Expression of Schwann cell markers by mammalian neural crest cells in vitro

During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.     

Observations on the ultrastructure of nucleated erythrocytes and thrombocytes,with particular reference to the structural basis of their discoidal shape

Summary The marginal band of nucleated erythrocytes in the toadfish is found, in electron micrographs, to be composed of about twenty-five microtubules approximately 200 Å in diameter. These form a bundle that encircles the erythrocyte just beneath the plasma membrane. These observations support the interpretation of Meves 1904, that this relatively stiff equatorial band may contribute to the maintenance of the discoid shape of nucleated erythrocytes in fish, amphibians, reptiles and birds.Similar microtubules form an annular bundle encircling the nucleus in fish thrombocytes. The number of tubular elements involved here is in excess of one hundred and they are located deep to the ectoplasmic layer instead of immediately beneath the plasmalemma. The term endoplasmic ring is therefore proposed for this structure.Comparative observations on nucleated erythrocytes of various species are presented showing that the density and fine structure of the material occupying the interchromosomal areas of the nucleus, always matches the cytoplasm and is related to the hemoglobin concentration of the species. These ultrastructural observations are consistent with the optical absorption and biochemical findings of other investigators indicating the presence of intranuclear hemoglobin in nucleated erythrocytes. Crystalline order is occasionally found in electron micrographs of the hemoglobin rich areas of the nucleus in toadfish erythrocytes but is not found in the cytoplasm.This research was supported by grant G-12916 of the National Science Foundation.     

A Model for Sclerotinia sclerotiorum Infection and Disease Development in Lettuce,Based on the Effects of Temperature,Relative Humidity and Ascospore Density

The plant pathogen Sclerotinia sclerotiorum can cause serious losses on lettuce crops worldwide and as for most other susceptible crops, control relies on the application of fungicides, which target airborne ascospores. However, the efficacy of this approach depends on accurate timing of these sprays, which could be improved by an understanding of the environmental conditions that are conducive to infection. A mathematical model for S. sclerotiorum infection and disease development on lettuce is presented here for the first time, based on quantifying the effects of temperature, relative humidity (RH) and ascospore density in multiple controlled environment experiments. It was observed that disease can develop on lettuce plants inoculated with dry ascospores in the absence of apparent leaf wetness (required for spore germination). To explain this, the model conceptualises an infection court area containing microsites (in leaf axils and close to the stem base) where conditions are conducive to infection, the size of which is modified by ambient RH. The model indicated that minimum, maximum and optimum temperatures for ascospore germination were 0.0, 29.9 and 21.7°C respectively and that maximum rates of disease development occurred at spore densities >87 spores cm⁻². Disease development was much more rapid at 80–100% RH at 20°C, compared to 50–70% RH and resulted in a greater proportion of lettuce plants infected. Disease development was also more rapid at 15–27°C compared to 5–10°C (85% RH). The model was validated by a further series of independent controlled environment experiments where both RH and temperature were varied and generally simulated the pattern of disease development well. The implications of the results in terms of Sclerotinia disease forecasting are discussed.     

Non-enzymatic formation of sulfenyl iodide by solubilized thyroid particulate protein with peroxidase activity

Thyroidal particulate protein with peroxidase activity has been studied to determined wetherr it could be induced to form a sulfenyl iodide, postulated as a reactive intermediate in the iodination of tyrosine. The protein was solubilized with digitonin and purified by tryptic digestion and filtration through Sephadex G-200. When supplemented with H₂O₂ it catalyzed the oxidation of guaiacol iodide or thiourea at 37°C. With iodide as substrate the product was an iodoprotein. In the absence of H₂O₂ the protein did not bind ¹³¹I^? or thio[¹⁴C] urea unless it first had been dialyzed against [³⁶Cl] chlorinated buffer. During dialysis a portion of the ³⁶Cl from the dialysis medium was bound by protein. Subsequent binding of iodide or thiourea was accompanied by loss of protein-bound ³⁶Cl.Addition of iodide to dialyzed protein at 4°C resulted in formation of a yellow compound with maximum absorbance at 355 nm. It was postulated to be a sulfenyl periodide on the basis of its absorption spectrum and its behavior with thio[¹⁴C] urea and 2-mercaptoethanol. The stability of the colored species was dependent on temperature and concentration of iodide. Disappearance of color as the solution was warmed was accompanied by formation of iodo-protein. Predialysis of the protein against p-chloromercuribenzoate, but not 2-mercaptoethanol or bisulfite, prevented the formation of the yellow proteiniodide species, indicating that a reactive sulfhydryl group was involved in the reaction. It was concluded that a particulate protein closely asscociated with thyroid peroxidase could be induced by non-enzymatic means to form a species which has properties consistent with those of a sulfenyl iodide. Further investigation will be required to determine whether the same protein-iodine species can be identified during the peroxidase-catalyzed oxidation of iodide.     

