Extraction of [14CI Vitamin a from Rat Liver and Kidney During Processing for Transmission Electron Microscopy

The retention of radio isotope-labekd vitamin A during processing for electron microscopy was investigated using the livers and kidneys of vitamin A deficient rats. [15-¹⁴C]Retinol (3μCi/animal) was administered by esophageal intubation to male rats which had been maintained on a vitamin A deficient diet for five or sir weeks postweaning. Glutaraldehyde- or osmium-fixed tissue was processed by three methods: a) routine (a graded series of ethanols, propylene oxide and epoxy), b) rapid (75% and 95% ethanol with three changes of epoxy), or c) water-soluble embedding (70% and 80% hydrorypropyl methacrylate). Water-soluble embedding retained the highest percentage of label in the tissue (liver: 96.31%; kidney: 98.68%). Inclusion of osmium tetroxide in the processing sequence and minimal exposure of tissue to lipid solvents were necessary for good retention of labeled vitamin A in tissues.

The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

In conducting the research described in this report, the investigators adhered to the “Guide for Laboratory Animal Facilities and Care,” as promulgated by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences—National Research Council.     

Extraction of [14CI Vitamin a from Rat Liver and Kidney During Processing for Transmission Electron Microscopy

The retention of radio isotope-labekd vitamin A during processing for electron microscopy was investigated using the livers and kidneys of vitamin A deficient rats. [15-¹⁴C]Retinol (3μCi/animal) was administered by esophageal intubation to male rats which had been maintained on a vitamin A deficient diet for five or sir weeks postweaning. Glutaraldehyde- or osmium-fixed tissue was processed by three methods: a) routine (a graded series of ethanols, propylene oxide and epoxy), b) rapid (75% and 95% ethanol with three changes of epoxy), or c) water-soluble embedding (70% and 80% hydrorypropyl methacrylate). Water-soluble embedding retained the highest percentage of label in the tissue (liver: 96.31%; kidney: 98.68%). Inclusion of osmium tetroxide in the processing sequence and minimal exposure of tissue to lipid solvents were necessary for good retention of labeled vitamin A in tissues.

The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

In conducting the research described in this report, the investigators adhered to the “Guide for Laboratory Animal Facilities and Care,” as promulgated by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences—National Research Council.     

A serological comparison of the three morphological phases of Coccidioides immitis; complement fixation tests with anti-Histoplasma capsulatum serum

Summary The results of this study indicated that antigens prepared from the three morphological phases ofCoccidioides immitis differed in their complement fixing activity with anti-Histoplasma capsulatum pooled serum. Spherule antigens were serologically less active in tests with the anti-H. capsulatum pooled serum than antigens prepared from arthrospores and from mycelium.Antigenic determinants which are common toC. immitis andH. capsulatum appeared to be located on the intact arthrospore cellular surface but not on the surface of spherule cells.Part of a dissertation submitted to the Graduate School of Duke University in partial fulfillment of requirements for the Ph. D. degree.This work was supported by contract with the Department of the Army, Fort Detrick, Frederick, Maryland.In conducting the research reported herein, the investigators adhered to Guide for Laboratory Animal Facilities and Care established by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, NAS-NRC.     

A novel antigen is common to the dome epithelium of gut- and bronchus-associated lymphoid tissues

