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.     

The diffuse diplotene stage of meiotic prophase in Neurospora

The prophase stages of meiosis in Neurospora crassa are re-examined following McClintock (1945) and Singleton (1953). A diffuse chromosome stage occurring between pachynema and diakinesis is described. It is proposed that the diffuse stage does not necessarily represent a condition of intense gene activity in the sense of directing the metabolic activity of the ascus.Supported by U. S. Public Health Service grants AI-01462 and GM-14263.     

Growth of a water strain ofLeptospira in synthetic media

Pertinent parameters of growth of a water strain ofLeptospira in synthetic media are described. Growth rates as a function of aeration, temperature, and substrate concentration were determined. Evidence is presented for the production of acetate and CO₂ from long-chain fatty acids. A marked drop in pH was shown to result from the accumulation of acetate during growth in synthetic medium. Growth was initiated at pH values from 5.2 to 7.7. An absolute CO₂ requirement for initiation of growth was demonstrated. Growth is also described with acetate as the sole carbon and energy source, demonstrating that leptospires are capable of fatty acid synthesis.This investigation was supported by Public Health Service grant AI-05440 from the National Institute of Allergy and Infectious Diseases, and by Public Health Service predoctoral fellowship (1-Fl-GM-40, 062-01) to the senior author from the National Institute of General Medical Sciences. Technical assistance of Mr. Howard Young is gratefully acknowledged.     

Calcium carbonate precipitation by heterotrophic bacteria isolated from biofilms formed on deteriorated ignimbrite stones: influence of calcium on EPS production and biofilm formation by these isolates

Heterotrophic CaCO₃-precipitating bacteria were isolated from biofilms on deteriorated ignimbrites, siliceous acidic rocks, from Morelia Cathedral (Mexico) and identified as Enterobacter cancerogenus (22e), Bacillus sp. (32a) and Bacillus subtilis (52g). In solid medium, 22e and 32a precipitated calcite and vaterite while 52g produced calcite. Urease activity was detected in these isolates and CaCO₃ precipitation increased in the presence of urea in the liquid medium. In the presence of calcium, EPS production decreased in 22e and 32a and increased in 52g. Under laboratory conditions, ignimbrite colonization by these isolates only occurred in the presence of calcium and no CaCO₃ was precipitated. Calcium may therefore be important for biofilm formation on stones. The importance of the type of stone, here a siliceous stone, on biological colonization is emphasized. This calcium effect has not been reported on calcareous materials. The importance of the effect of calcium on EPS production and biofilm formation is discussed in relation to other applications of CaCO₃ precipitation by bacteria.     

Development of a chemically defined medium for growth ofNeisseria gonorrhoeae

A chemically defined medium satisfactory for growth of a number of laboratory strains and recent isolates ofNeisseria gonorrhoeae has been devised. It contains inorganic salts, dextrose, guanine, cytosine, B-vitamin supplement, and the following amino acids:l-arginine,l-aspartic acid,l-cystine,l-isoleucine,l-leucine,l-proline,l-threonine, andl-valine.Nine of the eleven strains grew satisfactorily in this medium without being provided supplemental CO₂ during incubation, and a tenth strain grew in the medium supplemented with glutamine. No single B-vitamin or purine or pyrimidine base was essential for growth of any of the strains, but some combinations of them were stimulatory. Riboflavin, however, was inhibitory. The strains showed variations in requirements for amino acids. The amino acids which were either essential or stimulatory for one or more of the strains were included in the medium. Those to which the strains responded differently were used at concentrations intermediate between those optimal for growth of one strain and inhibitory for another. Conventional agar was inhibitory, but a purified agar, having a gel strength twice that of conventional agar, was satisfactory. An aqueous solution of 0.1% cysteine and 0.86% NaCl was satisfactory for preparation of inocula.This investigation was supported by a Public Health Service Predoctoral Fellowship (F-FI-GM-24-755-01A1) from the National Institute of General Medical Sciences of the United States Public Health Service to the senior author.     

Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Mechanism of calcium accumulation in acetate-fed aerobic granule

High calcium content has been widely reported in acetate-fed aerobic granules, but the reason behind this is unclear yet. By SEM–energy dispersive X-ray mapping analysis, this study showed that the majority of calcium was presented in the central part of the acetate-fed aerobic granule, and the granule shell part was nearly calcium-free. The elemental analysis of calcium ions coupled with the chemical titration of carbonate further revealed that the calcium ions that accumulated in the acetate-fed aerobic granule mainly existed in the form of calcium carbonate (CaCO₃). The formation of the CaCO₃ appeared to be highly dependent on the size of the aerobic granule, i.e., the CaCO₃ precipitation was found only in aerobic granules with radiuses larger than 0.5 mm. These experimental observations with regard to the formation of CaCO₃ in the acetate-fed aerobic granule were further confirmed by the model simulation, which was based on the principles of mass diffusion and carbonate dissociation in liquid phase. This study for the first time showed that the size of the acetate-fed aerobic granule would indeed play an essential role in the CaCO₃ formation, and provided experimental evidence that a crystal CaCO₃ core was not necessarily required for granulation.     

