Overexpression of Prdx6 reduces H2O2 but does not prevent diet-induced atherosclerosis in the aortic root.

The mammalian 1-Cys peroxiredoxin (Prdx6) is a unique member of the peroxiredoxin family of proteins capable of protecting cells from metal-catalyzed oxidative damage. We recently identified Prdx6 as a candidate for the quantitative trait locus Ath1, a gene responsible for a difference in diet-induced atherosclerosis susceptibility in mice. To investigate the role of Prdx6 in atherosclerosis, we generated transgenic mice that overexpress the Prdx6 allele from the Ath1-resistant 129/SvJ strain on an Ath1-susceptible C57BL/6J background. These mice expressed significantly elevated levels of Prdx6 mRNA and protein in multiple tissues including liver, aorta, and peritoneal macrophages, which accumulated significantly lower levels of hydrogen peroxide, revealing an enhanced antioxidant activity in these mice. However, overexpression of Prdx6 had no protective effect on LDL oxidation in vitro, and transgenic mice fed an atherogenic diet for 10 weeks did not possess an increased resistance to atherosclerosis nor did they maintain the high prediet plasma HDL levels consistent with the Ath1-resistant phenotype. In addition, the Prdx6 allele from the susceptible strain was shown to have a higher antioxidant activity than that of the resistant strains. These data suggest that the increased peroxidase activity attributable to Prdx6 overexpression in transgenic mice is not sufficient to protect mice from atherosclerosis, and that Prdx6 is not likely to be the gene underlying Ath1.

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis.

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

Immunocytochemical distribution of the GABA(B) receptor splice variants GABA(B) R1a and R1b in the rat CNS and dorsal root ganglia.

The anatomical distribution of the GABA(B) receptor (GBR) splice variants GBR1a and 1b in the CNS has not previously been studied. In the present study, distribution of the splice variants was mapped using immunohistochemistry. Polyclonal antibodies against splice variant unique epitopes were raised in rabbits. Affinity purified antibodies were used according to routine immunohistochemical procedures in sections from the rat CNS or dorsal root ganglia (DRG). The staining intensity was high in the cerebral cortex but lower in basal ganglia and the hippocampus. In the cerebellum, there was a marked difference in the distribution of GBR1a- and 1b-like immunoreactivity (LI). GBR1a-LI was preferentially localised in the granule cell layer whilst GBR1b-LI was mostly found in Purkinje cells and in the molecular layer. Cell bodies of the deep cerebellar nuclei stained for the GBR1a antibody while terminals surrounding the cell bodies were strongly labelled with the GBR1b antibody. A similar pre- vs postsynaptic pattern was seen in several nuclei ventral or caudal to the cerebellum (e.g. the cochlear nucleus, the facial nucleus, the spinal cord) but not in regions rostral to the cerebellum. In the spinal cord, strong labelling for both antibodies was seen in the dorsal horn. The GBR1b but not the GBR1a antibody stained tanycytes in the epithelium of the 3rd ventricle and in the central canal at the brain stem level. DRG neurons were positive for both the GBR1a and 1b antibody, but the former stained the cells much more intensely. Satellite cells were labelled with the GBR1b antibody. The most important aspect of these findings is that in some nuclei, GBR1b may mediate inhibition of transmitter release while in the same regions, GBR1a may mediate postsynaptic inhibition. Further, the observations support previous findings that GBR1b is the predominant splice variant in Purkinje cells.

Carbohydrate specificity of the Escherichia coli P-pilus papG protein is mediated by its N-terminal part.

The adherence of pyelonephritic Escherichia coli isolates to mammalian host cells is mediated by the P-pili structures on the bacterial surface. The protein constituting the distal part of the pili structure, papG, interacts with glycan receptors on the host cell. Variation in specificity for different glycoconjugates between the isolates, that may reflect variation in host tropism, has been correlated to three different classes of papG. Truncated variants of the class I, II and III papG adhesins were produced as fusion protein in E. coli and analysed for carbohydrate binding. The results showed that both carbohydrate binding and specificity of the papG adhesin resided in a linear part of the N-terminus of the protein.

Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase.

