A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells

We have shown that the cultured Sertoli cell from the immature rat contains a fibroblast growth factor (FGF)-like factor. It behaves as a cationic peptide, is a potent competence factor for BALB/c3T3 mouse embryo fibroblasts, and displays a high affinity for heparin. Both bovine basic FGF and Sertoli cell FGF-like factor rapidly increase c-fos mRNA in cultured Sertoli cells. FSH, serum, and phorbol esters individually stimulate c-fos in cultured Sertoli cells whereas platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-I have little affect. However, unlike FSH, basic FGF does not stimulate an increase in cAMP and unlike either serum or phorbol esters, basic FGF does not stimulate phosphoinositol turnover or intracellular calcium changes. When Sertoli cell protein kinase C activity is suppressed by preexposure to phorbol ester, basic FGF continues to be a potent stimulator of c-fos, indicating that the calcium/phospholipid pathway is not involved in FGF induction. Basic FGF and FSH also increase jun-B mRNA levels in cultured Sertoli cells. In response to FGF, jun-B is more transiently increased than c-fos. In contrast, in response to FSH, jun-B persists longer than c-fos. These results indicate that cultured Sertoli cells contain a FGF-like factor that increases c-fos mRNA via a mechanism not involving cAMP and the calcium/phospholipid pathways. The different responsiveness of c-fos and jun-B to FSH and basic FGF may explain differences in the ultimate actions of these two ligands.     

Intercellular Effects on Development of Competence in Bacillus subtilis

The intercellular transfer of competence during growth under the conditions specified by the transformation procedure of Spizizen was investigated with Bacillus subtilis 168. The rate of competence development as assayed uniformly in medium B was not affected by variations in the cell concentration, although the first appearance of transformants occurred earlier with high cell densities in medium A, approximately in proportion to the onset of the stationary phase in the culture. Growth in the presence of Pronase enhanced the frequency of transformation, but did not detectably alter the kinetics of competence development. The rate of competence increase in physiologically noncompetent cultures was not changed by mixing with competent cultures either in medium A or in medium B; however, an early appearance of transformants was noted in mixed cultures in which the proportion of competent to noncompetent cells prevented exponential growth of the noncompetent strain. These experiments indicate that the normal development of competence in B. subtilis is not mediated by a soluble or loosely bound protein factor capable of transmitting competence directly via cell contact. The onset of competence is thus a function of internal physiological changes which are induced by the overall metabolic state of the culture.     

Chemical signals that regulate mammalian oocyte maturation

The nature and functions of the chemical signals involved in the acquisition of competence to resume meiosis, and the maintenance of meiotic arrest in antral follicles are the subjects of this paper. Evidence indicating that gonadotropins are not required for the development of competence to undergo spontaneous maturation is discussed. However, gonadotropins may promote optimal conditions for oocyte development via an estrogen-dependent action on follicular cells. Evidence for the participation of cyclic AMP (cAMP), steroids and a putative maturation-inhibiting factor in the maintenance of meiotic arrest in mammalian oocytes is discussed. Cyclic AMP seems to play a critical role in the maintenance of meiotic arrest by actions in both granulosa/cumulus cells and oocytes. However, cAMP does not appear to equilibrate between cumulus cells and oocytes. In the granulosa/cumulus cells, cAMP may promote the generation/activation of a maturation-inhibiting factor which is transferred to the oocyte. Oocyte cAMP appears to be produced in the oocyte itself. The putative maturation-inhibiting factor may be maintained in an active form by a cAMP-dependent process in the oocyte. Alternatively, the putative maturation-inhibiting factor may play a role in maintaining oocyte cAMP levels. Some steroid hormones act synergistically with a cAMP-dependent process in the oocyte to maintain meiotic arrest. However, the physiological significance of this observation remains in question.     

Yeast competence for exogenous DNA uptake: towards understanding its genetic component

The demonstration of spontaneous yeast competence shows that artificial transformation rests on naturally occurring cellular processes. Such natural competence is either biologically mediated or environmentally induced. For instance, wild yeast might be transformed through conjugation by cell-to-cell contact mediated either by Escherichia coli or Agrobacterium tumefaciens. Moreover, natural competence can be enhanced by mechanical and physiological mechanisms. On the other hand, artificial yeast competence is usually achieved by biological, chemical and physical manipulations. These eliminate or weaken natural obstacles blocking the way of the transforming DNA in order to mitigate its entrance into the cell (biological and chemical approach) or simply to bridge it by either electrical or biolistic force (physical). Thus yeast competence is controlled by intrinsic (genetic) and external (environmental) factors. Both intrinsic and external parameters affecting yeast competence were scrutinized leading to the identification of genes and biological processes participating in the phenomenon. Therefore natural yeast competence is a complex, quantitative genetic trait which may have allowed yeast to better adapt over evolutionary times.     

Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O

Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.     

Signal transduction in Halobacterium depends on fumarate.

The isolation of a straight-swimming mutant of Halobacterium halobium is reported which has a defect in switching the rotational sense of its flagellar motor. Cells of this mutant strain could be complemented with an extract from wild-type cells by mild sonication and resealing of the cells in fresh medium. The switch factor responsible for restoration of wild-type behaviour was isolated from membrane vesicle preparations. Its chemical nature is proposed to be that of fumarate on the basis of chemical, chromatographic and mass spectrometric analysis. Since the switch factor (fumarate) was released from a membrane-bound state by heat and was accumulated into mutant cells that lack this compound, it is proposed that a membrane-bound protein exists which specifically binds the switch factor. Both the switch factor and fumarate cause stimulus-induced responses in cells at the level of one or few molecules.     

Analysis of the mechanism(s) of metaphase I-arrest in strain LT mouse oocytes: delay in the acquisition of competence to undergo the metaphase I/anaphase transition.

Fully grown oocytes of most laboratory mice progress without interruption from the germinal vesicle (GV) stage to metaphase II, where meiosis is arrested until fertilization. In contrast, many oocytes of strain LT mice arrest precociously at metaphase I and often undergo subsequent spontaneous parthenogenetic activation. Cytostatic factor (CSF), which prevents the degradation of cyclin B and maintains high maturation-promoting factor (MPF) activity, is required for maintenance of metaphase I-arrest in LT oocytes, similar to its requirement for maintaining metaphase II-arrest in normal oocytes. However, CSF does not instigate metaphase I-arrest since a temporary metaphase I-arrest occurs in MOS-null LT oocytes. This paper addresses the mechanism(s) that may instigate metaphase I-arrest and tests the hypothesis that there may be one or more defects in LT oocytes that delay their acquisition of competence to trigger the cascade of processes that normally drive entry into and progression through anaphase I. To test this hypothesis, MPF activity was artificially abrogated by treating oocytes with a general protein kinase inhibitor, 6-DMAP, at various times during the progression of meiosis I. This allowed a comparison of the time at which LT and normal oocytes become competent to undergo the metaphase I/anaphase transition even if oocytes were arrested at metaphase I when 6-DMAP-treatment was begun. There were no differences between LT and control oocytes in the kinetics of MPF suppression by 6-DMAP. However, it was found that LT oocytes do not acquire competence to undergo the metaphase I/anaphase transition in response to 6-DMAP until 50-60 min after normal oocytes. A similar delay was observed in strain CX8-4 oocytes, which also have a high incidence of metaphase I-arrest, but not in strain CX8-11 oocytes, which exhibit a low incidence of metaphase I-arrest. MOS-null LT oocytes also exhibit a delay in acquisition of competence to undergo the metaphase I/anaphase transition. Thus, a delay in competence to undergo the metaphase I/anaphase transition in response to 6-DMAP-treatment correlates with metaphase I-arrest. It is therefore hypothesized that the observed delay in acquisition of competence to enter anaphase I may instigate the sustained metaphase I-arrest in LT oocytes by allowing CSF activity to rise to a level that prevents cyclin B degradation and maintains high MPF activity before anaphase can be initiated by normal triggering mechanisms.     

Autogenous arteriovenous elbow fistula for haemodialysis and upper extremity ischemia

BACKGROUND: The autogenous brachiocephalic or brachiobasilic arteriovenous elbow fistula is not considered to be only the secondary haemodialysis access. In patients with an unsuitable forearm vessel bundle, it is indicated as primary access and it is the method preferred to the fistula creation using a vascular prosthesis. Its rather rare complication is the development of upper extremity ischemia. AIM: To summarise current knowledge of this fistula type and its associated complications METHODS: Review of the literature. RESULTS: The creation and maturation of the fistula and occurrence of the steal syndrome is influenced by a number of factors. The analysis and awareness of such factors will provide for creation of a suitable fistula as well as for timely complication diagnostics and treatment. CONCLUSIONS: The autogenous elbow fistula utilising the brachial artery and the cephalic or basilic vein in the upper extremity represents a high-quality haemodialysis access. Its potential complication is the occurrence of the steal syndrome. Its occurrence and manifestations do not constitute indications for ligation of the access. The gathered information shows that a suitable surgical procedure can help meet the basic rule for haemodialysis access--resolving the ischemia and maintaining the access.     