Summary The dome epithelium (DE) covering bronchus- and gut-associated lymphoid tissues (BALT and GALT) is composed of columnar cells, groups of lymphocytes, M cells, and pre-M cells. Although the cell biology and immunologic processes of this tissue are likely important in the afferent arm of secretory immune responses, virtually nothing is known about biochemical constituents of the DE. Therefore, a monoclonal antibody, 30E5, was used to study the distribution of a novel antigen, common to dome epithelia of GALT and BALT. 30E5 was secreted by a hybridoma, prepared by fusing murine splenocytes, immunized against dome epithelial cells, with P3×68/Ag8 myeloma cells. Reactivity of antigens was defined by indirect immunocytochemistry on sections of rabbit tissues or with dissociated epithelial cells. In situ, 30E5-reactive antigen circumscribed each group of dome epithelial lymphocytes, most or all of which were T cells, in rabbit appendix, sacculus rotundus, cecal patch, Peyer's patch, and BALT. In the DE this antigen was associated with the apical surface and the supranuclear or perinuclear regions of epithelial cells, but it was not associated with epithelial cells of villi, epithelium, or with individual lymphocytes. In peripheral lymph nodes, spleen, and in domes and follicles of GALT or BALT, 30E5-reactive antigen was visualized in linear wisps, primarily in regions populated by thymocytes. In other adult tissues, 30E5-reactive antigen was associated with involuntary muscle, myoepithelial cells of lactating mammary gland and with what appeared to be neural dendrites; but it was not found in epithelia other than DE. In neonatal rabbit appendix, this antigen first appeared in the upper dome epithelium two days after birth, a period coinciding with T cell infiltration and M cell maturation. The histologic distribution of 30E5-reactive antigen suggested that it might be a contractile filament, a receptor, or a differentiation antigen. Since 30E5 was associated with DE of both GALT and BALT, results support the concept of a molecule common to all mucosa-associated lymphoid tissues.In conducting the research described in this report, the investigators adhered to standards set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23) as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, USALimited quantities of ascites containing monoclonal antibody 30E5 will be distributed to interested investigators until such time as the hybridoma is available from American Type Culture CollectionAbbreviations ABC avidin-biotin-horseradish peroxidase complex - BALT bronchus-associated lymphoid tissues - DMEM Dulbecco's modified Eagle medium - GALT gut-associated lymphoid tissues - DE dome epithelium - DEL dome epithelial lymphocytes - MAb monoclonal antibody - MALT mucosal-associated lymphoid tissues The views of the authors expressed here do not purport to reflect the position of the Department of the Army or the Department of Defense Send offprint requests to: Department of Experimental Pathology, Division of Pathology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA     

Precocious development of lectin (Ulex europaeus agglutinin I) receptors in dome epithelium of gut-associated lymphoid tissues

Summary Dome epithelium (DE), the tissue covering lymphoid domes of gut-associated lymphoid tissues, was examined in both adult and neonatal rabbit appendix or sacculus rotundus to determine if dome epithelial cells matured earlier than epithelial cells covering adjacent villi. The localization of well-differentiated epithelial cells in rabbit gut-associated lymphoid tissues (GALT) was accomplished histochemically by use of molecular probes: fluorescein isothiocyanate or horseradish peroxidase conjugates of Ulex europaeus agglutinin I (UEA), a lectin specific for terminal L-fucose molecules on certain glycoconjugates. The villus epithelial cells of newborn and 2-, 5-, or 10-day-old rabbits did not bind UEA, but between the twelfth and fifteenth days of postnatal life, UEA receptors were expressed by well-differentiated villus epithelial cells. In contrast to villus epithelium, DE in appendix and sacculus rotundus of neonatal rabbits expressed UEA receptors two days after birth, a feature that distinguished the DE of neonatal GALT for the next two weeks. In adult rabbits, UEA receptors were associated with dome epithelial cells extending from the mouths of glandular crypts to the upper domes; in contrast to the domes, UEA receptors were only present on well-differentiated epithelial cells at the villus tips. Results suggested that in neonatal rabbits most dome epithelial cells developed UEA receptors shortly after birth, reflecting precocious development of DE as compared to villus epithelium. In adult rabbit dome epithelium UEA receptors appeared on dome epithelial cells as they left the glandular crypts, representing accelerated epithelial maturation.Abbreviations DE dome epithelium - DEL dome epithelial lymphocytes - FITC fluorescein isothiocyanate - HRP horseradish peroxidase - PBS phosphate-buffered saline - PBS-CaMg PBS containing calcium and magnesium ions - UEA Ulex europaeus agglutinin I The views of the authors expressed here do not purport to reflect the position of the Department of the Army or the Department of DefenseIn conducting the research described in this report, the investigators adhered to standards set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23) as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, USA     

A serological comparison of the three morphological phases of coccidioides immitis: Complement fixation tests with a Pooled antiserum obtained from rabbits with experimental Coccidioidomycosis

Summary A sonicated spherule preparation was more reactive than a sonicated arthrospore antigen in complement fixation tests with a pooled serum from rabbits with experimental coccidioidomycosis. The reactivity of the sonicated spherule approximated the reactivity of coccidioidin. When the sonicated spherule was separated into its supernatant and sediment fractions, both preparations exhibited serological activity.

