Silicon in rat liver organelles: Electron probe microanalysis

Summary Electron probe microanalysis of unfixed freeze-substituted rat liver tissue embedded in Spurr's low viscosity epoxy resin demonstrated the occurrence of Si as well as P, S, and Cl in the nucleus, nucleolus, mitochondria, and rough endoplasmic reticulum. Chemical analysis confirmed that the Si in the organelles did not originate from instrumental contaminants. This suggests that Si may be involved in the biochemistry of these subcellular organelles.Supported by Grant GM-08229-12-13 from the National Institutes of Health, USPHS.We are grateful to the Kevex Corporation and Mr. Glenn W. Meyer, Sales Engineer, for the use of the Kevex X-ray spectrometer; we wish to thank as well the Perkin-Elmer Corporation and their Western Branch Manager, Mr. Michael E. Mullen and Senior Microscopist, Mr. Minoru Shinorhara, for use of and assistance with the Hitachi HU 12 A transmission electron microscope. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and the X-ray photographs, and Darlene Lum for technical assistance in the laboratory.     

Strain distribution and linkage relationship of a mouse embryonic hemoglobin variant

Search for structural variants of three globin chains (x, y, z), synthesized only during mouse embryonic hematopoiesis, was carried out by electrophoretic analysis of blood from 12-day embryos, all with C57BL/6 mothers, and fathers from 115 inbred stocks selected for their diverse genetic origins. Structure of the -chains of adult hemoglobins differed among the tested strains, with 57 carrying the Hbb ^sallele, 56 the Hbb ^dallele, and two the Hbb ^pallele. The search revealed no x- or z-chain variants but confirmed and extended knowledge of a previously described y-chain variant. Blood of all embryos sired by males from the 57 Hbb ^sstrains contained only y¹-chains, while blood of all embryos sired by Hbb ^dor Hbb ^pmales contained y2-chains as well as the y¹-chains inherited from their C57 BL/6 mother. The locus controlling structure of the y-chain of mouse embryonic hemoglobins is thus extremely closely linked to the locus controlling structure of adult hemoglobin -chain, with maximum possible recombination frequency less than 0.019.This work was supported in part by Grants CA-01074 from the National Cancer Institute, USPHS, and GM 18684 from the National Institute of General Medical Sciences, in part by Grant ACS-VC58 from The American Cancer Society, in part by grants to the Jackson Laboratory from the Bushrod H. Campbell and Adah F. Hall Charity Fund and the Robert Sterling Clark Foundation, and in part by the Jackson Laboratory Endowment Fund. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Interaction of fibronectin-coated beads with attached and spread fibroblasts : Binding, phagocytosis, and cytoskeletal reorganization

After 15 min incubations, binding of 0.8-, 6-, and 16-microns fibronectin-coated latex beads occurred primarily at the margins of chick embryo fibroblasts that previously were attached and spread on fibronectin-coated glass coverslips. Extensive phagocytosis of the smallest beads and some phagocytosis of the larger beads occurred within 2 h. Following binding of the 16-micron beads, there were no changes in overall cell shape or in the distribution of several cytoskeletal proteins. There was, however, a local accumulation of actin and alpha-actinin patches adjacent to the sites where the beads were bound. The formation of alpha-actinin patches could be detected with 6- or 16-microns beads shortly after initial bead binding to the cells, but a similar reorganization of alpha-actinin in response to the binding of 0.8-micron beads was not detected. The patches of alpha-actinin appeared to be associated with membrane ruffles, since such structures were observed by scanning electron microscopy (SEM) to be sites of cell interaction with 6- but not 0.8-micron beads. Also, two other cytoskeletal proteins normally absent from membrane ruffles, tropomyosin and vinculin, were not detected at the sites of cell-bead interaction. No reorganization of vinculin at the cell-bead interaction sites was observed even when the 16-microns beads remained bound at the cell surfaces for up to 6 h. Nevertheless, prominent vinculin plaques were observed at the marginal attachment sites on the ventral cell surfaces. Consequently, formation of mature focal adhesions may be restricted to linear regions of cell-substratum interaction.     

