Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method

Survival and proliferation of mouse gonocytes was studied using a single cell clonogenic assay in vitro. The effect of growth factors and extracellular matrix on clonogenic development was quantitated. Fundamental requirements for growth of neonatal gonocytes included addition of fetal calf serum and coating culture wells with collagen IV alone or with added fibronectin. After 4-5 days, colonies ranged in size from four to > 128 cells, and some contained very elongated cells indicating migratory behaviour. Soluble stem cell factor did not have any effect on clonogenicity, although STO (subline of SIM mouse fibroblasts) cells, which produce membrane-bound stem cell factor, reduced colony formation from 79 +/- 5.9% to 20 +/- 3.3% without added growth factor. The majority of gonocytes and type A spermatogonia express the c-kit receptor according to in situ hybridization studies. However, the results indicate that the receptor may not be functional in neonatal gonocytes and their immediate progeny. The current assay for gonocytes can be extended to test new growth factors or proliferation-inducing agents directly, as well as to study cell-cell interactions. This assay and long-term propagation of neonatal germ cells will provide the much needed resources to enable greater understanding of the early development of germ cells.     

Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 microm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35 degrees C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H(2), formate, L-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H(2)) are oxidized to acetate and CO(2). When L-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g., Desulfitobacterium sp. strain PCE1 and Desulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 micromol of chloride released. min(-1). mg of protein(-1)). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.     

Roles for cysteine residues in the regulatory CXXC motif of human mitochondrial branched chain aminotransferase enzyme

The redox-active dithiol/disulfide C315-Xaa-Xaa-C318 center has been implicated in the regulation of the human mitochondrial branched chain aminotransferase isozyme (hBCATm) [Conway, M. E., Yennawar, N., Wallin, R., Poole, L. B., and Hutson, S. M. (2002) Biochemistry 41, 9070-9078]. To explore further the mechanistic details of this CXXC center, mutants of the Cys residues at positions 315 and 318 of hBCATm were individually and in combination converted to alanine or serine by site-directed mutagenesis (C315A, C315S, C318A, C318S, C315/318A, and C315/318S). The effects of these mutations on cofactor absorbance, secondary structures, steady-state kinetics, and sensitivity toward hydrogen peroxide (H(2)O(2)) treatment were examined. Neither the UV-visible spectroscopic studies nor the circular dichroism data showed any major perturbations in the structure of the mutants. Kinetic analyses of the CXXC mutant proteins indicated primarily a modest reduction in k(cat) with no significant change in K(m). The largest effect on the steady-state kinetics was observed with the C315 single mutants, in which substitution of the thiol group resulted in a reduced k(cat) (to 26-33% of that of wild-type hBCATm). Moreover, the C315 single mutants lost their sensitivity to oxidation by H(2)O(2). The kinetic parameters of the C318 mutants were largely unaffected by the substitutions, and as with wild-type hBCATm, reaction of the C318A mutant protein with H(2)O(2) resulted in the complete loss of activity. In the case of oxidized C318A, this loss was largely irreversible on incubation with dithiothreitol. Mass spectrometry and dimedone modification results revealed overoxidation of the thiol group at position 315 to sulfonic acid occurring via a sulfenic acid intermediate in the H(2)O(2)-treated C318A enzyme. Thus, C315 appears to be the sensor for redox regulation of BCAT activity, whereas C318 acts as the "resolving cysteine", allowing for reversible formation of a disulfide bond.     

Neural manifestations of memory with and without awareness

Neurophysiological events responsible for different types of human memory tend to occur concurrently and are therefore difficult to measure independently. To surmount this problem, we produced perceptual priming (indicated by speeded responses) in the absence of conscious remembering. At encoding, faces appeared briefly while subjects' attention was diverted to other stimuli. Faces appeared again in either an implicit or explicit memory test. Neural correlates of priming were identified as brain potentials beginning 270 ms after face onset with more negative amplitudes for repeated than for new faces. Remembered faces, in contrast, activated a different configuration of intracranial sources producing positive potentials maximal at 600-700 ms. We thus disentangled and characterized distinct neural events associated with memory with and without awareness.     

