcium, phosphorus and lead were then determined at various time periods after the injections. Serum calcium and phosphorus levels increased with time after Pb-Ac injection and the maximum values of calcium (17 mg%) were found after 1 h and of phosphorus (13.5 mg%) after 30 min. Both calcium and phosphorus levels reverted to the normal range after 12 h. The maximum net rates of increase of calcium and phosphorus were found immediately after Pb-Ac injection. At that time, deposition of lead at the calcifying sites of bone and incisor dentin was demonstrated by a histochemical examination. In other experiments the changes in the calcium and phosphorus contents in the medium after shaking bone powder in serum with Pb-Ac in an in vitro system were studied. It was confirmed that the calcium and phosphorus were displaced from the bone mineral, the extent of the displacement being correlated with the concentration of the Pb-Ac added to the medium, and that these displacements were very rapid reactions. These results suggest that hypercalcemia and hyperphosphatemia following Pb-Ac injection results from a direct action of lead on the bone mineral.

Notes: Summary The net rate of loss of45Ca from plasma was monitored in normal, thyroparathyroidectomized (TPTX), and nephrectomized rats. Also studied was the effect of a 24 h intravenous infusion of PTH in TPTX rats. The45Ca was injected after an overnight fast and 1 to 4 h prior to the time the first blood sample was obtained. In normal rats, the net rate of loss of45Ca from plasma was slower than in TPTX rats for at least 12 h after radionuclide injection. Nephrectomy did not affect this difference between control and TPTX rats until 8 h after45Ca injection or about 18 h after nephrectomy. At this time a reversal occurred and the net rate of plasma45Ca loss in nephrectomized rats with parathyroids became faster than in TPTX nephrectomized rats. When PTH was infused at a constant rate (3 mU/g body weight/h) plasma calcium concentrations rose gradually for approximately 12 h after which a constant plasma calcium level was maintained with continued PTH infusion. During the period when plasma calcium concentrations were rising, the net rate of loss of45Ca from plasma was slower than in TPTX controls infused only with saline. However, when plasma levels stabilized, plasma45Ca disappearance rates changed such that the rate loss of45Ca from plasma became faster in PTH-infused than in TPTX controls. It is concluded that PTH increases calcium efflux from the same compartment in bone which receives calcium from plasma, thereby returning some recently deposited45Ca to plasma. In the non-equilibrated state, this returns additional45Ca to plasma causing anet decrease in the rate of loss of45Ca from plasma. However, in the equilibrated condition, when both calcium efflux from bone and influx into bone are in balance but at higher flux rates, PTH stimulates the diffusion of45Ca into other bone compartments.

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: 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 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 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.