The coevolution of juvenile play–fighting and adult competition

Although play–fighting is widespread among juvenile mammals, its adaptive significance remains unclear. It has been proposed that play is beneficial for developing skills to improve success in adult contests (motor‐training hypothesis), but the links between juvenile play–fighting and adult aggression are complex and not well understood. In this theoretical study, we investigate the coevolution between juvenile play–fighting and adult aggression using evolutionary computer simulations. We consider a simple life history with two sequential stages: a juvenile phase in which individuals play–fight with other juveniles to develop their fighting skills; and an adult phase in which individuals engage in potentially aggressive contests over access to resources and ultimately mating opportunities, leading to reproductive success. The simulations track genetic evolution in key traits affecting adult contests, such as the level of aggression, as well as juvenile investment in play–fighting, capturing the coevolutionary feedbacks between juvenile and adult decisions. We find that coevolution leads to one of two outcomes: a high‐play, high‐aggression situation with highly aggressive adult contests preceded by a prolonged period of juvenile play–fighting to improve fighting ability, or a low‐play, low‐aggression situation in which adult contests are resolved without fighting and there is minimal investment in play–fighting before individuals mature. Which of these outcomes is favoured depends on the mortality costs and on the type of societal structure: societies with strong reproductive skew, favouring monopolization of resources, show high levels of adult aggression and high investment in juvenile play–fighting, whereas societies with low reproductive skew have both low adult aggression and low levels of play–fighting. A review of empirical evidence, particularly in the primate genus Macaca, highlights some limitations of our model and suggests that other, complementary functional explanations are needed to account for the full range of competitive and cooperative forms of play–fighting. Our study illustrates the power of evolutionary simulations to shed light on the long‐standing puzzle of animal play.     

Sensitive chiral high-performance liquid chromatographic assay for labetalol in biological fluids

The four stereoisomers of the combined α- and β-adrenoceptor antagonist labetalol were separated and quantified at therapeutic concentrations by normal-phase high-pressure liquid chromatography using a chiral stationary phase and fluorescence detection. Drug in plasma or urine was recovered by solid-phase extraction with 83±5% efficiency. Limits of detection from biological samples (3 ml) were between 1.5–1.8 ng ml⁻¹. Intra-day and inter-day variation at 25 ng ml⁻¹ were ≤2.7% and ≤5.80% respectively for all stereoisomers. The assay was applied to an examination of the disposition of labetalol stereoisomers after a single oral dose of racemate to a human volunteer. Labetalol appears to undergo enantioselective metabolism leading to relatively low plasma concentrations of the pharmacologically active enantiomers.     

Amiloride does not alter NaCl avoidance in Fischer-344 rats

Fischer-344 (F-344) rats differ from other common rat strains in that they fail to show any preference for NaCl at any concentration in two- bottle preference tests. Because 100 microM amiloride partially blocks the NaCl-evoked chorda tympani (CT) response in electrophysiological studies, we tested NaCl preference (0.068-0.273 M) in F-344 rats with and without 100 microM amiloride solution as the solvent. A third group was tested with unadulterated NaCl solutions following CT transection. Amiloride had no significant effect on the NaCl preference-aversion function, whereas CT transection significantly reduced NaCl avoidance. These results suggest that the amiloride-sensitive component of the NaCl response is not necessary for F-344 rats to display avoidance of NaCl, but the entire CT input is.      