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Zusammenfassung Ein mit Schallwellen hergestelltes Kügelchen-Präparat war activer in dem Komplement-Tests mit Blutserum von Kaninchen mit einer Coccidioidomycosisinfektion als ein mit Schallwellen hergestelltes Arthrosporantigen. Die Reaktivität der mit Schallwellen hergestellten Kügelchen war der des Coccidioidin ähnlich. Wurden diese Kügelchen in Niederschlag und Lösung getrennt, so hatten beide Präparate serologische Aktivität.

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Part of a dissertation submitted to the Graduate School of Duke University in partial fulfillment of requirements for the Ph.D. degree.This work was supported by contract with the Department of the Army, Fort Detrick, Frederick, Maryland.In conducting the research reported herein, the investigators adhered to Guide for Laboratory Animal Facilities and Care established by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, NAS—NRC.     

Endocardiosis in rhesus monkeys

Endocardiosis was diagnosed as an incidental finding in two rhesus monkeys. The gross and histologic appearance of the lesions was described, and the similarity of this lesion to lesions of endocardiosis as found in dogs and man was discussed.The animals used in this study were handled in accordance with the Guide for Laboratory Animal Facilities and Care established by the National Academy of Science, National Research Council.     

Development of dome epithelium in gut-associated lymphoid tissues: Association of IgA with M cells

Summary The dome epithelium (DE), which covers gutassociated lymphoid tissues (GALT) and provides both a protective barrier over lymphoid follicles and a route for antigen uptake from the gut, develops in rabbit appendix (caecum) during the first week of neonatal life. To determine if secretory immunoglobulins from maternal milk interact with this developing tissue, their interrelationships in neonatal rabbit appendix were examined by use of immunocytochemical techniques. The glycoprotein, secretory component, was not produced by neonatal rabbits less than 15 days old, since neither the membranous nor the free, secreted forms of maternal secretory component were associated with villi or DE of neonates. Immunoglobulin A (IgA), but neither IgG nor IgM, were noted on DE by light microscopy, even though IgG was abundant in the villus lamina propria and vascular spaces. The epithelial IgA was distributed, in a patchy pattern, across the upper dome surface of some two-day-old, and all five-and ten-day old nursing animals, but IgA was not on DE of rabbits prevented from nursing. Immunoelectron microscopy of appendix from nursed rabbits revealed IgA directly over the apical surface of M cells, where it formed a continous, thick coating without binding to adjacent immature absorptive cells; it was also within apical vacuoles of M cell cytoplasm. The distribution of IgA on the DE of rabbit appendices indicated that in differentiating GALT, maternal IgA reacted preferentially with M cells or pre-M cells, leading to speculation concerning a role for IgA in the development of GALT and in establishment of mucosal immune responses in neonates.In conducting the research described in this report, the investigators adhered to the standards set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23) as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, USAAbbreviations DE dome epithelium - GALT gutassociated lymphoid tissues - HRP horseradish peroxidase - IgA immunoglobulin A - SC secretory component The views of the authors expressed here do not purport to reflect the position of the Department of the Army or the Department of Defense     

Sulfur mustard-increased proteolysis followingin vitro andin vivo exposures

The pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Papirmeister et al. (1985) postulate enhanced proteolytic activity as a proximate cause of HD-induced cutaneous injury. Using a chromogenic peptide substrate assay, we previously reported that in vitro exposure of cell cultures to HD enhances proteolytic activity. We have continued our investigation of HD-increased proteolytic activity in vitro and have expanded our studies to include an in vivo animal model for HD exposure. In vitro exposure of human peripheral blood lymphocytes (PBL) to HD demonstrated that the increase in proteolytic activity is both time- and temperature-dependent. Using a panel of 10 protease substrates, we established that, the HD-increased proteolysis was markedly different from that generated by plasminogen activator. The hairless guinea pig is an animal model used for the study of HD-induced dermal pathology. When control and HD-exposed PBL and hairless guinea pig skin where examined, similarities in their protease substrate reactivities were observed. HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolytic activity that was time-dependent. The HD-increased proteolytic response was similar in both in vitro and in vivo studies and may be useful for elucidating both the mechanism of HD-induced vesication and potential treatment compounds.Abbreviations CPSPA chromogenic peptide substrate protease assay - HD sulfur mustard - PBL human peripheral blood lymphocytes - pNA p-nitroaniline In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, revised 1985.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