Studies on ribosomal mutants of Salmonella typhimurium LT-2

Summary Mutations conferring resistance to spectinomycin, resistance to neomycinkanamycin and cold-sensitivity of ribosome biosynthesis were located on the Salmonella typhimurium chromosome in relation to the genes cysG, strA, and aroC. The effect of temperature and growth medium on the expression of these mutations, and the interactions between the mutations was determined as a prerequisite to the mapping. On the basis of chromosomal location and dominance, it is concluded that cold-sensitivity of ribosome synthesis can result from mutations in several distinct genes.This work was supported by Public Health Service research grant AI-05526.     

Citramalate lyase of Clostridium tetanomorphum

Summary Assay methods and some properties of (+)-citramalate pyruvatelyase, an enzyme from Clostridium tetanomorphum that converts (+)-citramalate to pyruvate and acetate, are described. The enzyme is very active (0.8–1.2 units per mg protein) in freshly prepared extracts, but loses activity rapidly during storage. (+)-Citramalate is the only substrate found to be cleaved by the lyase; the equilibrium for the reaction permits almost complete cleavage at low substrate concentrations. A divalent cation is required as a cofactor. A sensitive and specific enzymic method for estimating (+)-citramalate is described.It is a pleasure to dedicate this paper to Prof. C. B. Van Niel who first awakened the interest of the author in the problems of bacterial metabolism and, more specifically, in the fermentation of glutamic acid.This work was supported in part by a research grant from the National Institutes of Health (AI-00563), U.S. Public Health Service, and by funds from the California Agricultural Experiment Station     

Inheritance and genetic linkage of transcobalamin II

Summary The genetic polymorphism of the vitamin B₁₂ transport protein transcobalamin II (TC II) was studied in a Caucasian population and in families. There are five codominent alleles of TC II which show a Mendelian mode of inheritance. No genetic linkage of TC II was found with gene loci for ADA, GLOI, Pi, HLA, AB0 and AK₁. TC II like proteints could be detected on autoradiograph of PAGE in two patients with congenital homozygosity for functional TC II deficiency. These vitamin B₁₂ binding proteins in the patients' serum were shown not to be normal R-proteins.Supported in part by grants from U.S. Public Health Service, NCI CA-22507, CA-19267, CA-08748, NIAID AI-07073. A portion of this work was conducted through the Clinical Research Center Facility of the University of Washington (RR-37)     

Sarcolemmal calcium binding sites in heart: I. Molecular origin in “gas-dissected” sarcolemma

Summary Calcium in the myocardial cell is highly compartmentalized and a fast, an intermediate, a slow and a nonexchangeable calcium pool have been described. The fast pool contains 66% of the total cell exchangeable calcium in cultured neonatal rat heart cells with a t _1/2 of < 1.5 sec. Though the cellular origin of this fast pool is unknown, its rapidity and its displacement by La³⁺ most likely places it at the sarcolemma or at least in rapid equilibrium with the sarcolemma.We isolated the sarcolemma of cultured neonatal rat heart cells using the gas-dissection technique, which yields a pure sarcolemmal preparation in less than a second, thereby precluding membrane changes which might occur during conventional plasma membrane isolation. We determined the calcium binding characteristics of these membranes, using an on-line technique to monitor ⁴⁵Ca, which allows measurement of ⁴⁵Ca binding characteristics in the presence of unbound ⁴⁵Ca. Two classes of calcium binding sites were determined: (i) K _d of 13 m, capacity 7 nmol/mg and (ii) K _d of 1.1 mm, capacity of 84 nmol/mg. To assess the molecular origin of the sarcolemmal calcium binding we treated the membranes with a variety of enzymes. Protease or neuraminidase treatment did not cause large changes in these parameters. Simultaneous treatment with two different phospholipases C or the extraction of the lipids with isopropanol resulted in a dramatic loss of the low-affinity binding sites.These results, in association with previously defined sarcolemmal phospholipid distribution, places the low-affinity binding sites at the cytoplasmic leaflet. The physiological implication of this localization as it pertains to cellular calcium exchange is discussed.We thank Eloise Andrews-Farley for culturing of the neonatal cells. This study is supported by U.S. Public Health Service grants HL 28539-07 and 08, the Laubisch Fund, the Castera Foundation and the American Heart Association, Greater Los Angeles Affiliate.     