25-hydroxycholesterol is produced by testicular macrophages during the early postnatal period and influences differentiation of Leydig cells in vitro

Leydig cells develop inappropriately in animals lacking testicular macrophages. We have recently found that macrophages from adult animals produce 25-hydroxycholesterol, an oxysterol involved in the differentiation of hepatocytes and keratinocytes. Therefore, we hypothesized that testicular macrophages also produce 25-hydroxycholesterol during the early postnatal period and that this oxysterol plays a role in the differentiation of Leydig cells. We assessed the production of 25-hydroxycholesterol and 25-hydroxylase mRNA by cultured testicular macrophages from rats at 10, 20, and 40 days of age. We also tested the long-term effects of 25-hydroxycholesterol on basal and LH-stimulated testosterone production, and 3beta-hydroxysteroid dehydrogenase activity as end points of Leydig cell differentiation in vitro. We found that testicular macrophages from animals at all ages produced both 25-hydroxycholesterol and 25-hydroxylase mRNA, with macrophages from 10-day-old animals having the highest steady-state levels of message. We also found that chronic exposure of Leydig cells to 25-hydroxycholesterol increased basal production of testosterone but decreased LH-stimulated steroidogenesis at all ages. Finally, 25-hydroxycholesterol increased 3beta-hydroxysteroid dehydrogenase activity in both progenitor and immature Leydig cells. These findings support the hypothesis that testicular macrophages play an important role in the differentiation of Leydig cells through the secretion of 25-hydroxycholesterol.     

Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid aminotransferase.

Branched chain amino-acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU x mg(-1) protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41-kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43-kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41-kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Genetic interrelationships of proteolyticClostridium botulinum types A,B, and F and other members of theClostridium botulinum complex as revealed by small-subunit rRNA gene sequences

The phylogenetic interrelationships of members of theClostridium botulinum complex of species was investigated by direct sequencing of their 16S rRNA genes. Comparative analysis of the 16S rRNA sequences demonstrated the presence of four phylogenetically distinct lineages corresponding to: i) proteolyticC. botulinum types A, B, and F, andC. sporogenes, ii) saccharolytic types B, E and F, iii) types C and D andC. novyi type A, and iv) type G andC. subterminale. The phylogenetic groupings obtained from the 16S rRNA were in complete agreement with the four divisions recognised within the species complex on the basis of phenotypic criteria.     

Spatial patterns in population conflicts

We consider a dynamical model for evolutionary games, and enquire how the introduction of diffusion may lead to the formation of stationary spatially inhomogeneous solutions, that is patterns. For the model equations being used it is already known that if there is an evolutionarily stable strategy (ESS), then it is stable. Equilibrium solutions which are not ESS's and which are stable with respect to spatially constant perturbations may be unstable for certain choices of the dispersal rates. We prove by a bifurcation technique that under appropriate conditions the instability leads to patterns. Computations using a curve-following technique show that the bifurcations exhibit a rich structure with loops joined by symmetry-breaking branches.     

Considerations for establishing the validity of immunocytological studies.

Immunocytology has wide spread applications for localizing tissue antigens, as evidenced by the recent exploitation of this technique in biological studies. Documenting the immunological specificity of the staining reaction is one of the most important technical considerations in validating the accrued data in immunocytological studies. The purpose of this report is to discuss and emphasize the need for conducting physiological studies in addition to the traditional immunological method and specificity controls. The ability of antibodies to bind molecules other than those molecules used as the immunizing material is a well documented fact. Hypothetically, preabsorption of the primary antibody with its specific antigen, could reduce subsequent binding of this antibody to a cross reactive tissue antigen, thus providing false confirmation of staining validity. The results of our experience with a cross reacting system in addition to other previously reported examples are discussed.