Autochthonous fungi are central components in microbial community structure in raw fermented sausages

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.     

Alteration of poly(adenosine diphosphoribose) metabolism by ethanol: mechanism of action

We present evidence that ethanol alters intracellular poly(adenosine diphosphoribose) metabolism and we further describe the mechanism by which ethanol exerts its effect on polymer synthesis. One percent ethanol stimulates polymer accumulation as much as 2.5-fold but does not alter polymer degradation in intact cells following DNA damage. Ethanol directly stimulates polymer synthesis following low doses of DNA damage induce by deoxyribonuclease I in a nucleotide-permeable cell system that does not possess a functional polymer turnover system. Ethanol has no measurable effect on polymer synthesis in undamaged nucleotide-permeable cells or in permeable cells treated with high doses of deoxyribonuclease I. Ethanol concentrations that stimulate poly(adenosine diphosphoribose) polymerase activity in vitro specifically lower KDNA without affecting KNAD or Vmax. The results clearly show that ethanol alters the binding of this enzyme to the DNA component of chromatin and that this altered binding is responsible for the activation of the enzyme. Altered affinity of poly(adenosine diphosphoribose) polymerase and perhaps other regulatory proteins for chromatin may play an important role in the pathology of alcohol.     

Modification of an arginine residue essential for the activity of NAD-malic enzyme from Ascaris suum

Purified NAD-malic enzyme from Ascaris suum is rapidly inactivated by the arginine reagent, 2,3-butanedione, and this inactivation is facilitated by 30 mM borate. Determination of the inactivation rate as a function of butanedione concentration suggests a second-order process overall, which is first order in butanedione. A second-order rate constant of 0.6 M-1 s-1 at pH 9 is obtained for the butanedione reaction. The inactivation is reversed by removal of the excess reagent upon dialysis. The enzyme is protected against inactivation by saturating amounts of malate in the presence and absence of borate. The divalent metal Mg2+ affords protection in the presence of borate but has no effect in its absence. The nucleotide reactant NAD+ has no effect on the inactivation rate in either the presence or absence of borate. A dissociation constant of 24 mM is obtained for E:malate from the decrease in the inactivation rate as a function of malate concentration. An apparent Ki of 0.5 mM is obtained for oxalate (an inhibitor competitive vs malate) from E:Mg:oxalate while no significant binding is observed for oxalate using the butanedione modified enzyme. The pH dependence of the first-order rate of inactivation by butanedione gives a pKa of 9.4 +/- 0.1 for the residue(s) modified, and this pK is increased when NAD is bound. The arginine(s) modified is implicated in the binding of malate.     

Growth line deposition and variability in growth of two circumpolar bivalves (Serripes groenlandicus, and Clinocardium ciliatum)

Growth patterns of two common circumpolar bivalves, the Greenland cockle (Serripes groenlandicus), and the hairy cockle (Clinocardium ciliatum) have been used in previous studies to reconstruct environmental conditions in the arctic. To date, there has been no direct determination that growth lines in either species are deposited periodically, and there has been no examination of factors affecting growth. We placed calcein-marked individuals of both species on oceanographic moorings in two fjords (Rijpfjord and Kongsfjord) in the Svalbard archipelago for one and two (Kongsfjord only) years. Growth patterns were compared with concurrent in situ temperature and fluorescence data in order to assess environmental controls on growth. Dark growth lines are evident on the outer shell surface and internally in shell cross section in both S. groenlandicus and C. ciliatum, and both species deposited only one line per year, unequivocally confirming that internal lines are deposited annually. Growth line deposition in both species began in late summer to early fall, before the seasonal decline in temperature. There was no difference in growth of S. groenlandicus between the two fjords despite differences in water temperature (3°C), fluorescence (nearly threefold) and the onset and duration of the winter season. C. ciliatum, however, grew approximately 2.8 times faster in the warmer, more food-rich Kongsfjord than in Rijpfjord. Subannual lines were counted in two individuals of each species from each fjord, but deposition of these lines was not clearly related to number of growing days estimated by temperature and fluorescence.