ALBERT

Notes: Four glands of the house sparrow, chicken and turkey were examined histologically and for their content of amylase. These were the external and intermediate mandibular glands, the maxillary gland and glandula anguli oris of the sparrow and the anterior and posterior mandibular, maxillary and anguli oris glands of the chicken and turkey. Amylase was determined by a starch substrate slide method and by biochemical assay. General morphology and mucopolysaccharide staining are described.All four glands of the sparrow demonstrated significant amylolytic activity by the assay. In the external mandibular and anguli oris glands this activity could be traced to mucous and seromucous cells of origin by means of the starch substrate slide procedure. None of the glands of the chicken or turkey displayed significant amylolytic activity.

Notes: A cinematographic analysis of the unrestrained walking, trotting, galloping, jumping and landing movements of 11 adult cats was undertaken to provide previously unavailable information concerning the demands imposed on the nervous system for the control of low and high speed movements and the demands imposed by such natural movements on muscle performance and proprioceptive response.With due regard for the swing (F and E1) and stance (E2 and E3) phases of the step cycle of an individual limb, single frame analysis of the film permitted measurement of instantaneous angles of the lower spine, hip, knee, ankle and metatarsophalangeal joints. Appropriate lever arm measurements were also made on 50 freshly dispatched cats and 25 cadavers such that the Law of Cosines could be used to calculate instantaneous lengths of select hind limb muscles that would apply to the natural movements of adult cats of small (1.5-2.5 Kg), intermediate (2.6-3.5 Kg) and large (3.6-4.5 Kg) size. Muscle displacements were analyzed relative to maximum and minimus in situ lengths and the lengths associated with quiet standing. Use was also made of a previous electromyographic analysis of hind limb muscles during unrestrained locomotion (Engberg and Lundberg, '69).The sequential relations between the four phases of the step cycle are maintained as forward speed increases from walking ( 〈 2 mph) to high speed galloping ( 〉 16 mph). There are significant differences in the time consumed by each phase, however, with a greater reduction in the E3 phase, little reduction in the E2 and E1 phases and virtually no reduction in the F phase. When each phase is expressed as a relative percentage of the duration of the total step cycle, the greatest reduction is again in E3 with little change in the E2 phase. In contrast F and E1 phases increase in the percent of time they occur in each cycle, with the greatest increase in the F phase. For all speeds, analysis of the phase relations between movements of various sections of the hind limb revealed a remarkable unity of knee and ankle joint movement. The hip joint is largely out of phase with the knee and ankle during E1 and E2, all three joints being in phase in F and E3. The digits are essentially out of phase with the other joints except in the stance phase of the gallop.Rates and extents of muscle displacement during natural movements are greater than might be anticipated when expressed in absolute mm's and mm/sec but not when considered in relation to maximum and minimum in situ length and the length associated with quiet standing (Ls). During stepping a progressive increase in forward speed results in: (a) a greater usage of muscles at lengths between Ls and maximum in situ length; (b) for knee and ankle extensors, pronounced increase in the lengthening contraction associated with the E2 (yield) phase of step; and, (c) for both flexor and extensor muscles, an increased active phase of lengthening or near isometric contraction immediately prior to periods of active shortening. In contrast to these changes in active muscle status, the change from walking to galloping has little effect on the extent and rate of passive muscle displacements, particularly the F phase stretch of extensors.For the soleus muscle, calculations were made of the relation between changes in overall muscle length during natural movements and the length of the average muscle fiber and the tendon of insertion. These measurements revealed that the increases in fiber length when passive and decreases in length during active shortening are less than would be anticipated from the extensive liteature on extirpated fibers. In contrast, the increase in fiber length when active is greater than would be expected from the admittedly sparse literature on this subject.The results of this study are discussed largely in relation to two points of neurophysiological interest: the physiological range of muscle stretch as it pertains to the responsiveness of muscle spindles and tendon organs; and those mechanical aspects of lengthening contractions that give insight into the neural control of stepping. For exciting both spindles and tendon organs passive muscle stretch and shortening contractions are shown to be relatively ineffective and lengthening and isometric contractions particularly effective movements. It is suggested that, just as recent literature has emphasized the co-activation of efferent alpha and gamma motoneurons as a muscle becomes active, so too is there a synchronous activation of afferents, particularly the Ia and group II endings of muscle spindles and Ib endings of tendon organs. Finally the thesis is advanced that, while it has been convenient to separate E2 from E3 in the description of the stance phase of the step cycle, extensor muscles are actually undergoing a single mechanical event: an active stretch-shorten cycle for knee and ankle extensors and an active isometric-shorten cycle for hip extensors. This hypothesis has significant implications for the neural control program that regulates the stepping sequence in that it emphasizes the extent to which appropriate changes must be preprogrammed in the mechanical properties of muscles for the smooth execution of stepping.

Notes: Cytological and cytochemical methods were used to identify and characterzie six distinct regions of the crayfish kidney: coelomosac, labyrinth I and II, and nephridial canal I, II, and III. Cells of the coelomosac possess cytoplasm which is strongly PAS-positive, but poor in RNA and protein. Their nuclei possess unusual projections which extend to the basal plasmalemma. Labyrinth I contains columnar cells rich in glycogen. Labyrinth II is characterized by a distended lumen and by shorter cells with high cytoplasmic RNA, many possessing a large intracellular vacuole. A PAS-positive brush border is unique to the two portions of the labyrinth. Cells in the nephridial canal show strong reactions for RNA and Mg++-dependent ATPase. In nephridial canal I and II, cells are flattened to cuboidal with the lumen being more distended in nephridial canal I than anywhere else in the tubule. In nephridial canal III, the cells are large and columnar, and the cytoplasmic RNA concentration is greatest apically. Nuclei in all regions of the tubule epithelium, except coelomosac, are large and react strongly for protein. Coelomosac nuclei and those in blood cells are condensed and contain little protein. The cytoplasm of blood cells displays a significant amount of RNA, and traces of polysaccharide material.These observations demonstrate the presence of highly specialized morphological and histochemical alterations along the length of the kidney tubule and indicate sequential modification of urine in the lumen. Evident morphological and cytochemical likenesses between analogous regions of the mammlian nephron and the crayfish kidney tubule suggest that basic physiological similarities may also exist.

Notes: In the tiger salamnder the distribution of optic fibers, as revealed by the Fink-Heimer method, is compared with the localization of acetylcholinesterase, as revealed by histochemical methods. AChE activity coincides with optic nerve axons in the optic fiber layer of the retina, in the optic nerve, in the optic tracts and in every optic projection. Reginons of optic fiber terminals show heavier activity than optic fibers of passage. Comparison with other vertebrates is also made.

Notes: Light and electron microscope studies indicate that the old ependyma just proximal to the plane of amputation in early lizard tail regenerates shows a sequence of morphological changes which suggests that it as well as the new ependyma growing into the regenerate may play an active role in the initiation and maintenance of early tail regeneration.The old ependyma close to the plane of amputation undergoes hypertrophy and/or hyperplasia causing a partial closure of the central canal and pseudostratification. Its nuclei shift from an original apical position to a basal one. The ependymal processes become more prominent and extend to the pia, a condition not found more rostrally. There is also a significant increase in the amount of Golgi substance and a moderate increase in the rough endoplasmic reticulum. These observations lead to the thought that these cellular changes may be an expression of enhanced secretion and other activities in the old and new ependyma just proximal or distal to the plane of amputation.

Notes: The dorsal proctodeal wall of the Coturnix contains a gland which is especially well-developed in the sexually active male and which secretes a foamy exudate. This gland has not been recorded in any other genus of birds and appears to be unique to members of the genus Coturnix. On the basis of its location in the adult we have named this gland the proctodeal gland (glandula proctodealis). Structure of the gland does not conform to any curently defined category of glands because it is an aggregate of individual glandular units each of which opens independently into the proctodeal cavity but with the entire structure enclosed by a connective tissue capsule. We have thus characterized it as an aggregate gland. Observations on the arrangement and composition of the fibrous capsule and its intimate relationship to the perimysium of M. sphincter cloacae indicate that release of the secretion probably results from contraction of the cloacal musculature. Preliminary data suggest that development of glandular tissue in the female may be a function of age rather than of androgen sensitivity which characterizes the male gland. It is concluded that the chemical nature of the glandular secretion and its function in the sexually active bird, as based on presently available information, remains unknown.

Notes: Wound healing in the integument of the sea cucumber, Thyone briareus, was studied for up to 50 days after inflicting wide excisional wounds and for 14 days after producing incisional wounds. Rapid re-epithelialization of the wound was effected by the migration of epidermal cells and pigment cells from the periphery of the wound margin. This occurred without apparent evidence of concomitant mitotic activity. Dermal wound healing was completed by the fourteenth day in the incision wounds but occurred very slowly in the broad excision wounds. Morula cells seem to be involved in both epidermal and dermal wound healing, although their precise role is unknown. In excisional wounds the integument was never completely restored to its normal appearance during 50 days of observation.

Notes: Some of the cytological and histochemical characteristics of hemocytes of Thyropygus poseidon were studied. Jone's system of classification of hemocytes was extended to millipede hemocytes. Seven classes were identified as prohemocytes, plasmatocytes, granular hemocytes, cytocytes, oenocytoids, spherule cells and adipohemocytes. Oenocytoids were rarely found. All cells except prohemocytes and oenocytoids sequestered oxidized products of catechol. Results of the histochemical tests for proteins also suggest that these cells sequester plasma proteins. Cytocytes do not bring about any visible coagulation similar to the hemocytes of some hemipteran insects. Granular hemocytes contain phenol-oxidase capable of oxidising tyrosine as well as catechol, similar to the hemocytes of crustaceans. In this respect millipede hemocytes differ from insect hemocytes. It is suggested that these cell types may represent distinct cell lines and may not represent transformation of one cell type to another.

Notes: Twenty-three stages in the embryonic development of the western painted turtle, Chrysemys picta bellii are described. The staging is based primarily on morphological changes, as well as age and size of the embryo.

