Molecular analysis of the locus responsible for production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10.

A 4.5-kb region of chromosomal DNA carrying the locus responsible for the production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10 (R. Jiménez-Díaz, J. L. Ruiz-Barba, D. P. Cathcart, H. Holo, I. F. Nes, K. H. Sletten, and P. J. Warner, Appl. Environ. Microbiol. 61:4459-4463, 1995), has been cloned, and the nucleotide sequence has been elucidated. Two genes, designated plsA and plsB and encoding peptides alpha and beta, respectively, of plantaricin S, plus an open reading frame (ORF), ORF2, were found to be organized in an operon. Northern blot analysis showed that these genes are cotranscribed, giving a ca. 0.7-kb mRNA, whose transcription start point was determined by primer extension. Nucleotide sequences of plsA and plsB revealed that both genes are translated as bacteriocin precursors which include N-terminal leader sequences of the double-glycine type. The role of ORF2 is unknown at the moment, although it might be expected to encode an immunity protein of the type described for other bacteriocin operons. In addition, several other potential ORFs have been found, including some which may be responsible for the regulation of bacteriocin production. Two of them, ORF8 and ORF14, show strong homology with histidine protein kinase and response regulator genes, respectively, which have been found to be involved in the regulation of the production of other bacteriocins from lactic acid bacteria. A third ORF, ORF5, shows homology with gene agrB from Staphylococcus aureus, which is involved in the mechanism of regulation of the virulence phenotype in this species. Thus, an agr-like regulatory system for the production of plantaricin S is postulated.

Transplanted CG4 cells (an oligodendrocyte progenitor cell line) survive, migrate, and contribute to repair of areas of demyelination in X-irradiated and damaged spinal cord but not in normal spinal cord.

In this study, we have examined the behavior of a lac-Z-transfected O- 2A progenitor cell line, CG4, following transplantation into normal and X-irradiated adult rat spinal cord, and we have also addressed the issue of whether CG4 cells transplanted remotely from ethidium bromide-induced demyelinating lesions in both X-irradiated and nonirradiated spinal cord are able to contribute to their repair. Following transplantation into X-irradiated spinal cord, CG4 cells survive, divide, and migrate extensively. The migration occurs mainly within the parenchymal tissue of the cord without preference for white or gray matter. Moreover, CG4 cells migrating away from their point of introduction are able to enter areas of demyelination and remyelinate the demyelinated axons therein. In contrast, when CG4 cells are transplanted into nonirradiated spinal cord, their survival is limited to areas of damage created by the injection procedure. The CG4 cells do not survive in undamaged, nonirradiated spinal cord. When transplanted remotely from areas of demyelination they are unable to traverse intervening areas of normal white matter, although they may enter lesions if transplanted into their close vicinity. These results have important implications for the development of potential therapeutic strategies for the treatment of multifocal demyelinating disorders that are based on glial cell transplantation.

Differentiation of the O-2A progenitor cell line CG-4 into oligodendrocytes and astrocytes following transplantation into glia-deficient areas of CNS white matter.

The in vitro properties of the CG4 cell line have led to its increasing use as a cell line with which to study the behaviour of the O-2A progenitor cell. In this study we have examined the in vivo behaviour of the CG4 cell line following transplantation into areas of adult rat spinal cord white matter which have been permanently depleted of glial cells by the combination of local X-irradiation and direct injection of 0.1% ethidium bromide. Twenty-one days after transplantation, both myelin-forming oligodendrocytes and glial fibrillary acidic protein-positive astrocytes were identified within the lesion, indicating that the CG4 cell line has bipotential differentiation properties when introduced into a pathological environment consisting of demyelinated axons but devoid of oligodendrocytes or astrocytes. In this respect, the CG4 cell line resembles other glial progenitor cell lines that have been transplanted into similar lesions. In some areas of the lesion, remyelination was observed that was similar in extent to that achieved by growth factor-expanded populations of O-2A progenitor cells. The transplant origin of the cell types within the lesion was confirmed by retroviral incorporation of the lacZ marker gene, the expression of which allowed their identification by histochemistry. In conclusion, the in vivo properties of the CG4 cell line make it a highly suitable line with which to study the behaviour of O-2A progenitors following transplantation into normal and damaged CNS.

Transfection of rat dermal papilla cells with a gene encoding a temperature-sensitive polyomavirus large T antigen generates cell lines retaining a differentiated phenotype.

The dermal papilla is a discrete group of cells at the base of the hair follicle and is implicated in controlling the hair growth cycle. Early passage dermal papilla cells can induce hair growth in vivo, but, upon further culturing, this property is lost. In order to study the events occurring in hair induction, a representative dermal papilla cell line was required. We have transfected passage 1 rat vibrissa dermal papilla cells with a polyomavirus large T gene encoding a temperature-sensitive T antigen, and generated permanent cell lines in which the immortalizing function can be switched off by temperature shift. The cells established without crisis, resembled cells in the starting population, and retained the aggregative properties of early passage dermal papilla cells. Growth studies were performed on the immortalized cell lines, which showed that transferring the cells to the restrictive temperature for the large T gene product resulted in cell senescence or quiescence, and changes in morphology. Implantation of cell pellets into the ears of immunologically compatible rats showed that the immortal cells retained hair-inductive ability. Cytokines are believed to have an important role in the control of hair growth. The pattern of cytokine gene expression in the immortal cell lines was compared with early passage dermal papilla cells and a non-hair-inducing dermal papilla cell line, using reverse transcriptase-polymerase chain reaction. Epidermal growth factor, tumour necrosis factor, and interleukin-1a were detected in the immortalized and non-hair-inducing dermal papilla cell lines, but were absent in passage 2 dermal papilla cells. All other cytokines examined were detected in all the cell types under study. These results demonstrate that the polyomavirus large Ttsa-immortalized dermal papilla cell lines are very similar to passage 2 dermal papilla cells and thus provide a good model for hair growth studies. Cytokine expression profiles indicate that the expression of several cytokines may be implicated in hair induction. Further studies are under way to investigate the relationship between cytokine expression and the hair growth cycle.

