Isolation of a rat mitochondrial release factor. Accommodation of the changed genetic code for termination

A single release factor has been isolated and partially purified from rat mitochondria. It requires ethanol in addition to the specific termination codon when assayed in a heterologous system with Escherichia coli ribosomes. The factor recognizes the codons UAA and UAG but not UGA, and therefore it has been designated mtRF-1. A factor of the bacterial RF-2 type, which in E. coli recognizes UGA, or of the mammalian type, which recognizes all three termination codons, has not been detected in mitochondria. The absence of a factor responding to UGA accommodates the use of this codon as a signal for tryptophan in the rat mitochondrial genetic code. The mtRF-1 could translate all of the known termination codons in the rat mitochondrial genome. It does not respond to AGG and AGA which in bovine and human mitochondrial DNA code for termination but which in rat mitochondria may not code for either an amino acid or for termination.     

Microsomal and cytosolic epoxide hydrolases, the peroxisomal fatty acid beta-oxidation system and catalase. Activities, distribution and induction in rat liver parenchymal and non-parenchymal cells

A number of structurally unrelated hypolipidaemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in rodent liver, but there is relatively limited data regarding the specific effects of these drugs on liver non-parenchymal cells. In the present study, liver parenchymal, Kupffer and endothelial cells from untreated and fenofibrate-fed rats were isolated and the activities of two enzymes associated with peroxisomes (catalase and the peroxisomal fatty acid beta-oxidation system) as well as cytosolic and microsomal epoxide hydrolase were measured. Microsomal epoxide hydrolase, cytosolic epoxide hydrolase and catalase activities were 7-12-fold higher in parenchymal cells than in Kupffer or endothelial cells from untreated rats; the peroxisomal fatty acid beta-oxidation activity was only detected in parenchymal cells. Fenofibrate increased catalase, cytosolic epoxide hydrolase and peroxisomal fatty acid beta-oxidation activities in parenchymal cells by about 1.5-, 3.5- and 20-fold, respectively. The induction of catalase (2-3-fold) and cytosolic epoxide hydrolase (3-5-fold) was also observed in Kupffer and endothelial cells; furthermore, a low peroxisomal fatty acid beta-oxidation activity was detected in endothelial cells. Morphological examination by electron microscopy showed that peroxisomes were confined to liver parenchymal cells in untreated animals, but could also be observed in endothelial cells after administration of fenofibrate.     

Postembedding immunogold labeling for electron microscopy using "LR White" resin

A method is described for performing postembedding immunogold immunocytochemistry on sections of LR White-embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high-resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications.     

Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11.

Extraction of Lactobacillus fermentum BR11 cells with 5 M LiCl yielded a preparation containing a single predominant polypeptide with an apparent molecular mass of 32 kDa. A clone encoding an immunoreactive 32-kDa polypeptide was isolated from a pUC18 library of L. fermentum BR11 DNA by screening with an antiserum raised against whole cells of L. fermentum BR11. Sequence determination of the insert in the clone revealed a complete 795-bp open reading frame (ORF) that defines a 28,625-Da polypeptide (BspA). N-terminal sequencing of the LiCl-extracted polypeptide from L. fermentum BR11 confirmed that it is the same as the cloned BspA. BspA was found to have a sequence similar to those of family III of the bacterial solute-binding proteins. The sequences of two ORFs upstream of bspA are consistent with bspA being located in an operon encoding an ATP-binding cassette-type uptake system. Unusually, BspA contains no lipoprotein cleavage and attachment motif (LXXC), despite its origin in a gram-positive bacterium. Biotin labelling and trypsin digestion of whole cells indicated that this polypeptide is exposed on the cell surface. The isoelectric point as predicted from the putative mature sequence is 10.59. It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction with acidic groups on the cell surface. It was shown that BspA could be selectively removed from the surface by extraction with an acidic buffer, thus supporting this hypothesis.     