Notes: Scanning and transmission electron microscopy were used to investigate the surface area and number per unit area of microvilli from jejunal villus epithelial cells in the rat, hamster and dog.The calculated mean microvillus surface area was 0.419 μ2, 0.573 μ2, 0.751 μ2 for the rat, hamster and dog respectively.The largest number of microvilli per square micron freeze dried villus surface was measured in the rat with a mean value of 65. Hamster and dog freeze dried specimens had lower mean values of 54 and 34 microvilli respectively.The total microvilli surface area in square micron per square micron villus surface was more closely related for the three species with values of 27.23 for the rat, 30.94 for the hamster and 25.53 for the dog.These data indicate an inverse relationship between the mean microvillus surface area and population density in the species studied. However, the total microvilli surface area per unit villus surface is relatively similar for the three species.The observed number of microvilli per unit villus surface was shown to vary depending upon the dehydration technique employed for preparation of scanning electron microscopic specimens. This variation probably reflects shrinkage artifact and should be considered in soft tissue studies involving the scanning electron microscope.

Notes: In Esox americanus and Esox niger (Esocidae) there are two types of surface pits, sensory and nonsensory. Both types of pits are morphologically very similar and are associated with modified (cardioid) scales. The pits are distinguished by the presence and absence respectively of neuromasts. Ultrastructure of the neuromasts indicates that they are basically similar to previously described neuromasts. However, the hair cells have crystalline-like deposits in the nuclear region. The nonsensory pit contains a small group of distinct epidermal cells, called central cells. These cells occupy a central position in the pit, similar to that of the neuromasts in the sensory pit. The central cells are unlike any cell types described for fishes, and on an ultrastructural basis no function can be ascribed to them and the significance of the nonsensory pit is unknown.

Notes: Esox americanus and Esox niger (esocidae) possess two types of surface pits, sensory and nonsensory. The pits are morphologically very similar and both are associated with modified (cardioid) scales. The pits are distinguished by neuromasts in the sensory and central cells in the nonsensory pit.Neuromasts and central cells are responsible for the formation and maintenance of respectively the pit and cardioid scale. This supports an extension of the epidermal co-participation hypothesis that epidermal cells control the form of dermal structures in addition to contributing to composition.The central cells are similar in structure to induced primordial cells of regenerating neuromasts.The development of primordial cells may be arrested and the cells maintained in a static condition during which time they still influence the dermal tissues and maintain a pit and cardioid scale. Essentially this represents the experimental conversion of a sensory pit into a nonsensory pit. This evidence is taken in support of the hypothesis that the nonsensory pit and central cells represent an aplasic condition of the lateral line system.

Notes: The gular gland of the bat Tadarida brasiliensis is a specialized sebaceous gland located in the skin of the suprasternal region of adult males. It consists of an aggregation of simple branched tubulo-acinar gland units, the number of which varies seasonally. Each acinus is composed of densely packed sebaceous cells at various stages of differentiation. Acinar basal cells and cells of the epithelium of the ducts can differentiate into sebaceous cells. Two main changes appear in the cytoplasm concurrent with the sebaceous transformation: the differentiation of cytoplasmic organelles and the deposition of lipid material. The appearance of a different type of mitochondrion and the development of large numbers of ribosomes and polyribosomes can be recognized in the cytoplasm at an early stage of differentiation. Concomitant with the deposition of significant numbers of lipid droplets, the cells develop abundant agranular endoplasmic reticulum occurring mainly as scattered tubular cisternae. These at times form whorls surrounding lipid droplets. At later stages, the cisternae of the agranular endoplasmic reticulum often occur in crystalline arrays between secretory oil droplets. The roles of the different cytoplasmic organelles, especially in relation to the production of sebum, are discussed.

Notes: The mutant gene srn produces sireniform abnormalities in the mouse. The effects of this gene on normal prenatal development were examined histologically by retrograde analysis from day 10 through day 18 of gestation.The sireniformphenotype is characterized by the fusion of the hindlimbs and accompanying defects of the posterior appendicular skeleton. Visceral defects involve the urogenital system and the lower intestinal tract. A single umbilical artery arises from the dorsal aorta and controls the fetal circulatory pathway normally mediated by the lateral umbilical arteries. This artery is believed to have resulted from the fusion of the paired primitive umbilical arteries.It is suggested that the srn mutation causes a narrowing of the posterior amnion early in development. The resulting abnormal pressure causes a dorsal displacement of the hindgut, allowing fusion of the hindlimb buds. Abnormal structural relationships and pressure in the narrowed posteriior region disrupt normal urogenital and intestinal development and increase the proximity of the primitive umbilical arteries.

Notes: The retinofugal projections of the snake Thamnophis sirtalis were studied by the method of experimentally induced Wallerian degeneration stained by the Fink-Heimer method. The retinal ganglion cells project to all parts of the contralateral lateral geniculate complex, nucleus lentiformis mesencephali, nucleus geniculatus pretectalis, nucleus posterodorsalis, basal optic nucleus and superficial layers of the optic tectum. In addition, the retinofugal projections were observed terminating in portions of the ipsilateral lateral geniculate complex and nucleus posterodorsalis.Examination of the morphology of the retinal terminal areas stained for Nissl substance with cresyl violet led to the conclusion that these regions are well differentiated and should not be considered poorly developed when compared with other reptilian forms such as turtles.

Notes: The course of the statoblast formation in Pectinatella gelatinosa was divided into four stages and studied histologically and histochemically. The bottom of the cystigenous cup is a center of cystigenous cell differentiation and the peripheral zone of the inner cystigenous layer turns to the outer cystigenous layer as the cystigenous cup grows. The annulus is formed by migration and transformation of the outer cystigenous cells.During early stages, the yolk cells have an intensely pyroninophilic or RNA-rich cytoplasm. The cytoplasmic pyroninophilia then diminishes as the amount of yolk granules increases.Several kinds of yolk substances occur in the mature statoblast. During statoblast formation glycogen appears first, then glycoprotein and finally neutral unsaturated lipid.Acid phosphatase activity is associated with granular structures in the cytoplasm. In the cystigenous vesicle, acid phosphatase activity is low and confined to the apical extremity of the cell. Histochemically detectable alkaline phosphatase activity is not involved in the formation of the statoblast.

Notes: The anatomy and physiology of feeding in miniature swine were studied, using gross dissection, electromyography, cinematography, and cineradiography.Small particles are preferred for ingestion, and large items are usually broken down outside of the oral cavity. The particles are initially picked up with the lower lip and then retrieved by the tongue; the tongue very rarely leaves the oral cavity. Geniohyoid, mylohyoid and digastric are the most active muscles during food collection.Mastication is fairly rapid (3 cycles/sec) and involves a transverse component which may be either medially or laterally directed. The direction of motion is generally reversed with every chew. Electromyography indicates that the transverse rotation is caused by a force couple consisting of protrusors (masseter, medial and lateral pterygoids) on one side and retrusors (zygomaticomandibularis and temporalis) on the other. The direction of the rotation is not necessarily related to the side containing the bolus. Mandibular depression is electromyographically biphasic. Concurrent tongue and hyoid movements complicate the interpretation of activity patterns in the oral floor.Both masticated food and liquid are stored between the tongue and the soft palate and epiglottis before being swallowed. Swallowing involves very strong activity in all of the hyoid and tongue muscles and very low activity in the adductors. Liquid is taken in by suction rather than lapping.Comparisons with other mammals are presented.

Notes: The wall of the gametolytic gland in Aplysia and Phyllaplysia consists of a muscular layer lined with an epithelium incorporating four distinct cell types, some of which show intense secretory activity. The cytoplasm of these cells, rich in RNA, has a peripheral layer of densely-packed mitochondria in association with a smooth endoplasmic reticulum. Lipid globules, perhaps resulting from absorption of lumen contents by microvilli, appear in the cytoplasm in the course of growth and reproductive activity. A basal band of fibrillar material appears to serve as a cytoplasmic skeleton. The elaboration of cell types, extensive areas of cytoplasmic RNA, and vigorous secretory activity suggest important roles for this organ such as development of food reserves, recycling of sexual materials of high physiological value, and interaction with the bag cells of the nervous system in coordinating ovulation with the availability of capacitated, exogenous sperm.

Notes: The predaceous neotropical characoid fish Ctenolucius has an essentially homodont dentition, the number of teeth increasing linearly with age. The basic manner of tooth replacement suggests that Ctenolucius is a primitive characoid. Tooth replacement continues throughout life and is similar to that of tetrapods, involving replacement waves which pass from the back to the front of the jaws. The waves containing the greatest number of teeth are found just anterior to the middle of the jaws. In the upper jaw the increase in the number of teeth is restricted to the anterior portion (premaxillary) whereas the number on the posterior part (maxillary) remains constant. In specimens measuring from 68-230 mm in standard length the posterior portion of the upper jaw doubles in length whereas the anterior portion triples. It is suggested that the area immediately anterior to the middle of the jaw, where replacement waves are longest, is where most of the increase in tooth numbers occurs.During growth of the teeth the absolute height is always greater than the absolute width as the shape changes. The final shape of the recurved conical teeth is determined only in the last stages of tooth formation when the main axis of growth abruptly changes.

Notes: Improved histological procedures have revealed in some detail the form and structure of auditory receptor organs in the frog. These organs, the papilla amphibiorum and papilla basilaris, are located in extensions of the saccular cavity, and lie in a path of vibratory fluid motion beginning at the columellar footplate and extending in a continuous circuit through the perilymph spaces and the round window back to the middle ear region.These organs exhibit great uniformity, and for the amphibian papilla there is a high degree of complexity in the tectorial structures that overlay the ciliated ends of the hair cells. In both papillae a “sensing membrane,” which is a derivative of the tectorial body, lies in the path of fluid flow and aids in the stimulation process.The mode of stimulation of the hair cells in the amphibian ear is peculiar, and in principle is the reverse of the familiar one of reptiles, birds, and mammals.

