Karyometric and cytophotometric study of hepatocyte nuclei of frogs exposed to cold and prolonged starvation

Summary Prolonged starvation of Rana pipiens at room temperature results in a decrease in the volume of the hepatocyte nuclei from that of recently fed individuals. A converse increase in the volume of the liver nuclei over the normal was noted in frogs exposed to low temperature for 10 days. The present work was conducted in an effort to determine which nuclear fraction is responsible for the observed changes in nuclear size.The amount of DNA, RNA and protein-bound sulfhydryl groups per nucleus was measured cytophotometrically and was found to be independent of changes in nuclear size. Total nuclear protein content, on the other hand, was found to vary in proportion to nuclear volume. It was concluded that the variation in nuclear size resulting from starvation and cold-treatment is due to differences in non-chromosomal protein content and cannot be attributed to changes in ploidy.Dedicated to Prof. Berta Scharrer in honor of her 60th Birthday. — Supported by U.S. Public Health Service Grant HD-1499-04 to Dr. Cowden.Work completed while a Postdoctoral Trainee under U.S. Public Health Training Grant to the Department of Pathology, University of Florida, NIH-GM-1142-03.Supported by a Career Development Award from the National Institute of Child Health and Human Development, K 3-HD-6176-04.     

Transition from polyteny to polyploidy in salivary glands of Cecidomyiidae

Summary Quantitiative cytochemical studies of Rana catesbeiana liver cell nuclei indicated that these populations consist entirely of diploid non-proliferating nuclei. While DNA values were stable, nuclear size varied over a considerable range. These size variations were directly related to total nuclear protein content, but protein SH and S-S content appeared to be related to ploidy rather than volume. Protein bound tyrosine represents an intermediate case. The latter two fractions appear to be partially bound to nucleic acids, since removal of either class of nucleic acid leads to an increase in absorption values.This investigation was supported by a grant from the U.S. Public Health Service, GM-10003-03.Post-Doctoral Trainee under U.S. Public Health Training Grant to the Department of Pathology, University of Florida, 5 Tl GM 1142-03.Supported by a Career Development award from the National Institute of Child Health and Human Development, K 3-HD-6176-03.     

Quantitative cytophotometric studies on polyploid liver cell nuclei of frog and rat

Summary Nuclei were isolated from fixed liver tissue of diploid and triploid Rana pipiens and of rat. While the frog liver nuclei present a single ploidy class on the basis of Feulgen absorption measurements, rat liver contained diploid, tetraploid, and some octoploid nuclei. Nuclear areas within single ploidy classes varied over wide ranges, especially in the frog material. The mode of this variation was dependent on ploidy. Microspectrophotometric measurements of several protein components were compared to ploidy and nuclear volume. General protein methods indicated a linear relationship to nuclear volume. Protein-bound sulfhydryl and disulfide groups were not related to nuclear volume, but could be related to ploidy. Protein tyrosine values showed a partial dependence on both factors.This investigation was supported by a grant from the U.S. Public Health Service, GM-10003-03.Post-Doctoral Trainee under U.S. Public Health Training Grant to the Department of Pathology, University of Florida, 5T1 GM 1142-03.Supported by a Career Development award from the National Institute of Child Health and Human Development, K3-DH-6176-03.     

A histochemical study of chondroid tissue in Limulus and Octopus

Summary In a comparative histochemical study of Octopus and Limulus chondroid tissues and mouse tracheal cartilage, it was demonstrated that both invertebrate chondroids behave as less acid mucopolysaccharides than those in mouse cartilage. Octopus chondroid was less reactive than Limulus chondroid. Cetyl pyridinium chloride blockage of toluidin blue metachramasia could be unblocked in mouse cartilage by an hour's treatment in 0.5 M KCl, but even extended periods in 2.0 M KCl failed to accomplish this in the invertebrate chondroids. With a number of methods for demonstrating proteins, Octopus chondroid was less reactive than Limulus chondroid. Limulus chondroid matrix was intensely stained by methods for demonstrating protein thiol groups, but essentially no staining was observed in Octopus chondroid matrix. These studies indicated that the composition and/or structure of matrix material in the invertebrate chondroids differ from one another, and in turn differ from conditions in vertebrate hyaline cartilage.This work was supported by a grant (HD-1499-04) and a Career Development Award (5-K3-6176-04) from the National Institute of Child Health and Human Development of the U.S. Public Health Service.A contribution of the Sea Horse Key Marine Laboratory of the University of Florida.     

