Expression of the Distalless-B gene in Ciona is regulated by a pan-ectodermal enhancer module

The Ci-Dll-B gene is an early regulator of ectodermal development in the ascidian Ciona intestinalis (Imai et al., 2006). Ci-Dll-B is located in a convergently transcribed bigene cluster with a tandem duplicate, Ci-Dll-A. This clustered genomic arrangement is the same as those of the homologous vertebrate Dlx genes, which are also arranged in convergently transcribed bigene clusters. Sequence analysis of the C. intestinalis Dll-A-B cluster reveals a 378 bp region upstream of Ci-Dll-B, termed B1, which is highly conserved with the corresponding region from the congener Ciona savignyi. The B1 element is necessary and sufficient to drive expression of a lacZ reporter gene in a pattern mimicking the endogenous expression of Ci-Dll-B at gastrula stages. This expression pattern which is specific to the entire animal hemisphere is activated preferentially in posterior, or b-lineage, cells by a central portion of B1. Expression in anterior, or a-lineage cells, can be activated by this central portion in combination with the distal part of B1. Anterior expression can also be activated by the central part of B1 plus both the proximal part of B1 and non-conserved sequence upstream of B1. Thus, cis-regulation of early Ci-Dll-B expression is activated by a required submodule in the center of B1, driving posterior expression, which works in combination with redundant submodules that respond to differentially localized anterior factors to produce the total animal hemisphere expression pattern. Interestingly, the intergenic region of the cluster, which is important for expression of the Dlx genes in vertebrates, does not have a specific activating function in the reporter genes tested, but acts as an attenuator in combination with upstream sequences.     

Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain

Aims:  The aim of this study was to purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a strain previously isolated from human gut.
Methods and Results:  Purification of the bacteriocin was performed by cationic exchange chromatography followed by a reverse phase step. Biochemical and mass spectrometry analysis showed homology with pediocin PA-1. To verify if P. acidilactici MM33 carried the pediocin PA-1 gene, total DNA was used to amplify the pediocin gene. The PCR product obtained was then sequenced and the nucleotide sequence revealed to be identical to that of pediocin PA-1. Treatment of P. acidilactici MM33 with novobiocin resulted in a plasmid-cured strain without bacteriocin-producing capacity. Antimicrobial assay and molecular analysis demonstrated that this strain was ped ⁻ suggesting that the ped cluster is plasmid encoded. Antimicrobial assay revealed that pediocin was bactericidal against Listeria monocytogenes , showing a minimal inhibitory concentration (MIC) of 200 AU ml⁻¹.
Conclusions:  A two-step purification procedure was elaborated in this study. The bacteriocin secreted by the human strain P. acidilactici MM33 is carried on a plasmid and the amino acid sequence is identical to pediocin PA-1.
Significance and Impact of the Study:  Pediococcus acidilactici MM33 is the first human pediocin-producing strain reported and could be used as probiotic to prevent enteric pathogen colonization.     

Effect of gamma radiation on heat shock protein expression of four foodborne pathogens

Aims: The effects of gamma radiation on three heat shock proteins (Hsps) (GroEL, DnaK and GroES) synthesis in two Gram-negative (Escherichia coli and Salmonella serotype Typhimurium) and two Gram-positive (Staphylococcus aureus and Listeria monocytogenes) bacteria were investigated. Methods and Results: The bacterial strains were treated with three radiation doses to induce cell damage, to obtain a viable but nonculturable state, and to cause cell death. Western blot analysis and quantification of Hsps in bacteria were performed immediately after irradiation treatment. In the four foodborne pathogens, GroEL was strongly induced by gamma rays in a dose-dependent manner, confirming the involvement of this protein in the cellular response to the stress generated by ionizing radiation. In addition, it was found that E. coli exposed to gamma radiation showed a significantly induction of DnaK and GroES proteins when compared with nonirradiated bacteria, whereas a GroES slight induction and a DnaK inhibition were observed in Salm. Typhimurium. Conclusions: The gamma rays influence the synthesis of Hsps in foodborne pathogen in a way that critically depends on the radiation dose. Significance and Impact of the Study: The study of stress response to several radiation doses was undertaken to elucidate how bacteria can survive in harsh conditions and cope with gamma radiation used to control foodborne pathogens and to characterize their adaptative response to this treatment.     

Quantitation of macromolecular binding using size exclusion filters: application to a fucosyltransferase assay

A method to quantify the covalent attachment of small radiolabeled substrates to macromolecules on the basis of molecular weight using size exclusion filters in an Amicon Centrifree micropartition system is described. GDP-[14C]-L-fucose was covalently attached to asialofetuin in a fucosyltransferase reaction catalyzed by mouse spermatogenic cell extracts. Radiolabeled product was separated from unreacted substrate by centrifuging 200-400 microliter of cell extract through a 10-kDa size exclusion filter at 1000 g for 10 to 20 min. After 10 washes with an appropriate buffer, no detectable radioactivity was found in the eluant and the membrane-bound radiolabeled product was counted in a scintillation vial. Using this method the fucosyltransferase activity of mouse spermatogenic cells was approximately 17 pmol/mg protein/min which is essentially identical to values obtained using size exclusion chromatography. This technique provides a rapid, efficient, and inexpensive alternative for the isolation and detection of acceptor-substrate complexes.     

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.     