Isoenzymes of hypoxanthine-guanine-phosphoribosyl transferase in a family with partial deficiency of the enzyme

The isoenzymes of hypoxanthine-guanine-phosphoribosyl transferase (HGPRT; E. C. 2.4.2.8) were studied by polyacrylamide gel disc electrophoresis in the erythrocytes of a family in which there was a partial deficiency of this X-linked enzyme. Hyperuricemic males, in whom HGPRT activity was 4% of normal, were found to have a variant enzyme which had altered kinetic and electrophoretic properties. In acrylamide gel, this variant migrated about 15% faster than the normal enzyme, and its K _m for hypoxanthine was twice that of the normal. The sister of two patients had 34% of normal activity in her erythrocytes and was thought to be a heterozygote. Electrophoresis of her hemolysate yielded profiles in which there were two zones of HGPRT activity. The more slowly migrating isoenzyme behaved electrophoretically like the normal isoenzyme. The faster-migrating isoenzyme had a mobility identical to that of the variant enzyme found in hemolysates from her hyperuricemic siblings. However, in her profile the activity of the variant enzyme was three times greater than that of the HGPRT found in the boys. This increased activity appears to be due to an interaction of the variant enzyme with the normal enzyme. Electrophoresis of a mixture of normal enzyme and the variant from a hyperuricemic male yielded a profile similar to that observed in this girl and a dramatic increase in the amount of activity in the variant zone.Aided by U.S. Public Health Service Grants No. HD04608 and GM 17702 from the National Institute of Child Health and Human Development and from the National Institute of General Medical Sciences, respectively, National Institutes of Health. Presented in part at the 1971 Annual Meeting of the Western Society for Pediatric Research, Carmel, California.     

MEMBRANE DIFFERENTIATIONS IN FREEZE-FRACTURED MAMMALIAN SPERM

A correlated thin-sectioning and freeze-fracturing study has been made of guinea pig and rat spermatozoa. In sections, the cell membrane over the acrosome has a concanavalin A and ruthenium red reactive glycocalyx which exhibits an ordered pattern related to the lattice of crystalline domains within the plane of the membrane revealed by freeze-fracturing. The cleaved acrosomal membrane also shows a finer linear periodicity in some areas. The membrane over the equatorial segment of the guinea pig acrosome is marked by a palisade of oblique ridges not observed in the rat. The plasmalemma of the postacrosomal region is rich in membrane intercalated particles, many randomly dispersed, others clustered in rectilinear arrays. A particle-poor zone is found just anterior to the posterior ring. The fold of redundant nuclear envelope posterior to the ring has many nuclear pores in close hexagonal array. The nuclear envelope lining the implantation fossa is devoid of pores. When cleaved it has a particle-free central area surrounded by a broad zone of large, closely packed, hollow particles. The membrane of the mid-piece in the guinea pig (but not the rat) contains linear strands of 6–8-nm particles oriented circumferentially. The membrane investing the principal piece exhibits the usual randomly distributed particles but in addition, a double row of larger (9 nm) particles runs longitudinally within the membrane over outer dense fiber 1. In the corresponding position in thin sections a local thickening of the membrane is discernible. These observations form a basis for further studies on the functional correlates of these regional specializations of the sperm membrane.     

AN ELECTRON MICROSCOPE STUDY OF SPERMATID DIFFERENTIATION IN THE TOAD, BUFO ARENARUM HENSEL

The differentiation of the spermatids of Bufo arenarum has been described from a study of electron micrographs of thin sections of testis. The development of the acrosome from the Golgi complex takes place in much the same manner as in mammalian spermatogenesis but no acrosome granule is formed. A perforatorium is described for the first time in this species. It is formed by a convergence of dense filaments that arise between the nuclear membrane and the head cap. During maturation of the spermatid the chromatin undergoes striking physicochemical alterations. Fine chromatin granules uniformly dispersed in the karyoplasm are replaced by larger and larger aggregates and these ultimately coalesce to form a very dense sperm head. Two centrioles of cylindrical form are situated very near the base of the sperm head. The longitudinal fibrils of the tail flagellum take origin from one, and the dense fibrous substance of the undulating membrane is closely related to the other. Phase contrast cinematographic observations on the swimming movements of living toad sperm, when considered in relation to the fine structural components of the tail, suggest that there is a contractile component in the undulating membrane as well as in the axial fibrils. The differences in the structure of mammalian and amphibian sperm tails are discussed in relation to differences in the character of their movements.