Ca2+-conductances in cultured rat retinal pigment epithelial cells

Membrane conductances for Ca²⁺ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamiland diltiazem-sensitive Ba²⁺ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba²⁺ currents indicating the presence of L-type Ca²⁺ channels. In order primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels. © 1994 Wiley-Liss, Inc.     

Involvement of an Intracellular Oligogalacturonate Hydrolase in Metabolism of Pectin by Clostridium thermosaccharolyticum

The enzymes pectin methylesterase and polygalacturonate hydrolase, which are responsible for the initial steps of pectin degradation by Clostridium thermosaccharolyticum, were shown to be induced on the polymeric substrates pectin and pectate, as well as on oligogalacturonates, and to be repressed in the presence of glucose. The digalacturonate and trigalacturonate produced by the extracellular pectin methylesterase-polygalacturonate hydrolase complex were transported across the cytoplasmic membrane and hydrolyzed by an inducible oligogalacturonate hydrolase to galacturonate. The oligogalacturonate hydrolase was separated from the polygalacturonate hydrolase and characterized. Its temperature optimum was 65°C, and its pH optimum was 6. The native molecular size was 90 kDa, and the enzyme was stable for more than 1 h at 65°C. The maximum reaction rate on oligomers decreased with the increasing degree of polymerization. Galacturonate was released by hydrolysis from the nonreducing end of the oligomer. The amounts of pectinolytic enzymes produced were all strictly correlated to the amount of biomass formed. Galacturonate was metabolized via a modified Entner-Doudoroff route.     

Cell type matters: competence for alkaloid metabolism differs in two seed-derived cell strains of Catharanthus roseus

Protoplasma - Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs)....     

Plant regeneration from mesophyll-protoplasts of four different ecotypes and two marker lines from Arabidopsis thaliana using a unique protocol

A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of four ecotypes (Col C24, Per-1, Bur-0, Landsberg erecta) and two marker lines (M4 and M10) of Ardbidopsis thaliana is described. The different lines showed plating efficiencies between 1.0 and 3.9% using Nitsch medium or this medium supplemented with coconut water. For the differentiation of callus into normal shoots a single shoot regeneration medium was applicable to all ecotypes, but depending on the line other regeneration media showed to be more suitable. The results indicated that the protoplast culture procedure is applicable, with minor modifications, to all tested genotypes but the most suitable shoot regeneration medium should be established for each A. thaliana line.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzyl-aminopurine - IAA indole-3-acetic acid - IPA isopentenyladenine - IPAR isopentenyladenosine - MES 2-[N Morpholino]ethanesulfonic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot

Multidrug-resistant strains of Mycobacterium tuberculosis have resulted in several recent outbreaks. Recognition of drug resistance is important both for treatment and to prevent further transmission. Here we use molecular biology techniques to study the basis of streptomycin resistance in single and multi-drug-resistant M. tuberculosis. We demonstrate that streptomycin resistance is associated with mutations implicated in ribosomal resistance. The mutations found either lead to amino acid changes in ribosomal protein SI2 or alter the primary structure of the 16S rRNA. The 16S rRNA region mutated perturbs a pseudoknot structure in a region which has been linked to ribosomal S12 protein.     

Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

Nucleosomes arrangement in chromatin

The spatial arrangement of nucleosomes in rat liver chromatin has been examined using the electric birefringence technique. All chromatin subunits studied (up to 9 consecutive nucleosomes) contain their full complement of the five histone types associated with about 200 base pairs repeat length DNA.     

A physical map of belomycin-specific fragmentation sites on PM2 bacteriophage DNA

Bleomycin treatment of PM2 DNA results in fragmentation of the genome at several specific sites. Application of restriction endonuclease digestion followed by bleomycin treatment has provided the basis for constructing a physical map of bleomycin fragmentation sites. Eleven sites have been located on the physical map relative toHpa II,Pst I, andHindIII cleavage sites. The fragmentation sites are not clustered in a particular region of the PM2 genome but 3 of the 11 sites do occur between theHpa II andPst I cleavage sites, a segment of DNA which comprises 14% of the PM2 DNA length.