Notes: To determine the prism sheath configurations in human cuspal enamel 80 teeth were initially ground to produce flat surfaces through the following planes: a horizontal series at successively greater distances from the dentinoenamel junction and longitudinally through the center of the cusps. Individual teeth were suspended in an acid-alcohol solution (1 cm3 conc. HCl in 100 cm3 95% ethanol) at 37°C for seven to ten days. The treatment “softened” the enamel to a depth of approximately 1 mm. The teeth were embedded in Epon and sectioned at 0.5 to 10 μm with a diamond knife. Thick and thin ground sections for phase contrast microscopy and acid-etched ground sections for Nomarski differential interference microscopy were prepared through the same regions. In thicker longitudinal sections, the prisms in gnarled enamel formed a zig-zag pattern which was unlike the twisting pattern generally observed in ground sections. The thinnest transverse sections showed the sheath outlines to be dramatically different from those seen elsewhere in the enamel. Some prism sheaths were circular, others were in the form of spirals. What could be described as sheaths within sheaths were also seen. In the thinnest longitudinal sections the prisms were seen to be elongated and discontinuous. Sheath outlines in enamel adjacent to the central core of gnarled enamel were similar to those described elsewhere in the body of the enamel. Keyhole, modified keyhole patterns and arcade forms were the dominant sheath patterns. Other atypical sheath configurations were seen scattered throughout this region.

Notes: The differentiation and degeneration of the cement gland in Xenopus laevis is described. The gland is first observed histologically at stage 19 (neural tube stage) as a packed group of apical ectoderm cells heavily laden with oocyte pigment granules, lying ventral to the cranial neural fold. By tailbud stage 35/36, the gland cells have increased in height and are approximately ten times taller than nonglandular apical ectoderm cells. The nuclei divide the gland cells into an apical region that is eosinophilic and contains oocyte pigment granules, and a basal region that contains clear droplets. The cells are decreasing in height by stage 40 (early tadpole) and begin to lose their pigment granules. Between stages 45 and 48, the pigment is extruded and the clear basal droplets diminish in number. From stage 48 to 49 the cells become vacuolated and the histotypic characteristics of the functional gland are lost. The gland is not vascularized, nor do phagocytic cells appear in its vicinity during any stage of its development. It remains bordered at its base by subjacent basal ectoderm during its entire life cycle of 10 to 12 days at room temperature.

Notes: The morphology of the rectal pads of two cockroaches, Blattella germanica and Blaberus giganteus is described and contrasted with that of Periplaneta americana, studied previously. The columnar absorptive cells of the three species are similar in structure. However, Blattella and Blaberus rectal pads have a second type of cell, termed the secondary cell, that is not present in Periplaneta. The secondary cells are embedded in the pad epithelium and have crypts of cavities opening into the subepithelial sinus. In addition, a multilayered sheath is present between the pad cells and the sinus. The sheath acts as a barrier, perhaps of low permeability, between the subepithelial sinus and the pads. It is interrupted only at the borders of the secondary cells. A mechanism for fluid absorption is presented and the possible role of secondary cells is discussed. It is proposed that the secondary cells reabsorb solute from the sinus and that this solute can then be recycled through the pad cells. Structures resembling neurosecretory terminals within the cavities of the secondary cells may be involved in regulation of recycling. The arrangement of the rectal pads in these insects has structural and perhaps functional similarities with other transporting systems, particularly the cryptonephric system.

Notes: Pyroantimonate precipitate indicates that the epithelium of the proximal tubule is the only segment of the tubular nephron of the fresh water lamprey where large accumlations of cations are distributed. Unusually large amounts of reaction product are located within the lateral intercellular spaces and within vesicles closely associated with the plasma membrane at the lateral and basal surfaces. This technique suggests the continuity of these vesicles with the plasma membrane and alludes to the possibility of an endomembranous system of vesicles and the intercellular spaces as vehicles for ion transport. Lateral intercellular spaces of proximal tubules of lower vertebrates may play a different role in kidney function that their counterparts in higher vertebrates.Osmium-zinc iodide has a specificity for certain cells within the proximal, intermediate, and distal segments, but no structural differences are noted when these cells are compared to unstained cells. Smooth endoplasmic reticulum remains unstained in the distal segment but the stain has a strong affinity for elements of the Golgi apparatus, lysosomes, and the nuclear envelope of all cell types. This technique does not suggest a structural or functional similarity between cells of the distal segment and the chloride cells of the gills of teleosts.

Notes: Anatomical analysis of the forebrain and midbrain of Anelytropsis, Dibamus and feyliniids reveals structural similarities with those of skinks and snakes. Skinks and feyliniids are probably derived from a common ancestral stock. This is suggested by mutual reduction of several telencephalic nuclei, by similar trends in the development of the dorsal thalamus, and by similarities in the lamination of their optic tecta. Anelytropsis, Dibamus, feyliniids and snakes show interdigitation of the periventricular gray zones of the optic tectum and enlargement of lamina 7 of the posterior colliculus. Of these three taxa, Dibamus is most similar to burrowing snakes and many of its brain characters are intermediate between skinks and burrowing snakes. These similarities may suggest common ancestry between Dibamus and snakes rather than parallelism.

Notes: It is possible to monitor the electrical activity of the motor neurons of Drosophila by recording the electrical activity of the muscle fibers. We have found that it is possible to specify the location of the subcuticular terminations and to describe the orientation within the thorax for the individual muscle fibers, because of the large size of the fibers and because the surface anatomy of Drosophila is known in detail. A map has been made to indicate the location of the muscle fibers with respect to superficial landmarks. The importance of the stereotaxic map for physiological studies is discussed.

Notes: The functional anatomy of pulmonary ventilation in Thamnophis elegans was studied by electromyography. Flow of air into and out of the lungs follows a triphasic pattern and occurs while the glottis is held open by a dilator muscle. Non-ventilatory rest periods characterized by a closed glottis and lack of ventilatory movements occur between breaths. Exhalation is caused by contraction of a pair of dorsolateral sheets of muscle that extends from the ventral surface of the vertebral column to the medial surfaces of the ribs and a pair of ventro-lateral sheets that extends from the medial surfaces of the ribs to the mid-ventral skin. Inspiration is powered by contraction of a series of rib levators and retractors. The last phase of the ventilatory cycle is a passive expiration that may be caused by elastic recoil of the walls of the filled lungs and relaxation of muscles used during inspiration. The site of respiratory pumping is shifted craniad after large prey items have been completely ingested. The significance of this shift and its anatomical basis are considered.

Notes: Histological studies of the neurosecretory system during the postembryonic development of a spider, Argiope aurantia, were made at the light-microscopic level.Neurosecretory cells which are found in all stages are classified into type I and type II cells. The type I cells are present in the aboral region of the brain and in pedipalpal, ambulatory and abdominal ganglia of the subesophageal mass. The type II cells which appear from the seventh stage are confined to the cheliceral ganglia. Three stages of secretory activity (poor, medium and full) based on stainability are described in these cells.In both types clear axonal transportation of neurosecretory material is observed. The discrete tracts and commissures formed by these neurosecretory axons are described in the brain and subesophageal ganglion. The complexity of some of these pathways is comparable to that of the ordinary neurons.One pair of nerves from the brain and four pairs of nerves from the subesophageal mass enter a neurohaemal organ, the Tropfenkomplex. This is a paired structure, situated dorsally, on either side of the subesophageal mass. The neurosecretory axons branch extensively within the organ and on their course they from sacs or pools filled with secretory material.The Tropfenkomplex is enveloped by a thin neural sheath which runs deep into the organ dividing it into a series of lobes. Glial cells are distributed within the organ. As in the neurosecretory cells, changes in stainability of secretory material were also observed in the Tropfenkomplex.During intermolt periods two peaks of stainability have been noticed. The first peak lasts for 24 hours after the molt, and this is followed by a low activity period between second and fifth day. From the sixth to the tenth day after the molt the second peak commences. It is suggested that the second peak may be responsible for bringing about molting.The cheliceral group appears (seventh stage) at a time when external indication of reproductive characters are visible. In the ninth stage, by the tenth day after the last molt, several of the type I and type II cells contain much secretion. This is followed by maturation of gonads and oviposition. Thus both type I and type II cells are believed to be involved in the reproduction of the animal.

Notes: Ovaries of the giant silkmoth Samia cynthia Drury have been studied histologically and histochemically during diapause, adult development, and after injury to the diapause animal. In addition to cellular changes involved in follicular growth and vitellogenesis, changes in the intermediate layer cells are very striking, showing a distinct pattern during ovarian development and after injury. Two types of granules, one periodic acid-Schiff (PAS) positive, the other paraldehyde-fuchsin (PF) positive, change in their number and distribution in the intermediate cells during development and after injury. The PAS-positive granules appear during the first one-third of development, increase in number and size until the tenth day, then gradually dwindle in number and size, and disappear in the adult cells. The PF-positive granules, present in moderate numbers in the diapause animal, decrease in number until the tenth day of adult development, then reappear, and are present again in adult intermediate cells. After injury to a diapause pupa, intermediate cells show a greatly increased number of PF-positive granules, but none that are PAS-positive.The PAS-positive granules are a neutral or mildly acidic mucosubstance, and may correspond to lysosomal activity, while the PF-positive granules may be a neurosecretory-like substance or the product of hormone-dependent protein synthesis, since the patterns of granule distribution in the intermediate cells seem to reflect both general metabolic and hormone-related events.