Studies on Transformations of Hemophilus influenzae : I. Competence

A procedure has been developed for obtaining Hemophilus influenzae of such competence that 1 to 10 per cent transform to any of several genetic factors by utilizing a period of aerobic growth followed by a non-aerobic period. Differences in levels of competence were not due to differences in genetic background. Competence was due to at least one factor intrinsic to the cell or site on the cell and was not transferable to non-competent cells. Competence was affected by salt concentration, pH, and temperature. Washing competent cells reduces their ability to transform, but not their capacity to bind DNA reversibly. The irreversible step could be restored with little or no accompanying growth. These facts suggest that reversible and irreversible binding represent separate biochemical steps. DNA initiates a reaction in cells leading to a loss of competence. In the absence of DNA the cells remain competent for at least an hour. Competence correlates quantitatively with predictability of multiple transformations. The observed and calculated values of multiple transformations are in closer agreement, the higher the frequency of transformation for single markers. The correction needed to bring the two figures into agreement is a measure of the fraction of non-competent cells.     

Effect of Competence Induction on Macromolecular Synthesis in a Group H Streptococcus

The effects of competence induction by competence factor (CF) on macromolecular synthesis in group H streptococcus strain Wicky were investigated. CF preparations (culture filtrates from competent group H streptococcus strain Challis) were either heated or partially purified to remove a bacteriocin. These preparations did not inhibit growth, although they induced high levels of competence in strain Wicky. The action of the CF preparations did not affect the overall rates of deoxyribonucleic acid and protein synthesis, but caused a reduction in the rates of ribonucleic acid (RNA) and peptidoglycan synthesis. When competence induction by CF was prevented, no alterations in RNA or peptidoglycan synthesis were observed, indicating that these changes are in fact related to the development of competence.     

Analysis of competence: receptors for fibroblast growth factor in early Xenopus embryos

Xenopus ectodermal cells have previously been shown to respond to acidic and basic FGF by differentiating into mesodermal tissue. In the present study, ectodermal explants from Xenopus blastulae were shown to have high affinity binding sites for 125I-aFGF (Kd = 1.4 X 10(-10) M). The total number of sites, determined by Scatchard analysis, was 3 X 10(8) per explant (surface area of approximately 1 mm2). Two putative receptors of relative molecular mass 130,000 and 140,000 were identified by chemical crosslinking to 125I-aFGF. Both acidic and basic FGF, but not TGF beta 2, could compete for affinity labelling of these bands. The receptor density at the cell surface parallels the developmental competence of Xenopus animal pole cells to respond to FGF. Receptors are present at highest density in the marginal zone but are not restricted to cells in this region.     

Natural transformation of Gallibacterium anatis

Gallibacterium anatis is a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function in G. anatis, we have established methods for transformation and targeted mutagenesis. The genus Gallibacterium belongs to the Pasteurellaceae, a group with several naturally transformable members, including Haemophilus influenzae. Bioinformatics analysis identified G. anatis homologs of the H. influenzae competence genes, and natural competence was induced in G. anatis by the procedure established for H. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies with G. anatis chromosomal DNA and with linearized plasmid DNA carrying G. anatis sequences. Both DNA types integrated into the G. anatis chromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10(-4) transformants CFU(-1). Transformation was also efficient with circular plasmid containing no G. anatis DNA; this resulted in the establishment of a self-replicating plasmid. Nine diverse G. anatis strains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. The G. anatis genome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, but G. anatis did preferentially take up its own DNA over that of Escherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation of G. anatis.     

Tumor-degenerating factor (TDF)]

A new human fibroblast-producing factor which degenerates human tumor cells in vitro was found and designated as tumor-degenerating factor (TDF). The characteristics of the TDF were as follows: 1) The molecular weight of the TDF was estimated to be 30k by SDS-PAGE. 2) It may be basic and glycosylated. 3) Its activity was inhibited by fibronectin and, vice versa, by binding TDF molecule with fibronectin molecule. 4) Its activity was enhanced by interferons. It is possible that the TDF is an important substance concerning the interaction between tumor cells and interstitial cells.     