Cytochemical studies on cytoplasmic RNA-associated basic proteins in oocytes,somatic cells,and ribosomes

Summary Cytochemical studies on oocytes from representatives of several animal groups, of somatic tissues known to possess high levels of cytoplasmic RNA, and of isolated ribosomes indicated that basic proteins associated with the ribosomes may be stained by the methods currently in use for demonstrating histones. These proteins were resistent to weak acid extraction in fixed material and were labile to acetylation, but pretreatment with hot 5% TCA caused a measurable increase in staining with a modified Sakaguchi reaction. Such proteins were not confined to oocytes, but could be demonstrated with the TCA-alkaline fast green method in mouse pancreas and liver cells, and by the ammoniacal-silver method in all cells with high levels of cytoplasmic RNA. Microspectrophotometric studies onAscidia nigra andClavelina picta oocytes indicated that the highest concentration of cytoplasmic basic proteins was found in smaller oocytes, but the concentration of alkaline fast green stainable cytoplasmic RNA-associated basic proteins decreased in a pattern that differed significantly from that of stainable levels of cytoplasmic RNA. The implications of these findings to current concepts in developmental biology were discussed.Nucleoplasmic basic proteins with high levels of arginine which are not associated with nucleic acids were encountered in oocytes of all the echinoderm species studied and in the mouse.This work was supported by a U. S. Public Health Service grant HD-1499-04 and a Career Development Award 5-K3-6176-04.Contribution number 382 of the Bermuda Biological Station.     

Ultrastructural changes in nurse and follicle cells during late stages of oogenesis in Drosophila melanogaster

Summary During stages 11 and 12, follicle cells surrounding the nurse cells produce lysosomes which presumably aid in the breakdown of the nurse cells. Accompanying a DNA reduction in nurse cell nuclei are several characteristic morphological changes including the appearance of intranuclear rod-like structures and nuclear granules about 300 Å in diameter. Similarities between structures seen in Drosophila nurse cell nuclei and those seen in other organisms are discussed.This research was supported by U. S. Public Health Service Grants 5TIGM903-3 and 1-F1-GM-33, 385-01 and National Science Foundation grant GB-7457.     

Vacuolar protein bodies in tomato leaf cells and their relationship to storage of chymotrypsin inhibitor I protein

Summary Electron microscopy of leaves of tomato has shown that tissue containing chymotrypsin inhibitor I protein has protein in the cell vacuoles. The vacuolar protein was found either as many small bodies or as few large bodies. The data indicate that the vacuole is a temporary storage site for protein which may play an important role in growth and development of the plant. This strongly suggests that the plant-cell vacuole is something more than a site for terminal deposition of waste products. The system offers an unusual opportunity to study the biochemistry and ultrastructure of synthesis, vacuolar deposition, and recall of a well-characterized plant protein.This work was supported in part by Washington State Medical and Biological funds, U.S. Public Health Service Grants 2K3GM17059 and GM12505, and by U.S.D.A. C.S.R.S. Grant No. 915-15-29. College of Agriculture scientific paper 3442, Projects 1791 and 1996.Program in Genetics and Department of Botany.Department of Agricultural Chemistry.Department of Agricultural Chemistry. Research Career Development Awardee of the U.S. Public Health Service.     

A cytochemical study of oocyte growth in four species of millipedes

Summary The cytochemistry of oocyte growth was investigated in four species of millipedes; Narceus americanus, Oxidus gracilis, Cheiropus plancus, and a Pleuroloma species, probably P. cala. The oocyte of all four species produced a yolk nucleus which arose in contact with the nuclear membrane, subsequently detached, migrated into the central ooplasm and disrupted coincident with the appearance of protein yolk granules in the oocyte cytoplasm. Since the follicular epithelium did not display any morphological or cytochemical manifestations of secretory activity, it is suggested that direct incorporation of exogeneous proteins into yolk may play a lesser role in vitellogenesis in these forms than in insects and many other animal groups. Ooplasmic RNA levels achieved a maximum before vitellogenesis was initiated and then decreased. Similarly the nucleoli increased in size and RNA content up to the point at which cytoplasmic RNA levels began to decline. Basic proteins associated with RNA were present in the oocyte cytoplasm and the nucleoli. These achieved a peak concentration in the same size oocytes as RNA, but the intensity of staining decreased more rapidly than that of RNA during subsequent growth. The concentration of nucleoplasmic protein in oocytes of all four millipede species increased in the germinal vesicles during the course of oocyte growth. Coincident with the initiation of vitellogenesis, a class of cytoplasmic inclusions was developed which have been called concentric ring bodies. These inclusions consist of concentric layers of organic matrix material with crystallized calcium salts sandwiched between. These probably represent calcium reserves which are utilized in the formation of the exoskeleton by the embryo.Presented in partial fulfillment of the requirements for the Master of Science degree to the Graduate School of Arts and Sciences of the University of Florida.This work was supported by the following U.S. Public Health Service grants: Pathology Training Grant 5-T1-GM-1142-03, and to R. R. C. HD-1499-04 and Career Development Award K-3-HD-6176-04.     

