Intracellular distribution of lysosomal enzymes in the mouse pigment mutants pale ear and pallid

Summary The size distribution of lysosomes was determined in kidney proximal tubule cells of two mouse pigment mutants, pale ear and pallid, which have an increase in kidney lysosomal enzyme content caused by a decreased rate of secretion of lysosomal enzymes into urine. Both mutations have larger lysosomes when compared with normal mice. However, neither mutant contains the giant lysosomes (up to 11 micron diameter) common to the well-characterized beige mutant, which has a kidney secretory defect similar to the pale ear and pallid mutants. Subcellular distribution studies, performed by the osmotic shock technique, likewise suggested differences among the pigment mutants. A very high content of soluble enzyme, indicative of lysosomal fragility during homogenization, was found in extracts from the beige mutation. By comparison, the percent of soluble enzyme became progressively lower in extracts of the pallid and pale ear mutants and was lowest in extracts from normal mice. All 3 pigment mutants had normal concentrations of osmotically resistant membrane-bound lysosomal enzymes. This indicates that the excess, non-secreted, lysosomal enzyme in all three pigment mutants likely is present in classical lysosomal organelles rather than in other non-lysosomal subcellular membrane fractions. The results also illustrate that mammalian mutants which exhibit decreased lysosomal secretory rates can have strikingly different effects on morphology of lysosomes.Supported in Part by National Science Foundation Grant PCM 77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM 007093-03.     

Lysosomal Dysfunctions Associated with Mutations at Mouse Pigment Genes

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.     

Deficiency of dihydroxy acid dehydratase in the mito-chondria of the iv-1 mutants of Neurospora crassa

The isoleucine-valine requiring mutants of Neurospora crassa which map at the iv-1 locus lack, or have a very low level of activity for, the enzyme dihydroxy acid dehydratase in the mitochondrial fractions derived from them. This enzyme is, however, present in the soluble fractions of the mutant homogenates. The enzyme is present in both mitochondrial and soluble fractions from homogenates of wild-type and from homogenates of iv mutants blocked at other steps in the isoleucine-valine pathway.The work reported here was supported in part by grants GM 12323 and 5TO1-GM-00337-09 from the National Institutes of Health, United States Public Health Service, and by a grant from the Robert A. Welch Foundation.Recipient of Research Career Award 4-K-6-GM-18,383 from the National Institutes of Health, United States Public Health Service.     

Theory of hemoglobin facilitated oxygen transport

The transport of oxygen in a hemoglobin-saturated medium is theoretically investigated using classical transport theory. It is found that all the chemical complexes can be expressed as a single function of oxygen pressure. A potential difference together with apH shift is predicted to occur across the medium. This research was supported by the United States Public Health Service Training Grant No. 5-T1-GM-833 from the National Institute of General Medical Sciences. This research was supported by a United States Public Health Service Research Career Program Award 5-K6-GM-18,420 from the National Institute of General Medical Sciences.     

The isolation and analysis of one functional type ofpyr-3 mutant inNeurospora

Twenty-seven pyrimidine-requiring mutants were isolated as suppressors of anarg-3 mutant. All 27 are deficient for ATCase activity and show linkage to thecol-4 marker located on linkage group IV. Analyses of prototroph frequencies resulting from crosses of the new mutants to previously mappedpyr-3 mutants indicate that this functional type ofpyr-3 mutant is restricted to one region of the genetic map. Complementation studies with 11 of the new mutants further extend and subdivide the complementation map of thepyr-3 locus.This work was supported in part by National Institutes of Health, Public Health Service Grant GM 15137-01 and by National Science Foundation Grant GB5998.     

A genetic analysis of linkage group I of Chlamydomonas reinhardi

Summary Crossing over in linkage group I ofChlamydomonas reinhardi was studied by means of the analysis of 1721 unordered tetrads. The data indicate that chiasma interference is positive, that chromatid interference is absent and that crossing over occurs at the 4-strand stage.