Gene expression analysis of the Streptococcus pneumoniae competence regulons by use of DNA microarrays

Competence for genetic transformation in Streptococcus pneumoniae is coordinated by the competence-stimulating peptide (CSP), which induces a sudden and transient appearance of competence during exponential growth in vitro. Models of this quorum-sensing mechanism have proposed sequential expression of several regulatory genes followed by induction of target genes encoding DNA-processing-pathway proteins. Although many genes required for transformation are known to be expressed only in response to CSP, the relative timing of their expression has not been established. Overlapping expression patterns for the genes cinA and comD (G. Alloing, B. Martin, C. Granadel, and J. P. Claverys, Mol. Microbiol. 29:75-83, 1998) suggest that at least two distinct regulatory mechanisms may underlie the competence cycle. DNA microarrays were used to estimate mRNA levels for all known competence operons during induction of competence by CSP. The known competence regulatory operons, comAB, comCDE, and comX, exhibited a low or zero initial (uninduced) signal, strongly increased expression during the period between 5 and 12 min after CSP addition, and a decrease nearly to original values by 15 min after initiation of exposure to CSP. The remaining competence genes displayed a similar expression pattern, but with an additional delay of approximately 5 min. In a mutant defective in ComX, which may act as an alternate sigma factor to allow expression of the target competence genes, the same regulatory genes were induced, but the other competence genes were not. Finally, examination of the expression of 60 candidate sites not previously associated with competence identified eight additional loci that could be induced by CSP.     

Two distinct functions of ComW in stabilization and activation of the alternative sigma factor ComX in Streptococcus pneumoniae

Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX (sigma(X)), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the sigma(X) protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of sigma(X). As sigma(X) is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of sigma(X) depends on ComW. The clpP comW double mutant accumulated an amount of sigma(X) higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for sigma(X) synthesis, it acts both in stabilization of sigma(X) and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of sigma(X), but only the N terminus is important for stimulating its activity.     

Persistence of the competent state in mouse fibroblasts is independent of c-fos and c-myc expression

Induction of competence in quiescent fibroblasts by platelet-derived growth factor (PDGF) is accompanied by a dramatic increase in the expression of c-fos and c-myc genes. However, the maintenance of the competent state and progression through G1 does not require high expression of these proto-oncogenes. These results suggest that the induction of c-fos and c-myc by growth factors in quiescent fibroblasts may be required to render the cells competent for progression.     

Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth

Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

What is the role of predation on stability of natural communities? A theoretical investigation.

A basic question in ecology concerns the role of species interaction on dynamics of natural communities. In this framework, ecologists have considered predation, competition, mutualism, the three most important interactions, highlighting their specific effects on distribution and abundance of species, providing knowledge about phenomena like coexistence and extinction. This paper seeks to identify the effects of predation on stability of natural communities by mathematical models. Simple multispecies community models, organized in trophic levels, are analyzed by means of a qualitative technique, loop analysis, combined with a computer calculation procedure. Results do not support the hypothesis of predation as a stabilizing factor. Rather, the outcomes of the analysis suggest that predation may or may not stabilize a community. This depends on the predator's behaviour and on the network of the community.     

Phosphorylation of basic fibroblast growth factor by a protein kinase associated with the outer surface of a target cell.

A protein kinase capable of phosphorylating basic fibroblast growth factor (FGF) can be localized on the outer cell surface of human hepatoma cells (SK-Hep cells). The addition of [gamma-32P]ATP, but not H3(32)PO4, results in a rapid (less than 10 min) incorporation of 32P into exogenously added basic FGF. The reaction is time and concentration dependent (apparent Km, 170 nM) and is stimulated by the addition of cAMP (EC50, 0.5 microM), but not the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. There is also no tyrosine protein kinase detected on the cell surface. The inhibition of basic FGF binding to its low and/or high affinity sites decreases the phosphorylation of basic FGF by the ecto-protein kinase. Accordingly, pretreatment of cells with heparinase for 30 min or coincubation with heparin (0.1-10 micrograms/ml) decreases phosphorylation in a dose-dependent manner. Furthermore, the addition of a nonphosphorylatable peptide analog of basic FGF ([Val112] basic FGF-(106-146)NH2) that can compete with basic FGF binding to cells prevents the phosphorylation of basic FGF. Together, these observations suggest that 1) exogenous basic FGF must associate with its low and/or high affinity binding sites to be phosphorylated, and 2) the kinase is cAMP dependent and associated with the outer cell surface, and support the hypothesis that phosphorylation may regulate the activity and/or bioavailability of the growth factor.