Anticonvulsants for soman-induced seizure activity

This report describes studies of anticonvulsants for the organophosphorus (OP) nerve agent soman: a basic research effort to understand how different pharmacological classes of compounds influence the expression of seizure produced by soman in rats, and a drug screening effort to determine whether clinically useful antiepileptics can modulate soman-induced seizures in rats. Electroencephalographic (EEG) recordings were used in these studies. Basic studies were conducted in rats pretreated with HI-6 and challenged with 1.6×LD₅₀ soman. Antimuscarinic compounds were extremely effective in blocking (pretreatment) or terminating soman seizures when given 5 min after seizure onset. However, significantly higher doses were required when treatment was delayed for more than 10 min, and some antimuscarinic compounds lost anticonvulsant efficacy when treatment was delayed for more than 40 min. Diazepam blocked seizure onset, yet seizures could recur after an initial period of anticonvulsant effect at doses 2.5 mg/kg. Diazepam could terminate ongoing seizures when given 5 min after seizure onset, but doses up to 20 mg/kg were ineffective when treatment was delayed for 40 min. The GABA uptake inhibitor, tiagabine, was ineffective in blocking or terminating soman motor convulsions or seizures. The glutamate receptor antagonists, NBQX, GYKI 52466, and memantine, had weak or minimal antiseizure activity, even at doses that virtually eliminated signs of motor convulsions. The antinicotinic, mecamylamine, was ineffective in blocking or stopping seizure activity. Pretreatment with a narrow range of doses of ₂-adrenergic agonist, clonidine, produced variable protection (40–60%) against seizure onset; treatment after seizure onset with clonidine was not effective. Screening studies in rats, using HI-6 pretreatment, showed that benzodiazepines (diazepam, midazolam and lorazepam) were quite effective when given 5 min after seizure onset, but lost their efficacy when given 40 min after onset. The barbiturate, pentobarbital, was modestly effective in terminating seizures when given 5 or 40 min after seizure onset, while other clinically effective antiepileptic drugs, trimethadione and valproic acid, were only slightly effective when given 5 min after onset. In contrast, phenytoin, carbamazepine, ethosuximide, magnesium sulfate, lamotrigine, primidone, felbamate, acetazolamide, and ketamine were ineffective.The animals used in studies performed in, or sponsored by, this Institute were handled in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals, proposed by the Committee to Revise the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, and published by National Academy Press, 1996, and the Animal Welfare Act of 1966, as amended. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as reflecting the views of the Department of the Army or the Department of Defense.     

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury

Summary The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anatomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.This study was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS20590. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23     

TASTE ACCEPTANCE IN SQUIRREL MONKEYS (SAIMIRI SCIUREUS)

Six squirrel monkeys were presented with solutions representingthe four primary tastes. The solutions included various concentrationsof glucose or sodium saccharine (sweet), sodium chloride (salty),citric acid (sour), and quinine sulfate or sucrose octaacetate(bitter). A 24 hr two-bottle choice technique was employed.Amount of food, water, and solution consumed every 24 hr wasrecorded. The results showed that the maximum intake for glucosesolution was with the 5.0% concentration, although maximum caloricintake was with the 1.25% concentration where there was a potentiationof food intake. Water was preferred over sodium saccharine atthree of the four concentrations which were tested, and waterwas preferred over or equally to the concentrations of sodiumchloride and citric acid that were used. However, quinine sulfateand sucrose octaacetate were preferred over or equally to waterat most of the concentrations which were tested. In conducting the research described in this report, the investigatorsadhered to the ‘Guide for Laboratory Animal Facilitiesand Care’, as promulgated by the Committee on the guidefor Laboratory Animal Resources, National Academy of Sciences- National Research Council.     

Immunocytochemical localization of a calcium-binding protein in the rat duodenum

Summary The cellular localization of the vitamin D-dependent calcium-binding protein (CaBP) in the duodenum of rat was studied using indirect immunofluorescence and immunoperoxidase staining methods. Specific positive reaction product, indicative of the presence of CaBP, was exclusively located within the villous part of the duodenal mucosa. Moreover, CaBP was detected mainly within the supranuclear region of the cytoplasm of absorptive cells and also at the level of their basal laminae. CaBP was not demonstrable either in the nuclei or associated with the brush border membrane of absorptive cells. Also, CaBP was neither detectable in goblet cells nor in sub-epithelial layers. When the specific anti-CaBP antiserum was replaced by nonimmune rabbit serum or when it was preabsorbed on a CaBP-Sepharose conjugate, no positive immunostaining was seen. Together with recent biochemical data our observations agree well with the view that CaBP may act as an intracellular buffer by protecting the cell against too high Ca²⁺ concentrations.     