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Zusammenfassung Mit Hilfe der Analyse von 1721 ungeordneten Tetraden wurde das crossing-over in der Koppelungsgruppe I vonChlamydomonas reinhardi untersucht. Aus der Häufigkeit der verschiedenen Tetraden-Typen wird geschlossen, daß crossingover im Vierstrang-Stadium geschieht, und daß eine positive Chiasma-Interferenz existiert, während eine Chromatiden-Interferenz nicht vorkommt.

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This work was supported by the United States Public Health Service (Grant No. E 1421) and the National Science Foundation (Grant No. G 2812).

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A portion of this work was conducted while a Public Health Service Research Fellow of the National Cancer Institute.     

The effects of interspecies infant interaction upon social behavior ofMacaca irus andErythrocebus patas

TwoMacaca irus and twoErythrocebus patas mother-infant pairs were used as experimental subjects to test the effects of interspecies interaction upon social behavior of monkeys. Cynomolgous and patas monkeys given intraspecies interation served as control animals. An unexpected result of this study is that mother-infant behaviors were more affected by interspecies interaction than were infant-infant behaviors. The results indicate that the importance one animal has for another in the development of social behavior depends upon two factors: (a) the behavioral repertoires of interacting animals must be similar and (b) each animal must respond appropriately to the other's behavior.This research was supported in part by National Science Foundation grants No. GB4925 and GB7601 and a grant from the Louisiana State University Graduate Research Council. Animal subjects were provided by the Delta Regional Primate Research Center, United States Public Health Service grant No. FR00164 from the Division of Research Facilities and Resources.The author received personal support while conducting this research as a United States Public Health Service Trainee.     

Maple syrup urine disease: Branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain -keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release ¹⁴CO₂ from the corresponding [1-¹⁴C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mMin both cell lines. Exposure of normal and mutant cells to a 10 mMload of leucine, isoleucine, and valine resulted in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, -ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.This investigation was supported in part by Grants AM-13622, AM-05646, and GM-17702 from the United States Public Health Service, Veterans Administration Grant M.R.I.S. No. 3181 to Dr. Nathan Gochman, and grants from the National Foundation and the Kroc Foundation. S. D. S. is a Postdoctoral Research Fellow supported by United States Public Health Service Training Grant AM-05646.     

Genetic variation in mouse salivary amylase rate of synthesis

Heterozygotes from matings of the mouse strains YBR/Cv and C3H/As have about 3 times more YBR-amylase than C3H-amylase in the saliva. The determinant for this quantitative effect is located on linkage group XVI close to or within the structural gene for salivary amylase. The quantitative effect is the result of an increase in the rate of synthesis of YBR-amylase, and the determinant is cis acting. Studies of other mouse strains suggest that regulatory genetic elements may modulate salivary amylase production.This work was supported by the Danish Natural Science Research Council and a grant from the United States Public Health Service (Grant GM-19521).     

Osmium tetroxide versus glutaraldehyde fixation in adrenomedullary tissue

Summary Osmium tetroxide and glutaraldehyde fixation of adrenomedullary tissue presents evidence that these two fixatives preserve the tissue in quite different manners. Not only is the type of fixative of importance, but also the osmolarity of the fixatives is a prime factor in producing an accurate pictorial account of catecholamines.Supported by United States Public Health Service Grants 5-Tl-GM-459, NB 05093-02, FR 0505-01, NB 00690-11 and National Science Foundation Grant GB 25 96.— With the technical assistance of John Yates, Earl Pitsinger and Brenda Ryker.     