Notes: The distribution of monoamine (catecholamine and 5-hydroxytryptamine)-containing nerve cell bodies in the brain stem and hypothalmus of the frog (Rana temporaria) was investigated with the help of the histofluorescence technique of Falck and Hillarp ('62). At the level of the hypothalmus of this amphibian brain, catecholamine-containing nerve cell bodies are found mainly within three areas of the periventricular gray substance, namely the peroptic recess organ, the paraventricular organ and the lateral recess region. On the other hand, most of the 5-hydroxytryptamine (serotonin)-containing nerve cell bodies of the brain stem of Rana temporaria appear to be concentrated within the midbrain tegmentum. This huge mesencephalic nerve cell collection can be subdivided into medial and lateral groups. More caudally, at the level of the isthmic tegmentum, another group of 5-hydroxytryptamine-containing perikarya located close to the midline, within the so-called raphae region, is clearly outlined. The latter group of neurons extends caudally as far as the level of the medulla oblongata. In addition, a small group of catecholamine-containing nerve cell bodies is also found in the ventromedial portion of the rostral midbrain tegmentum, whereas a few other catecholamine type neurons are scattered throughout the lower brain stem of the frog and more especially near the ependymal wall of the fourth ventricle. As a whole, the 5-hydroxytryptamine-containing neuronal systems of the brain stem of Rana temporaria are much more elaborated than the catecholamine neuronal systems of the same structure.

Notes: The guinea pig soleus muscle is a convenient model for the study of slow-twitch intermediate (STI) fiber ultrastructure because it is composed entirely of fibers of this class. Such fibers were compared with fast-twitch red (FTR) and fast-twitch white (FTW) fibers from the vastus lateralis muscle.FTW fibers are characterized by small, sparse mitochondria, a narrow Z line and, an extensive sarcoplasmic reticulum arranged primarily in longitudinal profiles at the A band and with numerous expansions at the I band. Abundant mitochondria with a dense matrix and subsarcolemmal and perinuclear aggregations are typical of FTR fibers. These fibers contain a plexus of sarcoplasmic reticulum at the A band and a less extensive network at the I band. The Z lines are wider (890 ± 74 Å) than those of FTW fibers (582 ± 62 Å). STI intermediate fibers are distinguished from other types by wide Z lines (1205 ± 58 Å), a faint M band, and a less extensive sarcoplasmic reticulum. Compared to FTR fibers, STI fiber mitochondria are usually smaller with less notable subsarcolemmal accumulations.FTW fibers have a more limited capillary supply, rarely contain lipid inclusions, and thus may be restricted to phasic activity. Extensive capillarity, mitochondrial and lipid context, and fast contraction times indicate possible phasic and tonic roles for FTR fibers. STI fibers, characterized by numerous lipid inclusions, extensive capillarity, relatively numerous mitochondria, but slow contraction-relaxation cycles, are morphologically suited for tonic muscle activity.

Notes: The haemocytes of Calpodes ethlius are described with the light and electron microscopes. Five fine structurally distinct types are distinguishable. However only three of these, the granular haemocytes, sphaerule cells and oenocytoids can be positively identified using a series of histochemical stains on smears and on thick sections of araldite-embedded material. The classification is based entirely on the structural features even though several suggestions concerning their function can be made from their fine structure. Intermediates having features of more than one cell type suggest developmental relationships.

Notes: Four hemocyte types have been identified in the late last larval instar of Galleria mellonella. Plasmatocytoids are round to spindle shaped cells, 10-20 μ long and 5-10 μ wide. The cytoplasm contains no distinguishing inclusions. Golgi complexes, rough endoplasmic reticulum and free ribosomes are abundant. Granular hemocytes are oval shaped cells, 10-20 μ long and 5-10 μ wide. The granules, their most characteristic feature, have a diameter of 0.2 μ, a microtubular sub-structure, and are made up of acidic mucosubstances. Lipid droplets may be present in these cells at some stage of development. These cells appear to be phagocytic. Spherule cells are oval shaped, 15-20 μ long and 5-10 μ wide. The spherules, approximately 2 μ in diameter, have a highly ordered substructure and are made up of acidic mucosubstances. Oenocytoids are the largest cells, 20 by 40 μ. The cytoplasm contains mostly free ribosomes and microtubules.

Notes: Formation of secondary palate in hamster was studied with electron microscopy. Prior to assuming horizontal position, the palatal shelves were covered by a two to three cell layer thick epithelium which was separated from the underlying mesenchyme by an intact basal lamina. Epithelial cells were attached to each other by desmosomes. Early hemidesmosomes could be identified as thickenings of the cytoplasmic membrane opposing the basal lamina. Epithelial cells, like other embryonic cells, contained only few organelles but were rich in polyribosomes. As the horizontal shelves approached each other towards the midline, lysosomes and tonofilaments appeared in the superficial and basal cells of the epithelia. Superficial cells showed degeneration and eventual lysis. Fusion of the opposing epithelia occurred between the deeper cells by means of newly formed desmosomes. The epithelial seam resulting from fusion of the epithelia was limited on each side by a continuous basal lamina. Its subsequent thining and eventual fragmentation resulted from the loss of cells by autophagy. There was no evidence of mesenchymal invasion of the epithelial seam. Mesenchymal macrophages appeared in the later stage of palatogenesis and were responsible for phagocytosis of cellular debris.Formation of the soft palate was basically similar to that of the secondary hard palate and occurred by fusion of the opposing shelves. Similarly, anterior closure of the palate occurred by fusion of the lower end of the nasal septum to the primary and secondary palates. Hyperplasia of the opposing epithelia, prior to their fusion, was often seen.It is suggested that formation of the palate occurs in predictable and coordinated fashion and that timely appearance of lysosomes causing lysis of intervening epithelia is of great significance in normal palatogenesis.

Notes: Gross anatomical, histological and histochemical studies of heart development in Mexican salamanders, Ambystoma mexicanum, are reported. Gross observation suggests that heart development in this urodele species is similar to other amphibians. Histological studies in early embryos show the ventromedially migrating sheets of precardiac mesoderm to be composed of two layers of cells. The right and left dorsal layers fuse and give rise to the myocardium, while the ventral layers form the pericardium. The endocardium arises from cells released by the leading edges of the migrating mesoderm mantles. In early myocardial cells, most of the proteins and carbohydrates are contained in yolk platelets; subsequently, these substances become distributed throughout the cytoplasmic matrixes. In early heart cells free lipid droplets are abundant but decline in size and number as development progresses. Concomitantly, there is an increase in bound lipids. Reticular fibers are detected in the endocardial-myocardial spaces simultaneously with trabeculae formation. Collagen appears somewhat later in development.

Notes: The description of the superficial veins of the face and neck was based upon the dissection of 28 male and female adult tufted or brown capuchin monkeys (Cebus apella), captured in southern Goiás, Brazil.Although the venous arrangement of these areas is basically the same in all primates, some characteristic differences were noted: 1. the anterior jugular and common facial veins were absent; 2. most specimens had a venous hyoid arch, and a connecting vessel between the external jugular and subclavian veins; 3. the inferior petrosal sinus drains into the external jugular v. through the postglenoid foramen; 4. the facial, lingual and cephalic veins open into the external jugular vein; 5. both the internal and external jugular veins have about the same diameter; 6. the vascular pattern appears to be quite stable; anatomical variations appear to be few and unimportant.

Notes: The photoreceptors of the adult Anableps anableps have been investigated by light microscopy, scanning and transmission electron microscopy. The fish is a surface swimmer and the eye is divided by the water meniscus. In general, the photoreceptors (rods, single cones, and double equal cones) resemble those of other vertebrates, but there are several unusual features: (1) The outer segment discs of the double cones differ in the two members. (2) All cones have a prominent accessory outer segment derived from the single connecting cilium, and there is no second centriole. (3) The exterior of the inner segments is ridged and grooved longitudinally, most markedly so in the cones. (4) A membranebounded oil droplet is present in the distal cone inner segment, formed from mitochondria which enlarge, fuse and transform in a vitreal-scleral gradient. (5) There are knob-like invaginations of rod cytoplasm into the cones immediately scleral to the external limiting membrane. (6) Subsurface cisterns underlie apposed plasma membranes of double cone inner segments and direct rod-cone inner segment contacts. (7) Fine “fins” on the cones interdigitate, with Müller cell cytoplasm between, just scleral to the external limiting membrane. (8) In the rod spherule there is a greater density of vesicles and the cytoplasm is darker than in the cone pedicle.The well-defined cone mosaic has a linear pattern peripherally and a square pattern centrally. The photoreceptors undergo photomechanical movements. Photoreceptor ultrastructure is alike in both dorsal and ventral parts of the retina, but the ventral retina contains more cells and is thicker than the dorsal retina.The adjustments necessary for simultaneous air and water vision are found mainly in lens shape, corneal thickness and curvature, and the greater number of cells in the ventral retina.

Notes: The ventral pelvic integument of anurans in hypervascular as compared to other skin regions. Recent studies indicate that this area is primarily responsible for water uptake and rehydration. Nine species representing four genera of terrestrial and aquatic Anura were dissected for comparison of vascularity in the pelvic area. Major differences in the ventral pelvic integumental vessels were found between aquatic and terrestrial genera. These variations are described, and names are suggested for previously unnamed vessels. Changes in vascularity are discussed and related to the literature on Anura pertinent to rehydration rates, tolerance of desiccation, resistance to desiccation, and habitat.Data from this study and previous studies suggest a correlation among the parameters discussed and habitat. The suggestion is made that when species are classified as xeric, mesic, or aquatic on the basis of the habitat in which they live, their responses to rehydration, tolerance to desiccation, and vasculatity should be considered, as these characteristics more accurately represent the micro-habitat.

Notes: The development of electric organ spindles of Gymnarchus niloticus has been investigated with respect to the exact time and place of origin and the process of formation of the adult plan. The results are compared with those of Dahlgren ('14). A common primordium for all the electroplates of of a spindle as held by Dahlgren ('14) is not supported by the present work.

Notes: Smooth muscle in the ovaries of estrus and anestrus cats was studied with the TP-Levanol Fast Cyanine 5 RN technique and adrenergic nerves were visualized with the Falck-Hillarp fluorescence procedure. Numerous bundles of smooth muscle fibers were observed in the mesovarium, and the hilar, medullary, and cortical regions of the estrus ovary. Also, theca externa of large vesicular follicles contained many smooth muscle fibers. Adrenergic nerves with varicosities were present in ovarian perifollicular tissue. Anestrus animals had a reduced number of cortical and perifollicular muscle fibers and the intensity and density of fluorescent nerves was reduced. It is suggested that contraction of ovarian smooth muscle facilitates ovulation. These contractions may be, at least partially, under local neural control.