The subcellular distribution of zinc in dog prostate studied by X-Ray microanalysis

Summary X-ray microanalysis of zinc in ultrathin sections of dog prostate was performed by electron microscope microanalysis using the potassium pyroantimonate method of preparation. Prostates of both mature and immature dogs were examined and the metal was found to be localised primarily in the nucleolus, nuclear chromatin and secretory granules of epithelial cells. Differences in zinc concentrations were observed between mature and immature tissues, particularly in the nuclear chromatin. The metal was also incorporated into epithelial secretions, lysosomes and fibromuscular stroma. Variable binding of zinc to tissue components was revealed by a combination of histochemical precipitation and subcellular analysis.The authors are grateful to the Tenovus Organisation for general financial support. This work was also supported by the Medical Research Council, Grant No. G974/304B and by a grant of the Austrian Bundesministerium für Wissenschaft und Forschung. One of them (F.S.) was financed by the British Council     

Modification of Lofland's colorimetric semiautomated serum triglyceride determination, assessed by an enzymatic glycerol determination

Erroneously high values for serum triglyceride levels obtained with the semiautomated method of Lofland were found to be due to contamination of the isopropanol extracts with glucose or other carbohydrate. Treatment of the isopropanol extracts with a mixture of copper sulfate and calcium hydroxide removed the contaminating glucose. Analysis of the glucose-free extracts by either the semiautomated or manual colorimetric method gave values in good agreement with each other and with those obtained by a new specific enzymatic method. The simple modification described in this paper obviates the necessity for the more expensive automated fluorometric apparatus.     

Fairy shrimps in distress: a molecular taxonomic review of the diverse fairy shrimp genus Branchinella (Anostraca: Thamnocephalidae) in Australia in the light of ongoing environmental change

Australia, and especially South-Western Australia, is a diversity hotspot for large branchiopod crustaceans. A significant proportion of this diversity is found in the anostracans (Crustacea, Anostraca) and particularly in the diverse genus Branchinella with at least 34 species. Members of this genus are found exclusively in temporary aquatic habitats which are increasingly threatened by secondary salinization and other anthropogenic pressures. The development of adequate conservation strategies is therefore considered a priority. To define conservation units, however, thorough knowledge of the taxonomy and phylogenetic position of extant lineages is essential. We reconstructed a large scale phylogeny of the Australian Branchinella by analyzing the 16S mitochondrial gene of 31 presumed species, complemented with analysis of morphological structures holding taxonomic information. Results revealed the presence of at least three new cryptic species. On the other hand, some Branchinella lineages, surviving in environments subjected to contrasting selection regimes, appeared to be conspecific. This suggests substantial physiological plasticity or important adaptive variation present in some species, potentially enabling them to better cope with environmental change, such as secondary salinization. Overall, these results further illustrate the benefits of combining molecular markers and classic morphological taxonomy and phylogeny to assess biodiversity and define conservation units in cryptic groups.     

Peptide immunization of guinea pigs against Chlamydia psittaci (GPIC agent) infection induces good vaginal secretion antibody response, in vitro neutralization and partial protection against live challenge

Immunization of female guinea pigs with a chimeric peptide consisting of variable domain IV (VDIV) and a region known as GP8 from the major outer membrane protein of Chlamydophila caviae, formerly Chlamydia psittaci guinea pig inclusion conjunctivitis strain, was performed to assess whether humoral immune responses could be elicited in the reproductive tracts of immunized animals. The C. caviae strain is able to cause a sexually transmitted infection in the guinea pig that closely parallels C. trachomatis infections in humans. The best anti-VDIV antibody response in vaginal secretions was achieved by intraperitoneal priming with subsequent intravaginal boosting (P < 0.001). Dot-blot analyses of vaginal secretions confirmed that these anti-VDIV antibodies, produced against a linear peptide, were able to recognize and bind to whole conformational C. caviae elementary bodies. Following live intravaginal challenge with C. caviae, a significant reduction in the intensity (P = 0.01) and an apparent reduction in the duration of the infection was evident between the guinea pigs immunized with VDIV-GP8 and non-immunized controls.