Genetics of human-mouse somatic cell hybrids: Linkage of human genes for isocitrate dehydrogenase and malate dehydrogenase

Based on somatic cell genetic analysis, autosomal gene linkage is reported for the supernatant enzymes of human isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) in human-mouse cell hybrids. The IDH, MDH linkage was not linked to the X and E ₁₇ chromosomes or to 12 additional human enzyme markers.This work was supported in part by grants from the U.S. Public Health Service (Child Health and Human Development) and the United Health Foundation of Western New York.     

New investigations on hematoxylin, hematein, and hematein-aluminium complexes. II. Hematein-aluminium complexes and hemalum staining.

The absorption spectra of hematein-aluminium solutions have been recorded at various concentrations and pH values; the solutions were prepared using analytically pure hematein and potassium alum as aluminium source. In aqueous solution, four different hematein-aluminium complexes could be distinguished by absorption spectroscopy. In weakly acidic media we observed the violet 1:1 and 1:2 complexes HmAl (VII) and HmAl2(3) (VIII), and in strongly acidic solution the red 1:1 complex HmAl2 (IX). Whereas, in weakly alkaline solution the blue 1:1 complex HmAl0 (X) was detected. By change of the pH value the complexes were mutual interconverted. The dye complexes were characterized by their absorption spectra and molar extinction coefficients. We have stained HeLa cells with the complex solutions under different experimental conditions. In all cases the nuclear staining was intense whereas the staining of the cytoplasm was weak. The microspectra of the stained nuclei were recorded and compared with the absorption spectra of the complexes in solution. Thus it was possible to identify the bound dye species. After staining in acidic media, the cells were red to red-violet depending on the reaction conditions. The three cationic dye species VII, VIII, and IX were bound in varying amounts. After blueing in weakly acidic media or in water, only the violet dye complex VII was detected whereas, after blueing in weakly alkaline media, only the blue complex X has been observed. Enzymatic digestion experiments have shown that the dye complexes in the nuclei were bound to DNA while those in the cytoplasm and nucleoli were bound to RNA. The binding between the dye complexes and the nucleic acids is discussed.     

Electrophoretic variation in human serum ceruloplasmin: A new genetic polymorphism

Through the application of a specific oxidase stain to results of starch gel electrophoresis of human serum, three different electrophoretic forms of ceruloplasmin—denoted CpA (fast), CpB (intermediate), and CpC (slow)—have been defined. The electrophoretic differences are small and were first recognized through a rare variant individual who had only the fast and slow forms. Five phenotypes displaying different combinations of the three electrophoretic forms have been defined in American Negroes; these are called CpA, CpAB, CpB, CpAC, and CpBC. Twin, family, and population studies have yielded evidence indicating that the A and B electrophoretic forms are controlled by a pair of autosomal codominant alleles, designated Cp ^A and Cp ^B , and suggesting that the C form may be determined by a third allele, Cp ^C , at the same locus. The variants constitute a genetic polymorphism in American Negroes, but occur only rarely in Caucasians.Supported by U.S. Atomic Energy Commission Contract AT(11-1)-1552, by U.S. Public Health Service Research Grants AM 09381 and HD 02083, and by U.S. Public Health Service Career Development Awards 6-K3-HE-24, 980 (DCS) and 1-K3-A-7959 (GJB).     

Phosphoglucomutase electrophoretic variants in the mouse

The phosphoglucomutase (PGM) electrophoretic phenotype of the mouse (Mus musculus) consists of several distinct components which can be grouped into two major zones designated PGM-1 and PGM-2. Evidence presented here indicates that each zone is controlled by a single genetic locus denoted Pgm-1 and Pgm-2, respectively. Two variant forms segregated at the Pgm-1 locus. They were codominantly expressed and inherited as alleles at an autosomal locus. The alleles were termed Pgm-1 ^a (fast) and Pgm-1 ^b (slow). These alleles were separately fixed in a number of inbred strains of mice. Preliminary evidence based on wild mouse phenotypes indicates that variant forms also exist for PGM-2 which are inherited as alleles at an autosomal locus. Genetic linkage relationships have not been determined for these loci. PGM-1 variants and PGM-2 were expressed in mouse fibroblasts in vitro.Supported by U.S. Public Health Service grants GM-09966 and GM-07249 from General Medical Sciences and 5 F2 HD-35,531 from Child Health and Human Development; and Atomic Energy Commission contract AT(30-1)-3671.Postdoctoral Fellow of the U.S. Public Health Service.     