Notes: The effect of hypophysectomy, growth hormone (GH) and an amino acid-glucose mixture on the regenerative ability of the hypophysectomized Triturus pyrrhogaster yielded the following results: 1The survival time of hypophysectomized newts can be prolonged substantially by the sulfamide application.2Although the limb regeneration in the hypophysectomized newt is retarded as compared with that of the pituitary intact control, it finally completes morphogenetic process under such conditions of prolonged survival.3The injection of 100 μg of GH restored the speed of regeneration of pituitary-deprived limbs to almost a normal level.4Injections of the amino acid-glucose mixture also promoted the limb regeneration in hypophysectomized newts. However, initial delay in regeneration to the time of bud appearance was not restored by the nutrients.

Notes: An anatomical analysis of the chicken trigeminal ganglion was made using light microscopy on specimens prepared by usual chemical fixation or freeze-drying methods and by electron microscopy. Two types of neurons were consistently seen, dark and light cells. Dark cells contained a dense cytoplasm with Nissl substance distributed evenly throughout, whereas light cells had a less dense cytoplasm containing clumps of Nissl substance. The Nissl bodies in light cells contained only a few small cisternae of granular endoplasmic reticulum as compared with many stacked cisternae in Nissl bodies of dark cells. The ratio of dark to light cells was approximately 62:38 in all regions of the ganglion. Dark cells were consistently smaller than light cells. In the seven-day old chick, the mean diameters of the dark and light neurons were 21.4 μ and 29.5 μ respectively; in the adult the values were 29.9 μ and 39.7 μ respectively. It is concluded that the dark and light cells belong to two distinct neuronal cell populations.

Notes: The ultrastructural features of cardiac muscle cells and their innervation were examined in the tarantula spider Eurypelma marxi Simon. The cells are transversely striated and have an A band length of about three microns. H zones are indistinct and M lines are absent. Thick and thin myofilament diameters are approximately 200 and 70 Å respectively. Eight to 12 thin filaments usually surround each thick one. Accumulations of thick and thin myofilaments occur perpendicular to the bulk of the myofilaments in some cells. The Z line is discontinuous and thick filaments may pass through the spaces in the Z line. Extensive systems of sarcoplasmic reticulum and transverse tubules are present; these form numerous dyadic junctions in both A and I band regions. Sarcolemmal invaginations form Z line tubules; lateral extensions of the plasma membrane portion of these invaginations form dyads. Nerve branches of the cardiac ganglion make multiple neuromuscular synapses with at least some of the cardiac muscle cells. Both large granular and small agranular vesicles are present in the presynaptic terminals. Intercalated discs similar to those present in other arthropod hearts occur between the ends of adjacent cardiac muscle cells.

Notes: The paddlefish (Polyodon spathula) is one of the most primitive and unique freshwater fishes of North America. It is adapted as a plankton filter-feeder. The wide mouth and greatly expansible pharyngeal cavity allows it to process a large quantity of water. Numerous setiform gillrakers, which operate against the flattened surfaces of the gill arches and opercula, are capable of filtering out small food particles. The abundance of taste buds and mucus secreting cells in the buccal cavity and pharynx probably aids in selecting and entangling the food. The first segment of the stomach is provided with peculiar, large longitudinal rods of fat which may help in selecting food from water by causing occlusion of the small lumen when the striated fibers of the muscularis contract. Teeth, unnecessary for microphagous fishes, are absent in adults.The digestive tube is one of the most complex present in fishes. It has two distinct divisions of the stomach and four distinct divisions of the intestine besides possessing a large caecum. Ciliated epithelial cells occur in many parts of the gut and may be an aid in moving or processing the small items of food.Besides the presence of cilia, other primitive features of the digestive tract include the lack of distinction between the esophagus and stomach, the presence of a spiral valve, and the presence of some of the diffuse pancreatic tissue within the wall of the gut.The numerous Peyer's patches in the wall of much of the intestine may be an immunological response to the heavy parasitic infestation.

Notes: The cranial glands of ten species of turtles were studied by the use of histochemistry applied to serial sections of whole heads. The majority were stenohaline species, but one brackish water form, Malaclemys, was included. The results show that all species have two major orbital glands, an anterior Harderian gland, and a posterior lachrymal gland. The latter is seromucous in all species except Malaclemys terrapin in which the gland shows little evidence or organic secretion. External and medial nasal glands are found in all species studied, and also are seromucous glands. With these reslts, combined with a review of the literature the following conclusions are made. The Harderian gland is by definition the orbital gland opening through the medial surface of the nictitating membrane at or near the anterior canthus. It is of constant occurrence, and histological appearance, probably serving the same function. However, despite much recent study this function remains unknown. The lachrymal gland is defined as the orbital gland which opens through the lateral surface of the nictitating membrane, or medial surface of the lower eyelid, at or near the posterior canthus. It is of variable occurrence, absent in many reptiles, and has a histological structure which is also variable. In the stenohaline species it is apparently involved in organic secretion, while in the brackish water Malaclemys it may be involved in salt secretion, as it is in Cheloniidae. The nasal glands in turtles are probably homologous with the nasal salt glands of lizards and birds, but they do not appear to subserve the same function. In all species of turtles studied the nasal glands are seromucous. They are perhaps involved in the maintenance of the epithelium of the olfactory cavity.

Notes: The duodenal glands of four marsupial species, the kangaroo, native cat, marsupial mouse, and bandicoot, form a narrow lobular collar immediately distal to the gastrointestinal junction. In two species, the koala and wombat, the duodenal glands are diffuse and scattered along the intestinal tract distal to the gastrointestinal junction for considerable distances. The glands of all species examined empty independently into the intestinal lumen. Histochemical studies indicate that the latter two species, the koala and wombat, elaborate an acid mucin whereas the former species produce a neutral mucosubstance. Ultrastructural examination shows that generally the duodenal glands are comprised of large pyramidal cells that show a definite polarity, with basally-placed nuclei and apically-situated secretory granules. Species differences, with regard both to the morphology and nature of the secretory granules and to the proportions of cell organelles, exist. The diversity of diet between species is discussed.

Notes: Cytodifferentiation of the myoepithelial cells (MEC) of the rat submandibular gland (SMG) was observed by studying the prenatal and postnatal development of the gland in vivo and in vitro by light and electron microscopy. The anlage of the SMG first appeared on the fourteenth day of gestation and, from its earliest inception, was surrounded by an intact basal lamina. Presumptive myoepithelial cells were first seen at 18 days of gestation coinciding with the onset of secretion in the rudiment. These cells were flattened, peripherally located and subjacent to the epithelial basal lamina. Initial deposition of cytofilaments in the MEC's was observed during the first three days following birth and fully matured cells were seen as early as one week after birth. Presumptive and immature MEC's were observed undergoing mitosis, but once cytofilament deposition had begun in the cells they did not divide. Myoepithelium developed in relation to embryonic secretory structures and were only observed surounding acini and intercalated ducts in the adult gland. New myoepithelial cells were formed as long as new acinar-intercalated duct units were formed. Myoepithelial cells did not produce secretory type granules at any time during their development or in their mature state.Development of the MEC's in vitro paralleled that in vivo and supported the above observations.

Notes: Larval compound (jointed) setae of the polychaete Nereis vexillosa Grube were examined by scanning and transmission electron microscopy and by polarization microscopy. Long-bladed spinigers and short-bladed falcigers are described. The proximal shaft of each of these types of setae flares distally into a serrated collar and encloses the proximal end of a toothed blade. The collar projects on one side as a boss. The blade and the cortex of the shaft have longitudinal channels. A large excentric cavity in the shaft (the medullary channel) contains a loose meshwork of trabeculae. In the distal part of the shaft these trabeculae are aggregated into diaphragms. The seta is invested with an electron dense layer of enamel. Juvenile setae contain both chitin and protein. With respect to the long axis of the seta, the blade and the cortex of the shaft are positively birefringent and the medullary diaphragms are negatively birefringent. KOH extraction renders the setae negative to a test for protein and reverses the sign of birefringence of the cortical material of the shaft.

Notes: The marine priapulid Priapulus caudatus has a voluminous body cavity filled with a blood-like fluid containing erythrocytes and leucocytes (amoebocytes). The hematocrit of animals weighing 0.5-14 gm was 2-10%. The erythrocytes contain a hemerythrin blood pigment. The structure of the coelomocytes was studied by light and electron microscopy. The erythrocytes are nucleated and contain marginal bands, vacuoles and occasionally crystals. The cytoplasm has few organelles. The leucocytes are amoeboid motile cells, the cytoplasm of which contains numerous organelles. The most conspicuous of these are oval particles, probably representing developmental stages of lysosomes. Most of these organelles contain tubules stretching from one pole to another. In the hind part of the animal, certain tissues, primarily the posterior warts contain large numbers of coelomocytes. The histological picture is complicated, showing some resemblance to the lymphoepithelial tissues of vertebrates.

Notes: The dissected tooth bearing bones of 20 specimens of Amia calva (Pisces:Holostei) ranging from the third to tenth season have been examined radiographically and in alizarin red S stained and cleared specimens. Although forms of alternate (1:1) tooth series replacement (sensu Edmund, '60) were frequently observed, even in the youngest, immature specimens, many examples of irregular replacement were recorded. In several bones, the maxillae in particular, series with every third (2:1) or fourth (3:1) tooth replacing were seen and possible patterns of 2:2, were recorded. It is concluded that these data are not consistent with the Zahnreihen concept but support a morphogenic field concept of tooth development.