The development and evaluation of immunodiffusion,immunofluorescence and physiological tests for the differentiation of atypical trichophyton rubrum and T. mentagrophytes isolates

Five laboratory procedures: 1) immunodiffusion, 2) immunofluorescence, 3) in vitro hair perforation, 4) pigment stimulation, and 5) a urease test were compared for their ability to differentiateT. rubrum fromT. mentagrophytes. Of the physiological tests, thein vitro hair perforation technique was the most reliable for differentiating the two species. With the serological tests, the organisms were not differentiated by immunodiffusion, but if appropriate dilutions of the conjugates were used in immunofluorescence testing, most isolates could be differentiated.A portion of a Dissertation submitted by the senior author to the University of North Carolina in partial fulfillment of the requirements for the degree of Doctor of Public Health in the School of Public Health. Training was provided by the Laboratory Director's Program which is supported by Training Grant TO1 GM 00567-07 from the National Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service. The laboratory research was performed at the Laboratory Division, Center for Disease Control, under the supervision of William Kaplan.     

Proline endopeptidase and exopeptidase activity in polymorphonuclear granulocytes

Summary Peptidases capable of releasing proline residues from polypeptides are present in the cytoplasmic fraction of rabbit polymorphonuclear granulocytes. This was shown with peptide substrates where proline is present either at the carboxy-terminal or within the polypeptide chain. Lysosomal and plasma membrane enzymes were inactive towards such polypeptides. The proline residue was hydrolyzed at either its amino end or its carboxy end. It is noteworthy that a Pro:Pro bond was cleaved both in the pentapeptide Thr-Lys-Pro-Pro-Arg and the dipeptide Pro:Pro.Supported by Grant AI-09116 from the National Institutes of Health, U.S. Public Health Service and Grant BMS72-01469 AO3 from the National Science Foundation.     

Morphogenesis and nature of the pigment granules in the adult human retinal pigment epithelium

Summary The pigment epithelial cells of the retina are a layer of highly specialized melanocytes. Beginning in the early embryonic period they produce melanin throughout the entire life. The Golgi apparatus plays a key role in the biosynthesis of melanin. The following steps can be distinguished morphologically: (a) Golgi-vesicles, (b) intermediate vesicles, (c) melanosomes, (d) melanin granules. Structures with a ringlike appearance that are described as lipofuscin granules in the literature prove to be altered intermediate vesicles and melanosomes.This investigation was carried out in part at the Francis I. Proctor Foundation for Research in Ophthalmology, San Francisco, California, U.S.A., and supported by United States Public Health Service Program Project Grant EY 00310, and Deutsche Forschungsgemeinschaft, Training Grant Nr. Sp 102/1.     

A histochemical study of lysosomal enzymes in the retina of the rat

Summary Sections of retinas from albino and pigmented rats were studied histochemically by the naphthol and lead methods for lysosomal enzymes (acid phosphatase, aryl sulfatase, glucosaminidase and -glucuronidase). Activity of the enzymes studied (except -glucuronidase) is demonstrable in granules which by their staining properties are identified as lysosomes. The matrix of the lysosome stains positively in PAS preparations and is diastaae resistant. The organelles are distributed mainly in the pigment epithelium, outer limiting membrane, inner nuclear layer, ganglion layer and inner limiting membrane of the retina.This investigation was supported by a generous grant from the Nuffield Foundation.     

Control of proliferation in two cell linesin vitro

Summary In two types of cells growing as monolayer cultures (an established line of Chinese hamster cells and a newly obtained strain of Rat Fibroblasts) no evidence is found for proliferation control by contact inhibition of mitosis or by cell-produced chemical inhibitors. Under the particular conditions of these experiments, the intracellular concentration of a multiplication-stimulating factor (MSF) associated with fetal calf serum appears to play a regulating role. This factor is degraded continually by proliferating as well as by non-proliferating, plateau phase cells. A model is proposed in which the intracellular concentration of MSF is governed by: the amount of MSF available to the cell from the culture medium; the permeability and amount of cell membrane accessible for diffusion (or transport) of MSF; and the rate of its degradation. It is hypothesized that mitosis only occurs if the concentration of MSF exceeds a critical value and that this critical value is a characteristic of the cell type. Consistency of the model with existing data concerningin vitro cell proliferation is discussed.This work was supported by Grants CA-04542 and CA-10372 from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service.Biophysics trainee under United States Public Health Service Training Grant GM-00712 from the National Institutes of Health.Holder of a Dernham Senior Fellowship in Oncology from the American Cancer Society, California Division.     