Rhesus monkey micro mixed lymphocyte and mitogen reactivity: Optimal conditions and normal variability

Optimal conditions for the rhesus monkey micro mixed lymphocyte system with multiple automated harvesting of samples were evaluated. Parameters studied were cell concentration, length of culture period, methods of inactivation of cell populations, supplementation of media, type of culture plates, and changes in the reactivity of cells from individual animals over an extended time period. This work was supported in part by Portland Veterans Administration Hospital, Portland, Oregon, and the General Research Support Branch of the U.S. Public Health Service Grant RR00163, the Bureau of Medicine and Surgery, Navy Department, Work Unit No. M4318. 01.007ABG2. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or the Naval service at large. The animals used in this study were handled in accordance with the provisions of Public Law 89–54 as amended by Public Law 91–579, “Animal Welfare Act of 1970,” and the principles outlined in the “Guide for the Care of Laboratory Animals,” U.S. Department of Health, Education, and Welfare Publication No. (NIH) 73-23.     

Studies on blood-brain barrier permeability after microwave-radiation

Summary Since the reported alterations of permeability of the blood-brain barrier by microwave radiation have implications for safety considerations in man, studies were conducted to replicate some of the initial investigations. No transfer of parenterally-administered fluorescein across the blood-brain barrier of rats after 30 min of 1.2-GHz radiation at power densities from 2–75 mW/cm² was noted. Increased fluorescein uptake was seen only when the rats were made hyperthermic in a warm-air environment. Similarly, no increase of brain uptake of¹⁴C-mannitol using the Oldendorf dual isotope technique was seen as a result of exposure to pulsed 1.3-GHz radiation at peak power densities up to 20 mW/cm², or in the continuous wave mode from 0.1–50 mW/cm². An attempt to alter the permeability of the blood-brain barrier for serotonin with microwave radiation was unsuccessful. From these studies it would appear that the brain must be made hyperthermic for changes in permeability of the barrier induced by microwave radiation to occur.The research reported in this paper was conducted by personnel of the Radiation Sciences Division, USAF School of Aerospace Medicine, Brooks AFB, Tex. 78235. The animals involved in the study were procured, maintained, and used in accordance with the Animal Welfare Act of 1970 and the Guide for the Care und Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council     

Helical structure in the apical tubules of several absorbing epithelia

Summary Unique and highly ordered structures were discovered in the so-called apical tubules of several absorbing epithelia (kidney proximal tubule, visceral yolk sac and ductuli efferentes) fixed in situ with a mixture of formaldehyde, glutaraldehyde and osmium tetroxide. The apical tubules were especially numerous in the apical cytoplasm, in addition to the invaginations of the apical plasma membrane, newly formed endocytic vesicles and large endocytic vacuoles. They showed a cylindrical structure (80 nm in diameter) limited by a smooth membrane. Helically wound parallel rows of particles (11 nm in diameter) were found in the apical tubules in close proximity to their limiting membrane. The structure of the helix was determined by following the rows through serial sections and semithin sections, and was found to be a left-handed quadruple helix. These particles surround an electron-lucent cylinder (35 nm in diameter), containing at its center a single row of particles (9 nm in diameter). The apical tubules with the luminal specializations were not seen in continuity with the apical plasma membrane, but were frequently connected with the large endocytic vacuoles, which were present in the deeper levels of the apical cytoplasm. From these observations, it is suggested that the apical tubules are not derivatives of the apical plasma membrane; rather, they represent an intracellular compartment, which is morphologically related to the large endocytic vacuoles.     