A serum factor cross-reactive with antibodies to a determinant of rabbit encephalitogenic sequence 65–74 of myelin basic protein

A serum factor. cross-reactive with antibodies to a defined determinant of myelin basic protein (residues 66–71), has been found in the sera of nine mammalian species where it may function as a specific neuroautotolerogen. In equilibrium competitive inhibition radioimmunoassays the factor appears to be completely competitive with synthetic peptide S24 (TTHYGSLPQKG) at high affinity and is therefore termed MBP-SF-24 (myelin basic protein serum factor of the S24 type). The bulk of the activity can be recovered by ammonium sulfate fractionation at 61.1% saturated ammonium sulfate (SAS), pH 7, (fractionE) after removal by precipitation at pH 7 of the 37.5, 42.6, 47.5, and 51.4% SAS fractions (fractionsA-D), including the immunoglobulins, and before removal by precipitation at pH 5 of the albumin fraction (fractionF). The factor, by its retention on XM300 during ultrafiltration of fractionE, can be purified 20-fold from serum proteins without much loss through a combination of SAS fractionation and ultrafiltration. The yield of MBP-SF-S24 in fractionE may range from a low 26 pmol S24 equivalents from 10 ml in sheep serum to a high 1.7 nmoles from 10 ml rat serum. The serum factor is reactive at high affinity with each of two populations of S24-reactive antibodies in one rabbit reagent antiserum and with one of two populations of S24-reactive antibodies in another. It appears to express a determinant involving residues THYGSL (66–71) of myelin basic protein with the same conformation as found in intact S24.This work was supported at Duke University Medical Center by Research Grant NS-10237 from the National Institutes of Health of the U. S. Public Health Service and the Medical Scientist Training Program Grant #5-T32-OMO-7171-08; at St. Luke's Hospital Center by RG-1197-B-6 from the National Multiple Sclerosis Society and by a grant from the M.T. Biddle Foundation; and at Northwestern University by Research Grant NS-06262 from the National Institutes of Health of the U.S. Public Health Service.     

Sex-limited effects of the expression of the db gene in mice during puberty

The autosomal recessive gene diabetes (db) produces a condition similar to human insulin-dependent diabetes mellitus in certain strains of inbred mice. In this investigation, the effects of expression of the db gene on the development of the submandibular glands, electrophoretic protein patterns in salivas, fasting blood glucose levels, and glycosylated hemoglobin levels were evaluated in mice undergoing puberty. Three sex-limited effects of the db gene were observed in diabetic male mice: (1) a compromise of the development of the specialized submandibular glands with the extensive tubular portion normally found in males, (2) failure to develop a salivary protein pattern unique to male mice, and (3) attainment of higher levels of fasting blood glucose than found in female diabetic mice. Since it has been documented that homozygous mice fail to develop functional gonads, apparently due to insufficient production of gonadotropin, it is likely that the compromised development of the specialized submandibular glands, and, consequently, the male salivary protein pattern, is a result of decreased testosterone production. Experiments in which diabetic mice were treated with testosterone support that conclusion, since testosterone caused transformation of the salivary protein pattern to one identical with that of normal male littermate controls and increased the tubular portion of the submandibular glands.This study was supported in part by Public Health Service Research Grant 5 R01 AM21177 and by the Indiana University Human Genetics Center (PHS P01 GM21054). The author was supported by Public Health Service Career Development Award 1 K04 AM00284. This is Publication No. 79-18 from the Indiana University Human Genetics Center.     

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. I. The radioimmunosorbent assay

A radioimmunosorbent technique is described which is capable of independently detecting both isozymes of carbonic anhydrase, CA I and CA II, in concentrations as low as 1 ng/ml. The technique is used to quantitate the different electrophoretic variants of red cell CA I as well as levels of CA II in the pig-tailed macaque, Macaca nemestrina.Supported by U.S. Public Health Service research grant GM-15419.U.S. Public Health Service Predoctoral Trainee (GM-71-14).     

Iron requirement for longevity ofPseudomonas cultures

Cultures of five out of six strains ofPseudomonas species grown in shaken flasks at 37 C needed tenfold more iron for longevity than for growth. At 30 C, the iron requirement was present in only two, and at 25 C in only one, of the six strains. The metal acted during early stationary phase and, to function, required protein synthesis. In a variety of microorganisms, qualitative and quantitative trace metal requirements for secondary metabolism and culture longevity are identical; we propose that successful completion of the former is a prerequisite of the latter.This work was supported in part by grant HD-03038-02 from the National Institute of Child Health and Human Development, U. S. Public Health Service.We express our appreciation to Miss Patsy Linn Person for performing preliminary experiments. A portion of the material was presented at the Annual Meeting of the American Society for Microbiology in Miami Beach, Florida, 4–9 May, 1969.     