Oriented migration of interstitial cells and nematocytes inHydra attenuata

Summary The migratory properties of hydra cells within the tissue were studied. The extent and direction of cell migration were examined in budding, non-budding, and regenerating animals. Nematocytes and a small number of single big interstitial cells (the multipotent interstitial cells) actively migrate preferentially in an apical direction. Basal migration of these cells occurs only when a bud is present and, in which case, the cells migrate into the developing bud. The regeneration of the hypostome and tentacles does not affect cell migration in either direction, except for apical migration of stenotele nematocytes, which was markedly reduced.This research was supported by National Science Foundation Grant (GB 29284), National Institute of Health Grant (HD 08086-01), and N.I.H. Public Health Service Training Grant (HD 00347).     

Interpolation coding: A representation for numbers in neural models

A central task of perception can be defined as one of computing hierarchies of invariants. One way of representing such invariants in intermediate levels of abstraction in this hierarchy is to use discrete units. These have been termed value units. A problem with such an encoding is that there has not been a good way to represent accurate numerical quantities using these units. This paper remedies the deficiency by describing a scheme that interpolates values between units representing fixed numerical quantities. The scheme has nice properties: it extends across functional mappings and it allows different sources of evidence to be combined.This work was supported in part by the National Science Foundation under Grant DCR-8405720 and the National Institutes of Health under Public Health Service Grant 1R01NS22407-01     

The role of the Golgi complex in the formation of secretion in the test cells of the follicle of the tunicate,Ciona

Summary Electron microscope studies were made on the test cells which comprise a part of the follicular envelope in the ovary of the tunicate Ciona. During development the cells become filled with secretory granules. The Golgi complex is well developed and usually centrally located in the cells. The morphological variations shown and described strongly suggest that the Golgi complex is mainly concerned with the origin of the secretion in these cells.Supported by research grants (GM-9229, 9230) from the National Institute of General Medical Science, United States Public Health Service.This investigation was supported by a Public Health Service Research Career Program Award (1-K3-GM-11, 524) from the National Institute of General Medical Sciences.     

Mitochondrion-desmosome complexes in human hepatocytes

Summary Mitochondrion-desmosome complexes similar to those seen in other epithelia were observed in hepatocytes from normal and diseased human livers of children and adults. Their occurrence could not be explained by random distribution of mitochondria in the cells. The close associations of mitochondria with desmosomes supported the hypothesis that the latter might be special areas of intercellular ionic diffusion between hepatocytes.This work was supported in part by United States Public Health Service Grants AI-1059 and TI AM-5384 from the National Institute of Arthritis and Metabolic Diseases, 5 MOl FR 000-50 from the General Clinical Research Center, HD 00674 from the National Institute of Child Health and Development and by a grant from the Life Insurance Medical Research Fund G-65-50.The author is very grateful to Dr. Alex B. Novikoff for the use of the facilities of his laboratory (supported by United States Public Health Service Grant CA-06576), to Mr. Nelson Quintana and Mrs. Julie Windsor for their superb technical assistance and to Miss Marianne Van Hooren for preparation of the photographs.     

Serologic and karyologic evidence of incorrect identity of an animal cell line (guinea pig spleen)

Summary A cell line labeled GPS and presumed to be guinea pig spleen has been shown to be mouse and to carry the H-2^k allele. Identification of characteristic chromosome markers suggests that the proper identity may be L-M mouse cells. The use of immunologic and karyologic identification procedures for routine checking of cell lines is discussed. This work was supported in part by United States Public Health Service Grant CA03720.