Immunofluorescence and ultrastructural localization of β2-microglobulin in human renal allografts

Summary The localization of 2-microglobulin (2m) was studied in renal biopsies from 18 patients with pathological transplanted kidney using immunofluorescence and electron-immunohistochemical techniques; the renal biopsies of 4 cases with normal kidneys were used as controls. Using immunofluorescence, 2m was not observed in the normal kidneys, 2m was found in the glomeruli (7 cases) and the tubular epithelium (16 cases) of the transplanted kidneys. Using immunoelectron microscopy, some labelling of the normal kidneys was observed mainly along the cell coat of foot processes and in tubular-epithelial lysosomes. In the glomeruli of transplanted kidneys, particularly in cases of acute or chronic rejection, 2m was most frequently localized on the outer layer of the basement membrane and along the cell coat of foot processes. The brush border of the proximal tubules and the lysosomal structures were intensely labelled. Although immunoelectron-microscopy studies are unable to discriminate between the localization of 2m in normal and transplanted kidneys, these findings nevertheless suggest the glomerular filtration of 2m and its metabolism in the tubular epithelium.     

Genetic control of glucokinase activity in mice

Inbred strains of mice were surveyed for liver glucokinase activity. Mice of all strains studied could be distributed into three groups with high, intermediate, and low levels of enzyme activity. Genetic analysis using crosses and backcrosses with prototype high (C3H/HeJ) and low (RF/J) strains revealed that glucokinase activity was controlled by a single gene. The name glucokinase and gene symbol Gk are suggested for this gene. The Gk ^a allele designates the strain with high glucokinase activity, while Gk ^b represents the allele in the strain with the low enzyme activity. The interaction of fasting and diabetes on the activity of glucokinase in these two strains is described.Supported in part by United States Public Health Service Research Grant CA 05873 from the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.     

Immunocytochemical demonstration of two vitamin D-dependent calcium-binding proteins in mammalian kidney

Distribution of vitamin D-dependent calcium-binding proteins (CaBPs) were studied in four mammalian species using monospecific antibodies raised against chick duodenal CaBP (D-CaBP), human cerebellar CaBP (L-CaBP), and rat duodenal CaBP (S-CaBP). The immunoperoxidase technique of unlabelled antibodies was employed. The distribution of D-CaBP/L-CaBP was identical in all the species studied except for the monkey. In the rat, pig, and human nephrons, D-CaBP/L-CaBP was seen in the cytoplasm of the cells of the distal convoluted tubules, initial segments of the collecting ducts and interspersed cells of the collecting ducts. Proximal convoluted tubules, glomeruli and maculae densae were negative. In the monkey, in addition to the cells of the distal convoluted tubules, the cells along the entire length of the collecting ducts were also strongly positive. S-CaBP was found to be species-specific, and hence positive results were obtained only in the rat nephron. The strongest positive reaction for S-CaBP was seen in the cells of the distal convoluted tubules. These same cells were also positive for D-CaBP/L-CaBP. S-CaBP was also detected in the cells of the thick ascending limb of the loop of Henle, along the entire length of the collecting ducts and in smaller amounts in cells of the macula densa. Intracellularly the S-CaBP was present only in the apical cytoplasm of positive cells. D-CaBP/L-CaBP stained the entire cytoplasm but the staining in the apical cytoplasm was denser.     

Enhanced differentiation of sexually dimorphic organs in L-thyroxine treated Tfm mice

Summary In the present study we examined the effects of L-thyroxine on the differentiation of three sexually dimorphic organs in mice affected by the testicular feminization mutation (Tfm): the submaxillary salivary glands, the kidneys, and the adrenal glands. In the salivary gland we compared the effects of L-thyroxine and testosterone in normal and Tfm mice on the morphology of the intercalated tubules and on the NGF content of this gland. In the kidney we examined thyroid hormone effects on the sexually dimorphic proximal convoluted tubules. In the adrenal gland we studied the effects of the same hormone on the weight of the organ and on the structure and ultrastructure of the zona reticularis in untreated and L-thyroxine treated Tfm mice. The results show that the thyroid hormone enhances differentiation of the dimorphic structures in all these organs and increases the secretory activity of the salivary and adrenal glands. The stimulatory effects of L-thyroxine are more pronounced on the weight of the adrenal than of the salivary gland and of the kidney. Our results raise the question whether the enhanced differentiation elicited by L-thyroxine in the three sexually dimorphic organs of Tfm mutants is due to a direct hormonal effect, or whether it is fully, or in part, mediated through the hypothalamo-pituitary-thyroid axis.This investigation was supported by the Progetto Finalizzato Biologia della Riproduzione of the Italian National Council of Research (CNR)