Myosin regulation and calcium transients in fibroblast shape change, attachment, and patching.

Following our study in Balb/c 3T3 cells and other cultured fibroblasts of the changes in myosin light chain phosphorylation associated with alterations in cell shape, attachment, and receptor patching, we have now determined the corresponding changes in cytoskeletal myosin distribution, and in the cellular calcium concentration, since this might, in part, mediate such responses. Immunofluorescence microscopy showed that myosin assembly into ordered forms such as actomyosin bundles and myosin sheath almost always correlated with previously shown high phosphorylation levels of myosin regulatory light chain, whereas diffuse distributions usually correlated with low or undetectable levels. An exception was observed in treatment to alter cellular cAMP levels when, in a biphasic response, assembly was correlated inversely with the phosphorylation states shown previously. Fluorescent indicators for intracellular calcium concentration, [Ca++]i, showed that myosin disassembly by trypsin or EGTA acting externally on the cells was preceded by a transient increase in [Ca++]i. For EGTA this was associated with transient recruitment of myosin into dorsal sheath structure as well as the transient enhancement of phosphorylation shown earlier. Blockage of EGTA-induced disassembly could be achieved by azide, which also caused an immediate increase in [Ca++]i and inhibited its subsequent decline. Trypsin-induced dephosphorylation did not appear to involve an eventual reduction of [Ca++]i. Therefore, in many but not all of the systems studied, correlated changes were observed in myosin assembly, [Ca++]i, and the myosin phosphorylation levels shown earlier.

Immortalization of rat keratinocytes by transfection with polyomavirus large T gene.

Transfection of primary rat keratinocytes with the polyomavirus large T gene promotes the establishment of cell lines. The keratinocytes express the large T protein and can be continuously cultured in medium containing a low concentration of calcium. The immortalized keratinocytes retain the ability to differentiate when the calcium concentration is increased to normal levels.

Myosin light chain phosphorylation in fibroblast shape change, detachment and patching.

The level of phosphorylation of myosin regulatory light chain in BALB/c 3T3 and certain other cultured substrate-attached fibroblasts has been shown to be altered by several agents which influence cell shape, attachment and/or surface receptors. This was investigated by metabolic labelling with [32P]orthophosphate, followed by exposure of the cells to the chosen conditions, rapid freezing to 'fix' phosphorylation levels, extraction and concentration in the presence of kinase and phosphatase inhibitors, and final analysis by two-dimensional gel electrophoresis. Gel patterns were interpreted by comparison with immunoprecipitates with antiserum to mouse nonmuscle myosin. Treatment of cells either with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or dibutyryl-cAMP suppressed light chain phosphorylation as predicted from the control mechanisms proposed previously from in vitro studies for Ca++ calmodulin and cAMP-dependent protein kinase respectively. Other effects were less easily explained: in BALB/c 3T3 cells, contrasting with previously reported behaviour of CHO cells, the cAMP-induced decline was small and transitory; and in at least one cell line (16C) the EGTA-induced decline was preceded by a strong pulse of enhanced phosphorylation. A striking and unexpected result was that azide, almost certainly acting on mitochondrial function, caused myosin light chain phosphorylation to be maintained over a long period even in the presence of EGTA which would otherwise bring about an immediate drop. The cleavage (by trypsin) or binding (by con A) of surface receptors was also shown to trigger the biochemical modulation of cellular myosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the proteins, glycoproteins and glycosaminoglycans of fibroblast adhesions to substratum.

The focal adhesion preparations which remain attached to a glass substratum when fibroblast bodies are removed by a gentle stream of buffer have been analysed by gel electrophoresis coupled with other selective methods of analysis. The results are consistent with the presence of three classes of macromolecular components. (i) Muscle and associated proteins amongst which actin was abundant with significant amounts of tropomyosin, some myosin and traces of alpha-actinin. Some vimentin was present but no vinculin. We detected a major new protein component, as yet unidentified, with a molecular weight in the region of 50000-55000 which is not desmin or tubulin and could have an important function at the focal adhesion. (ii) Glycoproteins which are a specialised subset of those in the whole plasma membrane and included a family which bind ricin and therefore contain beta-galactose end groups, together with a series having carbohydrate chains which bound neither ricin nor concanavalin A. The relative proportion of ricin-binding glycoproteins compared to concanavalin A-binding glycoproteins was higher than in whole plasma membranes. (iii) Glycosaminoglycans, with hyaluronate identified as the major component by column chromatography and its susceptibility to Streptomyces hyaluronidase.

Two modes of loss of the Tol function from Pseudomonas putida mt-2.

Some of a set of independently arising Tol- (non toluate-utilising) derivatives of Pseudomonas putida mt-2 have lost the unique plasmid present in the parent strain. In others this plasmid has suffered a deletion of a specific region of about 27 Md.

Molecular sizes and relationships of TOL plasmids in Pseudomonas.

Plasmid deoxyribonucleic acid was isolated from thirteen Pseudomonas strains judged on genetic criteria to carry plasmids coding for the degradation of toluene and m- and p-xylenes (TOL plasmids). Most strains carried a single species, but two strains carried two size classes, and cells of a third strain contained plasmids ranging in size from 25 X 10(6) to 202 X 10(6) daltons. Some plasmids could be transformed into a Pseudomonas putida strain to yield Tol+ progeny. Plasmids from 5 of the 13 strains were indistinguishable on the basis of size and gel pattern of fragments after endonuclease digestion.