C. parvum in germ-free mice

Summary The effect of previous sensitization to C. parvum, by cross-reacting antigens from other bacteria, on the immunostimulatory effects of C. parvum treatment were studied in germ-free and conventional mice. It was found that the development of splenomegaly and specific delayed hypersensitivity following C. parvum injection were similar in both germ-free mice and conventional mice.Supported by U.S. Public Health Service Grant No. 5S07 RR05705 and NIH Grant no. AM 18530Visiting Investigator. Present address: Department of Experimental Immunobiology, The wellcome Research Laboratories Beckenham, Kent, England.Recipient of a post-doctoral fellowship from the National Foundation for Ileitis and Colitis Inc.Recipient of Research Career Development Award No. AM 0073 from the National Institute of Arthritis, Metabolism and Digestive Diseases     

Mutations affecting levels of tetrahydrofolate interconversion enzymes in Saccharomyces cerevisiae. I. Enzyme levels in ade3-41 and ADE15, a dominant adenine auxotroph

Summary The ADE15, ade3-41 and ser2 markers have been mapped with respect to one another and with respect to the SUC1-MAL1 locus on chromosome VII of Saccharomyces cerevisiae. Three methods have been used for this mapping, viz. conventional tetrad analysis, analysis of single site conversion asci, and analysis of double site conversion asci. The physical distance separating ade3-41 and ADE15, two mutations affecting synthesis of tetrahydrofolate interconversion enzymes, has been estimated.Supported by Public Health Service Research Career Development Award 1K04 AM 36710 and Public Health Service Grant AM 14254 to E. W. J.Supported by Graduate Fellowship from Department of Biology, Case Western Reserve University.     

Fine structure of the pore-annulus complex in the nuclear envelope and annulate lamellae of germ cells

Summary Octagonal symmetry in the pore margin has been demonstratedin situ in annulate lamellae and the nuclear envelope of germ cells. The annular material is located to variable extent within the pore and also extends beyond the pore margin; in the latter case it may be continuous with extra-pore annular material of some adjacent pores. In thin sections of fixed material, the annular material of both the nuclear envelope and annulate lamellae appears to be composed of a matrix within which are embedded thin filaments and small granules, the disposition and interrelationship of which are described and discussed. The so-called intra-annular granule is described as consisting of a number of smaller units (similar to the granular component of the annular material) which become aggregated in the center of some pores in both the nuclear envelope and annulate lamellae. The possible significance of intra-annular granules is discussed in terms of binding and movement of macromolecules.This investigation was supported by research grants (HD-00699, GM-09229) and a Career Development Award from the National Institutes of Health, U. S. Public Health Service. The author acknowledges the skillful technical assistance of Mrs.Robert Decker.     

Immunochemical cross-reactivity between intact purified myelin basic protein (MBP) and the synthetic encephalitogenic peptide S49

Three antisera to myelin basic protein—a rabbit antiserum pool against rat myelin, a rabbit antiserum pool against rat myelin basic protein (MBP), and a monkey antiserum against bovine MBP—were found to contain detectable levels of antibodies that would bind radiolabeled S49 (GSLPQKAQRPQDENG). Strongly encephalitogenic in Lewis rat, S49 is a synthetic peptide representing residues 69–84 of bovine MBP with a deletion of glycine-76 and histidine-77 to make it analogous to rat and guinea pig MBPs. The rabbit antimyelin antiserum and the monkey anti-MBP antiserum contained antibodies directed against a non-sequential determinant that required asparagine 84, the glycine-histidine deletion, and residues 69–71 for maximal activity. S49-reactive antibodies from the rabbit anti-MBP antiserum were directed solely against a sequential determinant comprising residues 69–71. S49-reactive antibodies from all three antisera reacted in liquid phase with purified intact rat, guinea pig, and bovine MBP showing that the determinant is exposed for B cell recognition even in bovine MBP and can serve both as immunogen and reactant.This work supported at Duke University Medical Center by Research Grant NS-10237 from the National Institutes of Health of the U.S. Public Health Service and the Medical Scientist Training Program Grant #5-T32-OMO-7171-08; at St. Luke's Hospital Center by NS-15322 from the National Institutes of Health of the U.S. Public Health Service; and at Northwestern University by Research Grant NS-06262 from the National Institutes of Health of the U.S. Public Health Service.