Notes: The first description of vertebral development in a plethodontid salamander is presented. Eurycea bislineata has larvae that hatch at a rather early stage of development. Somites and the notochord appear early. Somitic differentiation is slight, and no distinct sclerotome can be found. As a result, there is no clear primary segmentation of the skeletogenous tissue. No evidence of a sclerocoele can be found. The amount of sclerotomal cells surrounding the notochord is very low, relative to other tetrapods. Yet discrete perichordal rings of cells do form, in nearly midsegmental positions, and these give rise to the intervertebral cartilages. Osteogenesis of the centra is initiated prior to hatching and is coincidental with ossification of the neural arch. There is no sign of a neurocentral suture. The centrum forms as a thin shell of bone directly from sclerotomal cells. The notochord is a prominent feature of the vertebral column throughout life, retaining its integrity until late in life when some disintegration occurs locally. The notochord is filled with cartilage midvertebrally in late larval stages, and some additional cartilage forms later in life. The intervertebral cartilage enlarges greatly in late larval life. An opisthocoelous joint forms in this cartilage, apparently as a result of differential changes in the cells of the perichordal ring rather than by an invasion of cells from an external source. The intervertebral cartilage is a dominant structural and highly important functional feature of the adult vertebra. In metamorphosed individuals it may become extensively mineralized, and it consists of many different structural kinds of cartilage.The cranio-vertebral joint seems to form in a single segment, contrary to the condition reported by some early investigators. It is complex, and consists of articulations between the odontoid process of the atlas and the occipital arch, as well as between the occipital condyles and atlantal cotyles. The notochord plays a dominant role in the early development of the odontoid, but then changes radically and is absent in the adult process.The anterior trunk region seems to be much more conservative than posterior parts of the column. The patterns of nerve routes and nature of development of the ribs and rib bearers differs greatly from conditions elsewhere in the column. The rib patterns are similar to presumed ancestral conditions. Rib development on the sacral and caudosacral vertebrae is in some ways more similar to that of the anterior vertebrae than of the central trunk vertebrae.Quantitative aspects of variation in the vertebrae of adult salamanders are presented. There is more regional variation and less site variation than would be expected from literature reports.Evolutionary aspects of the origin of the cranio-vertebral joint, transverse process and ribs, patterns of segmentation, and centrum development are considered in the light of the new information on Eurycea. There is no evidence that more than one vertebra is involved developmentally or evolutionarily in the cranio-vertebral joint. The most generalized condition of rib bearers in living salamanders is one in which the dorsal and ventral bearers are in cartilaginous continuity during development. There are many variations on this theme in living species. It is inappropriate to speak of a resegmentation of the sclerotome in Eurycca, even though the adult vertebra is a transsegmental structure, because there is no primary segmentation of the scanty sclerotome. The important feature found in vertebral development in all tetrapods is the perichordal tube and its subsequent differentiation. Questinos concerning precise homologies of the salamander vertebral centrum with those of other vertebrates cannot be answered by data from development sequences with currently used criteria of homology. On the other hand, it appears that all centra, regardless of subdivision, are homologous in all tetrapods.

Notes: The Oberhautchen of scales from the dorsal, parietal, and ventral regions of Sceloporus occidentalis (Iguanidae), Gerrhonotus multicarinatus (Anguinidae), and Anniella pulchra (Anniellidae) were examined with a scanning electron microscope. At low magnification, all scales of S. occidentalis exhibit well-defined outlines of cells belonging to the Oberhautchen layer and the previously overlying clear layer. The dorsal and parietal cells of this species exhibit a minutely dentate Oberhautchen that forms tooth-like spinules 0.2 to 0.5 μ long and arranged in irregular rows. Minute pits 0.1 to 0.3 μ in diameter characterize the Oberhautchen of a ventral scale.Cell outlines are not evident on the scales of G. multicarinatus. The Oberhautchen of dorsal and parietal scales of this species is prominently laminated. Laminae are less prominent on scales of the lateral fold, and no intrinsic surface structure is evident on a ventral scale. In contrast, the fossorial anguinomorph Anniella pulchra exhibits Oberhautchen surfaces with practically no intrinsic microornamentation. However, what appear to be outlines of Oberhautchen cells are visible on the dorsal and ventral scales.These observations suggest that modifications of Oberhautchen microornamentation may have evolved to reduce friction with the substrate or other scales. The lack of pronounced microornamentation of the Oberhautchen on some body scales may indicate that a complex interdigitation between clear layer and Oberhautchen cells is not essential to the sloughing process.

Notes: Stolonic growth has been compared among several genera of calyptoblastic (Campanularia flexuosa, C. calceolifera, Gonothyraea and Sertularia) and gymnoblastic (Pennaria, Bougainvillia, Eudendrium and Cordylophora) hydroids with respect to such features as the (1) complexity and uniformity of the tip movements, (2) constancy of growth cycle duration, (3) variability in growth among cycles, (4) growth rate, and (5) variability of retractions among cycles.The “growth cycle,” previously described in C. flexuosa, is the basis for elongation in all species observed. Its pattern is indistinguishable between the Campanularia species; however, at the generic level the tip movements show peculiarities which delineate each genus from all others. In addition, the movements in all calyptoblasts are uniform from cycle to cycle and comparatively simple, whereas those of the gymnoblasts are complicated by the variable appearance of one or more secondary forward thrusts between crests.A dichotomy is seen between the calyptoblasts and most gymnoblasts in other respects as well. Cycle time is very predictable and cycle-to-cycle variability in growth is relatively narrow in all calyptoblasts. However, in most gymnoblasts both features are much more loosely regulated: only Pennaria shows some degree of control to its timing mechanism, and only in Bougainvillia and Eudendrium does the uniformity of growth per cycle reach the calyptoblast level.On the premise that calyptoblasts are evolutionarily the more advanced group, simplicity and regularity of growth movements are concluded to be the evolved, and complexity and variability the primitive, conditions. On this basis the evolutionary relations among the four gymnoblasts, as deduced from their stolonic growth behavior alone, are consistent in many but not all respects with those interpreted from morphological considerations.

Notes: In order to clarify the morphology of the circulatory system of amphioxus the blood vessels were investigated using modern techniques of light and electron microscopy.The pattern of circulation in amphioxus is forward ventrally and backwards dorsally. In addition, circulating corpuscles, usually associated with the blood of higher chordates, are absent.The circulatory system of amphioxus consists of well defined contractile vessels and vascular spaces or sinuses within a connective tissue matrix. The contractile vessels have a discontinuous endothelial lining resting on a basal lamina and are enclosed by a simple layer of contractile myoepithelial cells. Discontinuous endothelial linings occur throughout the vascular tree, including major and minor afferent and efferent vessels and blood sinuses. This is in contrast to higher animals where the endothelium forms a more or less continuous lining along the inner surface of the boundary layer.It is suggested that the endothelial cells of amphioxus, like the endothelial cells in capillaries of higher chordates, most likely play a role in the physiology of the circulatory system by removing residues of filtration from the basal lamina, thereby facilitating an exchange of materials to and from the surrounding tissues.

Notes: The axillary sinus of G. variegata is formed from a perivascular lymphatic which locally invests the lateral vein. Within the sinus the wall of the vein is distended by lymphoid tissue which is itself supported by reticular fibres. Lymphocytes, reticular cells, macrophages and mast cells occur in the tissue. The overall appearance of the structure is lymph node-like. Although Cardianema sp. (Nematoda:Filarioidea) parasitised the lymphatic system of some geckos examined, the non-pathologic origin of the lymphoid tissue is indicated by its presence in both axillae of infected and uninfected geckos alike. Comparison is made with lymph nodes and node-like structures in other vertebrates.

Notes: The innervation of each of the muscles involved in mediating head movement in the desert locust Schistocerca gregaria is described in detail. The number of motor neurones to each muscle and the neutral pathway and ganglion of origin of each are deduced from both histological and electrophysiological evidence. Only two of the muscles are, on histological evidence, innervated by as few as four different neurones, while several receive more than ten, and one at least 13. Individual muscles are shown physiologically to receive, in a few cases, as many as six different motor neurones. At least six muscles are innervated by motor neurones originating in more than one ganglion. One group of four muscles consisting in total of less than 100 muscle fibres receives more than 20 different motor neurones from three different ganglia through three or four different nerve roots. In these muscles, many single muscle fibres receive innervation from at least two different ganglia. It is concluded that the segmental nature of an insect muscle can not be deduced solely from a knowledge of the ganglion of origin of the motor innervation to that muscle. The innervation patterns that exist today must reflect past evolutionary development, but changes in the peripheral distribution of motor neurones, or migration of motor neurone cell bodies from one ganglion to another, or the development of additional motor neurones, or several of these factors together, must have formed a part of that development.

Notes: Quite a number of phenomena having to do with cells' influences upon one another's movements have come to be regarded as expressions of “contact inhibition.” However, no single, central mechanism has been shown to underlie them all. Consequently, the term “contact inhibition” should not be used without operational modifiers. Inhibitions of individual cell movements imputed to be mediated by cell-cell contacts include inhibition of overlapping (which results in monolayering), of colony expansion, of cell speed (nuclear translocation), of ruffling, of orthogonal movement (proposed to explain spontaneous parallel alignment of cells), and of neighbor exchanges.The six inhibitions listed above are operationally distinct, and only two (overlapping and colony expansion) are known to result from a common mechanism. A seventh phenomenon, so-called “contact inhibition of cell division” (more operationally termed postconfluence inhibition of cell division) is in a separate category and is not considered here.Evidence eliminating action-at-a-distance is available only for the first three, and hence only these should at present be termed contact inhibitions. Inhibition of neighbor exchanges is yet hypothetical; at its extreme, it would immobilize cells in a confluent monolayer, but such immobilization has been found not to occur.Contact inhibition of overlapping, the most studied of the six, is not displayed by invasive cells with respect to normal cells; invasive tumor cells overlap freely upon normal cells, although not necessarily upon one another. Contact inhibition of overlapping, and its loss by invasive cells, can readily be interpreted, by means of the differential adhesion hypothesis, as consequences of cell-type-specific differences in cell-cell and cell-substratum “strengths of adhesion.” These strengths of adhesion are formulated as specific interfacial free energies, which are the only parameters of cellular adhesiveness that have been rigorously shown to determine equilibrium configurations of cell populations.