Role of poly-β-hydroxybutyric acid in cyst formation byAzotobacter

Several parameters associated with the growth ofAzotobacter vinelandii in liquid culture were examined in order to investigate the relationship between the accumulation and degradation of poly-β-hydroxybutyric acid (PHB), the development of viscous capsular components, and cyst formation. The amount of intracellular PHB, which increased markedly during the log phase of growth, reached a maximum during the early stationary phase and subsequently declined. During polymer degradation there was a concurrent increase in the extent of encystment in the cultures supplemented with CaCO₃. An increase was noted in the viscosity of culture supernatants during polymer degradation when CaCO₃ was deleted from the medium and the culture pH was controlled by the periodic addition of 0.1m KOH. The extent of encystment and the amount of PHB accumulated were directly proportional to the substrate concentration. The PHB was selectively labeled by the addition of sodium acetate-2-¹⁴C to late log-phase cells. During polymer utilization in either encysting or nonencysting cultures 20% of the label was evolved as CO₂. In the nonencysting cultures, 45% of the radioactivity was distributed between residual PHB and other cellular components, and 35% was in the supernatant polysaccharide-like material. Intact cysts retained 80% of the label. Experiments with ruptured cysts indicated that about 35% of the radioactivity was present in the intine material.     

Cryopreservation of encapsulated gentian axillary buds following 2 step-preculture with sucrose and desiccation

Alginate beads containing axillary buds of in vitro-grown gentian (Gentiana scabra Bunge var. buergeri Maxim.), were successfully cryopreserved following 2 step-preculture with sucrose and desiccation. The optimal preculture conditions were as follows: axillary buds were excised from in vitro-grown gentian plants and precultured on semi-solid Murashige and Skoog (MS) medium containing 0.1 M sucrose for 10 days (25 °C, 16-h photoperiod) (first step). This was followed by incubation on semi-solid MS media containing 0.4 M (1 day) and then 0.7 M sucrose (1 day) (second step). After preculture, the buds were encapsulated in alginate beads and desiccated aseptically on silica gel for 9 h to a water content of 10% (fresh weight basis), followed by immersion in liquid nitrogen (LN). With this protocol, 87% of the gentian buds survived exposure to LN and showed normal development of shoots and roots in vitro and in vivo. Depletion of NH₄NO₃ in the regeneration medium did not improve survival following desiccation and exposure to LN. The results show that 2 step-preculture with sucrose is effectively applicable in encapsulation–desiccation based cryopreservation of gentian axillary buds. This preculture can replace the conventionally used lengthy cold-hardening treatment and is useful for routine cryopreservation of gentian germplasm.     

Intragenic mapping of the Neurospora pyrimidine-3 locus by functional deletions

Summary On the basis of complementation between polar and non-polar pyrimidine-3 mutants of Neurospora crassa, a physical linear order of the polar mutants within the locus is derived. The basis of this derivation is that a polar mutant is an intragenic functional deletion extending from the site of the mutation to the translationally distal end of the locus.Supported by Public Health Service Research Grant AI-01462.     

The production of extracellular thermostable neutral proteinase and α-amylase byBacillus stearothermophilus

Summary Extracellular thermostable neutral proteinase was produced byBacillus stearothermophilus strains NCIB 8924 and NRRL B-3880 growing at 55°C. The formation and stabilization of this proteinase was found to be dependent on the concentration of free calcium ions. Therefore, procedures that removed free calcium ions from the medium, such as the use of phosphate buffer, resulted in a lower production of proteinase. The calcium-deficient proteinase was denaturated or adsorbed by calcium phosphate compounds. During the sterilization procedure of the culture medium, the CaCO₃ precipitation, caused by the removal of CO₂, influenced the amount of proteinase produced in a phosphate buffered medium made with tap water. An improved medium without phosphate buffer was used for 10 and 300 l batch cultivations and the calcium requirement for proteinase formation by the two strains was determined.     

Contribution of the allelicMtz 3 andMtz 4 allotype genes to the formation of individual rabbit serum α2-macroglobulin molecules