Benefits of increased colonist quantity and genetic diversity for colonization depend on colonist identity

Larger numbers of colonists can be more likely to establish and spread due to the benefits provided by either more individuals (quantity) or a greater diversity of genotypes or phenotypes (genetic diversity). However, the value of higher colonist quantity or genetic diversity varies widely across studies, leaving a great deal of uncertainty in how these respective mechanisms affect colonization success. This variability is potentially driven by differences in which traits are present in respective colonist pools (‘colonist identity’). Studies with high‐performing colonizers (e.g. genotypes pre‐adapted to the colonizing environment) may find increasing quantity or diversity to be beneficial because it increases the chance high‐performers are sampled, while studies with no high‐performers may find no effects of quantity or diversity. Alternatively, quantity and genetic diversity may play little to no role if the smallest populations already contain high‐performing colonists because there is no scope for a sampling effect to operate. We conducted a field mesocosm experiment to determine if variability in the benefits provided by increased quantity or genetic diversity relates to colonist traits. Nine distinct genotypes of Daphnia pulex characterized also by phenotype, were introduced in ‘single’ (one individual) or ‘many’ (nine individuals) introduction quantities and at ‘low’ (monoclonal) and ‘high’ (mixed genotypes) genetic diversities. We found that larger‐bodied D. pulex genotypes benefited less from increased colonist quantity, while increasing genetic diversity tended to have a lower effect on higher growth rate genotypes. Our results show that the trait values of the colonists can determine the benefits gained when colonist quantity or genetic diversity are increased, with potential applications to future research and practical efforts to promote, or prevent, population establishment.     

Mutational analysis of Glu-327 of Na+-K+-ATPase reveals stimulation of 86Rb+ uptake by external K+

A competition assay of⁸⁶Rb⁺uptake in HeLa cells transfected with ouabain-resistantNa⁺-K⁺-ATPasemutants revealed a stimulation of⁸⁶Rb⁺uptake at low external concentrations (1 mM) of competitor(K⁺). Of the models that weretested, those that require that two K⁺ be bound before transportoccurs gave the worst fits. Random and ordered binding schemesdescribed the data equally well. General models in which both bindingand transport were allowed to be cooperative yielded parameter errorslarger than the parameters themselves and could not be utilized. Modelsthat assumed noncooperative transport always showed positivecooperativity in binding. E327Q and E327L mutated forms of rat₂ had lower apparent affinities for the first K⁺ bound than didwild-type rat ₂ modified to beouabain resistant. The mutations did not affect the apparent affinityof the second K⁺ bound. Modelsthat assumed noncooperativity in binding always showed positivelycooperative transport, i.e., enzymes with two K⁺ bound had a higher flux thanthose with one K⁺ bound. Increasesin external Na⁺ decreased theapparent affinity for K⁺ for allmodels and decreased the ratio of the apparent influx rate constantsfor E327L.

     

The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.     

Stage-specific synthesis and fucosylation of plasma membrane proteins by mouse pachytene spermatocytes and round spermatids in culture

Little is known about the ability of mammalian spermatogenic cells to synthesize plasma membrane components in the presence or absence of Sertoli cells. In this study, purified populations (greater than 90%) of pachytene spermatocytes or round spermatids were isolated by unit gravity sedimentation and cultured for 20-24 h in the presence of [35S]methionine or [3H]fucose. Cell viabilities remained over 90% during the course of these experiments. Plasma membranes were purified from these cells and analyzed by two-dimensional gel electrophoresis. Qualitatively, the same plasma membrane proteins were synthesized by both cell types with the exception of the major Concanavalin A-binding glycoprotein, p151; the synthesis of p151 is greatly diminished or inhibited after meiosis. [3H]Fucose was incorporated into at least 6 common glycoproteins of both cells. Eight components fucosylated with molecular weights from 35,000 to 120,000 were specific to pachytene spermatocyte membranes. One fast-migrating fucosylated component may represent an uncharacterized lipid whose synthesis is terminated after meiosis. Round spermatids specifically fucosylated two components with molecular weights of 45,000 and 80,000. These results demonstrate the viability of germ cells of the male mouse in short-term culture and show that they are capable of synthesizing and fucosylating plasma membrane components in the absence of Sertoli cells.     

Immunochemical identification of multiple cell surface antigens appearing during specific stages of mouse spermatogenesis

Cell surface antigens that appear in a defined temporal sequence during mouse spermatogenesis were previously detected serologically, but not identified biochemically, with four heterologous antibodies prepared against purified populations of pachytene spermatocytes (AP), round spermatids (ARS), vas deferens spermatozoa (AVDS), and mixed seminiferous cells (ASC) [Millette and Bellvé, J Cell Biol 74:86–97, 1977]. These antigens have now been identified immunochemically on nitrocellulose blots from SDS polyacrylamide gels. Three antisera (AP, ARS, and ASC) recognize a similar subset of determinants on one-dimensional immunoblots of germ cells and plasma membranes prepared from a mixed population of late spermatogenic cells. Comparisons of minor bands to reveal differences among these antisera. AVDS exhibits the least complex binding pattern. The results indicate that at least ten surface constituents appear during the pachytene stage of meiosis, coincident with a period of maximal RNA and protein synthesis [Monesi, Exp Cell Res 39:197–224, 1965]. Furthermore, two-dimensional immunoblot comparisons of plasma membranes isolated from pachytene spermatocytes and round spermatids reveal differences between surface determinants detectable at these two spermatogenic stages. For example, ASC recognizes two newly described proteins that are restricted to pachytene spermatocytes (? M_r 57,000, pI 6.45) and to round spermatids (? M_r 39,500, pI 4.85), respectively.