Notes: The induction, isolation and some of the properties of serine-requiring mutants of Chinese hamster ovary cells have been described. These cells promise to be useful in biochemical genetic studies and cancer research.

Notes: An adenosine kinaseless (AK-) mutant of the mouse fibroblast line 3T6 has been obtained in cell culture by evolution of resistance to 6-thio-methylpurine ribonucleoside and tubercidin. The mutant excretes purines (xanthine and hypoxanthine) into the culture medium. Human or mouse cells lacking hypoxanthine-guanine phosphoribosyl transferase (HPT-) excrete increased amounts of purines, but a human cell mutant lacking both HPT and AK excretes considerably more hypoxanthine. The difference in hypoxanthine excretion between the HPT- mutant and the HPT- AK- mutant originates from the adenosine normally reutilized through the activity of adenosine kinase. The activity of adenosine kinase is essential to retard the adenosine cycle and to prevent cellular loss of purines.

Notes: Leukemic myeloblasts and cells derived from normal chick hematopoietic tissue produced colonies in soft agar. Colonies produced by leukemic myeloblasts differed from normal chick tissue in their morphological characteristics, in the greater initial number of cells required for colony formation and in their decreased dependence on conditioned medium for development. The colony forming cells for both types were enriched when allowed to grow for several days in liquid growth medium.In soft agar, myeloblasts differentiated into more mature granulocytic cells and macrophages. These differentiated cells accumulated between one and two weeks after seeding. When tested for release of avian myeloblastosis virus (AMV), 6 out of 18 colonies were releasing AMV at one week whereas 3 out of 39 were releasing AMV at two weeks. Five two week old colonies which were negative for AMV were producing myeloblastosis associated viruses (MAVs). Normal colony forming cells were present in leukemic buffy coat and although colonies made by these cells contained MAVs, no AMV could be detected.The data obtained with normal avian tissues were similar to those obtained by others with mammalian hematopoietic tissue. Colony formation by normal hematopoietic tissues was strictly dependent on factors present in conditioned medium. Tissues producing colonies included bone marrow, yolk sac, spleen and peripheral leukocytes. Colonies were not obtained from thymus and bursa. Furthermore, the colony origin did not appear to be erythroid in nature.

Notes: Data have been presented with respect to the widely varying pH optima for the growth of a number of normal and transformed human, mouse, monkey, hamster, rabbit and rat cell lines. At that optimum, differences between normal and transformed cells with respect to maximum population densities tended to become less prominent. Other differences (serum requirement, long-term viability of cultures) were not demonstrably pH-dependent.

Notes: We present evidence that the chemical requirements among all the bioluminescent coelenterates that have been examined are very similar or identical to those already described for Renilla by Cormier and associates. Components required for luminescence in Renilla were also found in a number of bioluminescent coelenterates examined such as Aequorea, Obelia, Cavernularia, Ptilosarcus, Stylatula, Acanthoptilum, Parazoanthus and Mnemiopsis. Depending on the organism these include one or more of the following: luciferyl sulfate, luciferase, and luciferin sulfokinase. These isolated components were found to be indistinguishable from those found in Renilla as evidenced by their reactivity in the Renilla bioluminescent system, by the spectral characteristics of the isolated luciferyl sulfates, by the molecular weights of the luciferases, and by the colors of the bioluminescence produced in vitro.

Notes: In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg 2 until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.

Notes: The effects of lanthanum ions upon amoeboid movement and upon the redistribution of dense cytoplasmic inclusions following centrifugation were investigated in Amoeba discoides. Treatment with low lanthanum concentrations produced a characteristic inhibited condition in which locomotion ceased while saltatory movements continued. The resorting of stratified cytoplasm by local internal cytoplasmic flow was unaffected. This contrasted with p-hydroxymercuribenzoate treatment which inhibited both locomotion and saltatory movements. The inhibitory effects of lanthanum ions were antagonised by calcium ions but not by magnesium ions. It is reasoned that lanthanum competes for a peripheral binding site thereby disrupting a calcium-mediated regulatory mechanism of amoeboid movement.

Notes: It is possible, by direct measurements, to demonstrate the presence of cyclic AMP in Tetrahymena. Its presence in a unicellular eukaryote suggests a fundamental role in cellular metabolism independent of its function as a “second messenger.” Caffeine, an inhibitor of phosphodiesterase, raises the intracellular concentration of cyclic AMP and causes an inhibition of cellular division. Dibutyryl cyclic AMP also causes a partial inhibition of proliferation and is potentiated by caffeine. Inhibited cells do not recover spontaneously during continued exposure to caffeine but the inhibition is reversible resulting in a synchronous wave of division peaking one generation time after return to fresh medium.Synchronous cells subjected to pulses of caffeine at various intervals of the cell cycle are set back in time as measured by the excess division delay. There is a linear relationship between the age of the cells at the pulse and the duration of the excess division delay such that the older the cells are the greater is the setback.Caffeine and dibutyryl cyclic AMP inhibit ciliary regeneration as does also deuterium oxide.

Notes: Calcium uptake by normal human lymphocytes was found to be a saturable process which was competitively inhibited by manganese indicating the existence of a carrier-mediated mechanism for calcium uptake. Exchange diffusion was not observed, Phytohemagglutinin (PHA) significantly stimulated calcium uptake within minutes after treatment. The increased uptake was attributed to a decreased Km for the proposed membrane carrier rather than to an increased Vmax. Also PHA did not stimulate a normally unused exchange diffusion process, nor did it affect calcium efflux. Uptake by both unstimulated and PHA-treated lymphocytes was not influenced by magnesium or by cycloheximide or actinomycin D.

Notes: Purified subcellular fractions containing melanosomes from B-16 mouse melanoma were treated with 2% sodium dodecyl sulfate or 0.5 M sodium hydroxide to dissolve protein. Quantitative measurements indicate that each melanosome contains 0.065×10-10 of protein or about 19% by weight. SDS acrylamide gel electrophoresis of proteins from purified melanosomes resolved six polypeptide bands of major density and about 15 minor bands. These results indicate that the melanosome may be more complex than previous genetic, biochemical or morphological evidence had suggested.

Notes: The temperature dependence of the uptake of glucose by exchange transport is investigated at two pH values (7.5 and 5). The Arrhenius's energy of activation, the heat of activation, the free energy of activation and the entropy of activation are calculated. The parameters are different at pH 7.5 and 5. For both pH levels the heat of activation and the entropy of activation change abruptly at 20°C. This finding leads us to assume that at this temperature a change in the structure of the membrane takes place.

Notes: Previous studies showed that after 5 μg of Salmonella typhosa endotoxin there was an increase in colony stimulating factor temporally related to a fall in murine marrow in vitro colony forming cells (CFC). This was followed by differentiation along the marrow granulocytic pathway. The present studies showed that after 5 μg of endotoxin the peripheral blood CFC fell by approximately 50% at one hour, rose to a level ten fold that of control at six hours and then returned to control values by 48 hours. There was a progressive increase in the number of splenic CFC to ten fold that of control from 24 to 72 hours after endotoxin. These data imply a migration of CFC from the marrow to the spleen along with an in-situ increase in splenic CFC. Thus, either migration or differentiation may explain the fall in marrow CFC after endotoxin.Spleen colony forming units (CFU) in the marrow were measured by a transplantation technique and the transplantation fraction (f Fx) determined. A decrease in marrow CFU at 24 hours after endotoxin was secondary to a change in the f Fx. from 11.1% to 7.6%. There was however, an increased percentage of CFU in DNA synthesis in the interval of 6-48 hours after endotoxin, as judged by the hydroxyurea technique. As the marrow CFC fell within 20 minutes of endotoxin administration, the data suggest the CFC may be affected initially and that changes in the generative cycle of the CFU may be of a secondary nature.

Notes: Previous studies with mammalian cultured cells have shown that volume regulation in hypotonic medium requires active Na transport. In the present study, determinations of intracellular Na and K content were made in cultured mouse lymphoblasts during the process of swelling and subsequent shrinking (volume regulation) in hypotonic medium. Na and K content were measured in cells in which the shrinking phase was inhibited by the cardiac glycoside, ouabain. In osmotically-shocked cells, an initial permeability increase to K, and not Na, was observed, which allowed K to diffuse out rapidly, down its gradient. Na, meanwhile, rapidly flowed inward with water entry during the swelling process, and was later lost with the same kinetics as the cell shrinkage. This loss of Na was prevented in the presence of ouabain. The results imply that volume regulation is achieved by pumping Na gained during swelling out of the cells, while any K taken up by the pump is rapidly lost through a more permeable membrane. The loss of osmotically active Na, presumably with accompanying anions, allows water to passively diffuse down its osmotic gradient, reducing cell volume subsequent to the initial passive swelling, during which K was rapidly lost.

Notes: Myoglobin synthesis was compared in cell cultures of leg (red) and breast (white) muscle of chick embryos. In leg muscle cultures a rapidly increasing amino acid incorporation into myoglobin begins two days after muscle cell fusion; in breast muscle cultures no comparable increase was observed. This qualitative difference in cultures of the two muscle cell types provides possibilities for the further study of the mechanism of myoglobin synthesis.

Notes: Temperature sensitive cells have been isclated from Syrian and Chinese hamster cells using a method based on selective detachment from a glass substrate. The Syrian hamster isolates occurred at a high frequency (about 1 in 103) and reverted rapidly; polyoma virus transformation conferred on cells the ability to grow, perhaps abnormally, in agar suspension. A slightly modified isolation technique was applied to Chinese hamster cultures and resulted in the isolation of at least one mutant (from a starting population of 5 × 108 cells) with a spontaneous reversion rate of less than one in 6 × 107. Treatment of the mutant with ethyl methane sulphonate induced reversion. It was concluded that selective detachment provided a useful method for the isolation of conditional lethal mutants of mammalian cells.