The cDNA encoding human stratum corneum chymotryptic enzyme (SCCE); an epidermal serine-proteinase which was recently purified from human stratum corneum, was isolated from a keratinocyte derived library. The obtained nucleotide sequence contained an open reading frame sufficient to encode a preproprotein consisting of 253 amino acid residues. Expression of two mRNA species hybridizing with SCCE cDNA, 1.2 and 2.0 kilobases, respectively, was detected to human skin. These two forms differ with respect to the length of the 3'-untranslated sequence. Analysis of mRNA derived from various human tissues showed that abundant expression of the SCCE gene was restricted to human skin. The cloned cDNA was introduced to a bovine papilloma virus-based expression system and recombinant protein was purified and characterized. The results show that recombinant SCCE is produced with a 22-amino acid residue signal peptide and a propeptide of 7 amino acid residues. Tryptic digestion removed this propeptide and yielded a proteolytically active protein with the same NH2-terminal amino acid sequence as native SCCE. The deduced amino acid sequence contains the conserved active site regions of serine proteinases. The calculated molecular mass of unglycosylated active SCCE was 24.4 kDa. The sequence indicates one tentative N-glycosylation site located near the C terminus. Recombinant SCCE was found to be heterogenous regarding glycosylation in a manner similar to that of the native enzyme.

Membrane protein acylation. Preference for exogenous myristic acid or endogenous saturated chains in Acholeplasma laidlawii.

Mycoplasmas are small bacteria without a cell wall, often found as surface parasites on eukaryotic cells. Of the more than 200 membrane proteins from Acholeplasma laidlawii resolved by two-dimensional PAGE, 23 were covalently modified with acyl chains. These acyl proteins had lower pI values than average and were all labelled by different exogenously supplied radioactive fatty acids attached by O-ester bonds. The fatty acids were selectively incorporated in the order myristic acid (14:0) greater than palmitic acid (16:0) greater than stearic acid (18:0) greater than oleic acid (18:1). However, endogenously synthesised saturated fatty acids, most of which were 16:0, were preferred over the supplied ones. A fraction of the exogenous 14:0 was elongated to 16:0. Absence of saturated fatty acids increased the incorporation of 18:1. The maximum extent of modification was one acyl chain for protein T2, on the exterior surface and two acyl chains for protein D12, spanning them membrane. Exogenously supplied fatty acids were incorporated into membrane lipids in proportion to their occurrence. However, the acylated proteins always contained 8-10 times more saturated chains than did the lipids. When exogenously supplied, all A. laidlawii polar membrane lipids could donate acyl chains to the acylated proteins but the neutral fraction (fatty acids and diacylglycerol) was most efficient. An incorporation into the acylated proteins of labelled cysteine, but not glucose or glycerol, was observed. Acylated proteins with different chains interacted similarly with a Triton X-114 detergent phase, and no full-size proteins (or acylated fragments) were released from cells by proteolytic enzymes. The results indicate an anchoring with peptide segments in addition to the acyl chains. Both 14:0 and 16:0 were attached at one end of both T2 and D12, but the N-terminal methionine of T2 was not acylated. The extent of modification and preference for saturated chains in the A. laidlawii membrane acylated proteins is more similar to eukaryotic than to eubacterial proteins.

Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii.

A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex.

Cloning and expression of Acholeplasma laidlawii membrane acyl proteins in Escherichia coli.

Many integral membrane proteins in Acholeplasma laidlawii are enriched in hydrophilic amino acid residues and covalently modified with fatty acids. In order to understand how these proteins are inserted and anchored in the bilayer, we have cloned several of the major A. laidlawii proteins in Escherichia coli: 900 recombinant clones containing 4-kbp DNA fragments, inserted into the BamHI site of the plasmid pAT 153, were screened with antibodies. With antimembrane antibodies, 26 positive clones were detected, and with a mixture of five different monospecific antibodies, another 7 clones were obtained. Immunological analysis of the colonies in situ verified that antigens for A. laidlawii membrane proteins D12, T2, T3, T4a, and unidentified proteins were produced in separate clones. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting showed several fragments [25 to 94 kilodalton (kDa)] for each of these proteins, some of which were even visible on Coomassie Blue-stained gels. It is concluded that A. laidlawii membrane proteins can be efficiently expressed in E. coli.