The contribution of the allelicMtz ³ andMtz ⁴ genes to the formation of individual rabbit serum α₂-macroglobulin (α₂M) molecules was examined by precipitation of α₂M from rabbits of known genotype with antiallotype antisera. The α₂M was isolated fromz ³z³ andz ⁴z⁴ homozygous andz ³z⁴ heterozygous rabbits, iodinated with I¹²⁵ and precipitated by sequential reactions with antiallotype antiserum and goat anti-rabbit IgG. Purified unlabeled α₂M or α₂M in serum was used to inhibit competitively the reaction of antiallotype antiserum and labeled α₂M. Nearly all α₂M molecules have z3 or z4 antigenic determinants; approximately 50% of α₂M molecules in heterozygotes have both. Altogether, the z3, z3,4, and z4 molecules in heterozygotes have approximately 60% of the number of z3 and 40% of the number of z4 determinants as compared to the respective homozygotes. Unlike all other known allelic blood protein systems of rabbits, allelic exclusion does not occur in α₂M molecules of heterozygotes; rather, hybrid molecules are formed. Presented in part at the Fifty-fourth and Fifty-fifth Annual Meetings of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 12–17, 1970, and Chicago, Illinois, April 12–17, 1971. This investigation was supported in part by U.S. Public Health Service Grants AI-09241 and AI-07043. B.H.B. performed this investigation in partial fulfillment of the requirements for the Doctor of Philosophy Degree in the Graduate College; he is supported by a postdoctoral fellowship from the Schweppe Foundation. K.L.K. is the recipient of U.S. Public Health Service Research Career Development Award AI-28687.     

Maltobionic acid enhances intestinal absorption of calcium and magnesium in rats

ABSTRACT

In Experiment 1, the effects of calcium maltobionate (MBCa) on calcium and magnesium absorption were examined using male rats. Four diets were designed in which 25%, 50%, and 100% of calcium carbonate (CaCO₃, Control) were substituted with MBCa and were designated as MBCa-25, MBCa-50, and MBCa-100, respectively. The cecal concentration of short-chain fatty acids was significantly higher in groups MBCa-50 and MBCa-100; however, pH of cecal contents did not significantly differ among the groups. Retention rates of calcium and magnesium were significantly higher in all MBCa groups as compared to the Control. In Experiment 2, the efficiency of calcium absorption was compared using everted sacs of jejunum and ileum with CaCO₃ and MBCa as calcium sources. More calcium from MBCa was absorbed as the concentration of calcium increased in comparison to CaCO₃. It was concluded that MBCa is a better calcium source than CaCO₃ in terms of both calcium retention and absorption.

Abbreviations: ANOVA: analysis of variance; Ca: Calcium; CaCO_3: calcium carbonate; ICP-OES: Inductivity coupled plasma optical emission spectrometer; Mg: magnesium; MBCa: calcium maltobionate; OCPC: o-cresolphthalein complexone; SCFAs: short-chain fatty acids; SE: standard error; TRPM6: transient receptor potential melastatin 6.     

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.     

Biochemical properties of yeast l-asparaginase

Only a single l-asparaginase has been found in the yeast Saccharomyces cerevisiae. The enzyme is synthesized constitutively, and its functioning is not controlled by the products of its activity. The apparent K_m for the yeast l-asparaginase reaction is 2.5×10^–4 m. Activity is greatest at pH 8.5 and is unaffected by the ionic strength of reaction mixtures. l-Asparagine can serve as the sole nitrogen source for cell metabolism but cannot serve as the sole supply of carbon. Active l-asparaginase is necessary for the use of l-asparagine as a nitrogen donor for cell growth. This requirement suggests a possible way in which l-asparaginase-deficient strains of yeast or other organisms might easily be selected.G.E.J. was supported by U.S. Public Health Service Predoctoral Fellowship No. 5 F01 GM36,437.     

l-lactate or pyruvate stimulated growth of novikoff rat hepatoma cells

Summary Novikoff rat hepatoma cells (subline N1S1-67) grew when 30mm l-lactate or pyruvate was substituted ford-glucose in Swim's medium 67 supplemented with dialyzed calf bovine serum. A 2.6-fold increase in cell number (1.34 generations) was obtained. RNA, DNA, protein and dry weight increased in proportion to the cell number. In control medium lackingl-lactate, pyruvate ord-glucose, cell growth of 0.42 generation was obtained. Growth withl-lactate was dependent on thel-lactate concentration up to 30mm at which the greatest increase in cell number occurred. Significant growth did not occur whend-lactate, glycerol, acetate, α-ketoglutarate, succinate or malate, each at 30mm, was substituted ford-glucose. Growth in the medium containingl-lactate was not due to the utilization ofd-glucose or some other substrate carried into the culture with the inoculum. Medium contamination byd-glucose was insufficient to explain the growth obtained in the medium containingl-lactate, but could have accounted for growth in the control medium. Throughout growth, the concentration ofl-lactate in the medium remained unchanged. The increase in cell number cannot be explained byl-lactate triggering the utilization of glycogen, nor by oxidation and degradation of protein, amino acids, fatty acids, or carbohydrate moieties of glycoproteins in the medium.l-Lactate does not serve as a significant carbon or energy source in the growth of these cells. This investigation was supported by grants from the National Institute of Allergy and Infectious Disease, the National Science Foundation, and the United States Public Health Service.