Notes: Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate.These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G0 phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G1 phase.

Notes: When Ehrlich ascites cells were cultivated in serum-free media their cellular protein synthetic rate declined to a new steady-state level and the cells stopped multiplying. On addition of serum the cellular protein synthetic rate increased to the level before serum starvation and cells resumed multiplication. The activity in serum stimulating protein synthesis was inactivated on incubation with cells. At cell concentrations of the usual culture conditions this inactivation took several hours; at very high cell concentrations it was complete in ten minutes. Serum-starved cells inactivated low serum (2%-6%) media in the same length of time. Studies of inactivation of high serum media demonstrated that cells had a limted capacity to inactivate. Cells grown in 10% serum were unable to inactivate. Inactivation was not due to accumulation in the medium of either low molecular or macromolecular cell products. Inactivation was strongly inhibited at 4° or by treatment of cells with fluoride or cycloheximide (long exposure): less inhibited by treatment with 2-deoxyglucose or glutaraldehyde; and slightly inhibited by treatment with cyanide or cycloheximide (short exposure). Inactivating ability was unaffected by trypsinization. These findings are best explained by the hypothesis that cells take up the serum activity by endocytosis.

Notes: Medium conditioned by excised whole lungs from endotoxin-injected C57BL mice was highly active in stimulating hemopoietic colony formation, particularly of granulocytic type, in agar cultures of mouse bone marrow cells. The colony stimulating factor (CSF) in this material had an α1-α2 electrophoretic mobility, was eluted from calcium phosphate gel by 0.04 M phosphate buffer and had an unusually low apparent S20W of 1.9. Sequestered polymor-phonuclear neutrophils were excluded as a major source of this CSF. The high specific activity and ease of preparation of lung conditioned medium make it valuable both for the large scale production of CSF and as a source of an unusual type of CSF.

Notes: Subpopulations of human lymphoid cells are capable of spontaneously fusing with fibroblasts of human or murine origin to form human-human or human-mouse hybrid cells. These cells were present in thymus, spleen, and bone marrow. After fractionation on discontinuous bovine serum albumin gradients, the cells were found in the less dense layers of the gradient. Cells of fetal origin, except for bone marrow, fused spontaneously at a higher rate than those of adult origin. The highest rate of fusion was found with adult bone marrow cells. These lymphoid cells appear to be “thymus-derived” cells.

Notes: Fetal rat hepatocytes in primary monolayer cultures multiply in arginine-deficient medium. Both the “recovery efficiency” and the final cell density of the cultured cells are proportional to the concentration (0-15%, v/v) of dialyzed fetal bovine serum in the medium. Stationary-phase cells divide again following addition of fresh serum to the culture. After two to three generations of growth, the chromosome number of these cells remains diploid [2N = 42].Cross-feeding (of a subpopulation of arginine-requiring liver-derived cells by parenchymal arginine-synthesizing cells) and cellular degradation of various serum proteins do not account for sources of arginine required for cell multiplication in this culture system. Because these cultured hepatocytes utilize ornithine for arginine biosynthesis, and because ornithine enhances the rate and the amount of cell multiplication, it is more likely that the multiplying cells are parenchymal arginine-synthesizing hepatocytes.At least two classes of serum factors are required for the growth of cultured fetal rat hepatocytes: one stimulates cell multiplication; the other is required for cellular survival and/or attachment to the culture dish. These factors have been partially separated by fractionation with ammonium sulfate; they are non-dialyzable, heat labile, and sensitive to changes in pH.

Notes: Trypsinized cells of newborn mouse cerebellum have been separated by velocity sedimentation at unit gravity in shallow gradients of Ficoll. The two main technical difficulties were formation of gels around the dissociated cells and clumping of cells before and during the sedimentation procedure. These were solved by adding DNase to the dissociation medium and with holding serum, respectively. Proliferating cells of the external granular layer separated according to size differences in the cell generation cycle. Identification of Purkinje or other early-forming neurons was made by labeling them with 3H-thymidine on their birthdays. Many of the fractions contain viable cells capable of aggregating in culture.

Notes: A substance promoting the growth of mammalian cells in vitro has been isolated from Bacto-peptone (Difco Labs., Detroit) and identified as 6,8-dihydroxypurine. This compound, which is isomeric with xanthine(2,6-dihydroxypurine), enhances the growth of one Chinese hamster and some other cell lines; xanthine itself is inactive. The biological action of the compound is discussed.

Notes: An apparatus is described for the continuous monitoring of the growth of suspension cultures of mammalian cells in normal conditions and following the administration of a drug.

Notes: Flow microfluorometry has been used to quantitate cell-surface binding of fluorescein-conjugated lectins. Frequency distributions of total surface binding of Concanavalin A per cell were prepared for a variety of cultured cell populations, including established cell lines, virus-transformed lines and non-transformed parental lines. In the case of growing Chinese hamster cells (line CHO), much of the variability of Con A binding per cell could be related to variability of cell size. Experiments with cells synchronized by mitotic selection indicated that the modal surface density of binding sites was almost constant throughout the cell cycle. However, as indicated by inhibition of binding with α-methyl mannopyranoside and by the effect of trypsin, the sites on each cell were heterogeneous in chemical structure and/or exposure. Agglutinability of virus-transformed cell lines or trypsin-treated parental lines was demonstrated but could not be correlated closely with binding.

Notes: The average speed of nuclear translocation of 3T3 cells, recorded in a time-lapse film of a perfused culture, was negatively correlated with the number of contacting cells, and, to a lesser degree, with the amount of a cell's perimeter in contact with other cells. When a cell was in contact with five or more other cells, its speed was reduced by 50%, on the average, although the variation in individual cell speed was considerable at each level of contact. A partial correlation analysis showed that any extracellular soluble factors governed by the local cell density had little or no effect on speed, relative to the prominent effect of the number of cell-cell contacts, and hence that 3T3 cells display true contact inhibition of speed. This confirms the original demonstration by Abercrombie and Heaysman (1952), who studied chick embryo heart fibrpolasts. In our study, the relationships between average speed and age of the culture was such that a possible independent contribution of a time-associated factor other than contact to the diminution in average speed, although not necessary to account for the data, could not be excluded.The same intercellular contacts found to inhibit speed in this study were previously reported to cause no immediate prolongation of individual cell generation times, despite the fact that the filmed culture was undergoing so-called “contact” inhibition of cell division. In the present study, moreover, no correlation was observed between the average speeds of individual cells and their generation times. Hence, postconfluence inhibition of cell division and contact inhibition of speed of cell movement seem to be independent phenomena.

Notes: Erythroid differentiation of Friend leukemia cells is enhanced when the cells are grown for four days in the presence of dimethylsulfoxide (DMSO). Dimethylformamide (DMF) has a similar though less marked effect. 5-Bromo-2′-deoxyuridine (BUdR) (10-5M) inhibits both DMF- and DMSO-stimulated differentiation. For maximum inhibition, BUdR must be present during the first two days of growth, during which time DNA synthesis is maximal. The addition of BUdR after the third day has no effect.Since BUdR is incorporated into DNA and thymidine prevents BUdR inhibition of DMSO-stimulated differentiation, it is likely that BUdR acts by virtue of its incorporation into DNA. Although BUdR alone had little effect upon cell multiplication, in combination with DMSO, cell growth was inhibited up to 40%. Since the BUdR-inhibition of the DMSO effect was approximately 70%, it is unlikely that its effect on differentiation is due to selective killing of those cells which are stimulated to differentiate.

Notes: Sedimentation velocity separation of Rhesus monkey bone marrow cells has demonstrated a reproducible but heterogeneous size distribution of cells capable of forming granulocytic colonies in agar culture (CFC's). This heterogeneity is shown to be due to the cell cycle status of the progenitor cell population. In vitro exposure of bone marrow cells to lethal doses of tritiated thymidine (H3TdR) either before or after separation restricts the size distribution of CFC's, greatly reducing the proportion of rapidly sedimenting cells. The calculation of the volume distribution of such cells before and after H3TdR exposure indicates that 55% of total CFC's in adult marrow are in G0 or G1 with a volume of 410 μ3, 42% are in S phase and of volume 450-950 μ3, and the remainder are in G2 and mitosis with a volume of between 600-950 μ3. CFC's in mid gestation fetal liver were larger than their adult counterparts and were of homogeneous volume indicative of a single non cycling population with no evidence of an S or G2 component. H3TdR exposure confirmed the non-cycling status of these fetal progenitor cells.

Notes: If the aging of diploid human fibroblasts reflects stable genetic or epigenetic changes which are few in number and different in different cells, then complementation could occur in hybrids between individual senescent cells. However neither pairs of aged fibroblasts nor even pairs of young and senescent fibroblasts produce viable hybrids under conditions known to promote cell fusion.

Notes: We have shown that the murine lymphoblast, L5178Y, requires extracellular magnesium or calcium for proliferation in suspension culture. Although both cations produce biphasic effects on growth, magnesium is the more potent since it: (1) stimulates proliferation at lower concentrations; (2) supports optimal proliferation over a wider concentration range; (3) maintains higher cell densities at stationary phase; and (4) produces less inhibition at high concentration.At suboptimal concentrations, calcium facilitates the effect of magnesium but at optimal concentration, no facilitation is evident. A concentration of 3.2 mM calcium or magnesium inhibits growth, while the same concentration composed of equimolar calcium and magnesium does not inhibit proliferation.Extracellular calcium and magnesium may influence proliferation by effecting changes in intracellular calcium and magnesium since: (1) reduction in extracellular concentrations sufficient to produce decreased growth rate is associated with decreased cell calcium and magnesium; (2) cell magnesium and growth rate are related such that an exponential decrease in relative doubling time occurs with decrease in cellular magnesium.Cells cultured at 37°C in magnesium and calcium-deprived medium will proliferate at a normal rate if either or both cations are replaced within six to 10 hours; however, with longer deprivation the doubling time in restored medium is progressively lengthened.