Regulation of muscle Cav1.1 channels by long-term depolarization involves proteolysis of the alpha1s subunit.

The effects of long-term depolarization on frog skeletal muscle Cav1.1 channels were assessed. Voltage-clamp and Western-blot experiments revealed that long-term depolarization brings about a drastic reduction in the amplitude of currents flowing through Cav1.1 channels and in the levels of the alpha1s subunit, the main subunit of muscle L-type channels. The decline of both phenomena was prevented by the action of the protease inhibitors E64 (50 microM) and leupeptin (50 microM). In contrast, long-term depolarization had no effect on beta1, the auxiliary subunit of alpha1s. The levels of mRNAs coding the alpha1s and the beta1 subunits were measured by RNase protection assays. Neither the content of the alpha1s nor the beta1 subunit mRNAs were affected by long-term depolarization, indicating that the synthesis of Cav1.1 channels remained unaffected. Taken together, our experiments suggest that the reduction in the amplitude of membrane currents and in the alpha1s subunit levels is caused by increased degradation of this subunit by a Ca2+-dependent protease.

Mg, Mn, and Co ions enhance the formation of Entamoeba histolytica cyst-like structures resistant to sodium dodecyl sulfate.

BACKGROUND: Entamoeba histolytica forms cyst-like structures (CLS) in PEHPS but not in TYS-33 medium. Sodium dodecyl sulfate [(SDS (0.1%)] dissolves most of them in 10 min, but not natural cysts. Chitin is responsible mainly for cyst wall resistance. Its synthesis depends on Mg(2)+, Mn(2)+, or Co(2)+, whose action is interactive. With the aid of the Simplex method, we analyzed the effect of 20 blends of these cations to find the one that, when added to PEHPS, produced the highest proportion of CLS resistant to 1% SDS (RCLS). METHODS: The concentration of Mg(2)+, Mn(2)+, and Co(2)+ was determined in PEHPS and TYI-S-33 with a flame atomic absorption spectrometer. The proportion of RCLS produced in PEHPS with each ion blend was tested. The CLS and RCLS affinity to fluorescein wheat germ agglutinin (WGA/FITC), which binds chitin, was determined. RESULTS: PEHPS contained a similar concentration of Co(2)+ (0.52 microM) and 3.4 and 1.6 times more Mg(2)+ (798 microM) and Mn(2)+ (3.15 microM) than TYI-S-33, respectively. The proportion of RCLS increased gradually in PEHPS until reaching 3.6 +/- 1.43% with MgCl(2) 1.22 mM, MnCl(2) 14.44 mM, and CoCl(2) 19.44 mM (ion blend No. 20). Both CLS and RCLS bound WGA/FITC. The RCLS formed in the presence of ion blend No. 20 appeared wrinkled. CONCLUSIONS: Mg(2)+, Mn(2)+, and Co(2)+ enhanced the ability of PEHPS to form RCLS, possibly because these ions stimulated their chitin synthesis. Although ion blend No. 20 produced the highest proportion of RCLS, this high ion concentration may be toxic for encysting amebas.

Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli.

We have cloned and determined the nucleotide (nt) sequences of the genes encoding peptidyl-tRNA hydrolase (Pth) homologues of Salmonella typhi (St) and the Lyme disease spirochaete, Borrelia burgdorferi (Bb). We also completed the nt sequence of a pth homologous gene contained in a Chlamydia trachomatis (Ct) clone identified in the databanks. The open reading frames (ORFs) of the Pth homologues encode putative polypeptides of 194 (St), 188 (Bb) and 194 (Ct) amino acids exhibiting significant identity with Escherichia coli (Ec) Pth. Together with the products of two previously unidentified ORFs from Bacillus subtilis and Saccharomyces cerevisiae, and the recently recognized Haemophilus influenzae and Mycoplasma genitalium pth genes, these seven putative polypeptides and the Ec Pth form a group of homologous basic proteins spanning eubacteria and eukaryota which can be defined by at least three conserved regions. Previously known Ec pth mutations were located in highly conserved residues.

Open reading frames flanking the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli.

The nucleotide (nt) sequences flanking the peptidyl-tRNA hydrolase-encoding gene (pth) of Escherichia coli were determined and analyzed. A coding open reading frame (ORF-3), identified just downstream from pth, had a deduced amino acid (aa) sequence homologous to a family of GTP-binding proteins. We found discrepancies between the ORF-3 sequence from a plasmid clone used in previous studies and another one derived from Kohara's phage collection. Two putative genes, ORF-4 and ORF-2, were also found upstream from pth. ORF-4 could code for a 393-aa peptide homologous to membrane-bound proteins. The nt sequence between ORF-2 and pth revealed the existence of a CAP-binding site correctly positioned to regulate the expression of ORF-2.

Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis.

Escherichia coli rap mutants do not support vegetative growth of bacteriophage lambda and die upon transcription of lambda DNA bar sites. Bacteria harbouring a pth(ts) mutation synthesize thermosensitive peptidyl-tRNA hydrolase (Pth) and die at 42 degrees C from a defect in protein synthesis. We present evidence that both rap and pth(ts) mutations affect the same gene: (i) peptidyl-tRNA hydrolase activity was found to be defective in rap mutants; (ii) at a threshold temperature, pth cells, like rap mutants, prevented lambda growth and were killed by transcription of cloned bar sites; (iii) sequencing a 1600 bp DNA fragment comprising both loci revealed an ORF located within the limits set by a complementation analysis and encoding a putative polypeptide of 21 kDa; (iv) cloning and sequencing of rap and pth(ts) mutant DNAs both revealed single nucleotide transitions from the wild type ORF sequence, resulting in Arg134 to His and Gly101 to Asp changes respectively. Analysis of plasmid-directed proteins identified a polypeptide of approximately 21 kDa; the N-terminal sequence, amino acid composition and isoelectric point of this protein match those expected from the ORF nucleotide sequence. We propose that Pth activity, directly or indirectly, is the target for lambda bar RNA leading to rap cell death.

[The epidemiology of silicosis in the El Escorial region]. / Epidemiología de la silicosis en la comarca de El Escorial.

In a sample of 186 stone workers who performed granite "tearing" and "stone work" (either manual or mechanical) we have found silicosis in 50.5% (simple silicosis 47.3%, and complicated silicosis 3.2%). The most commonly found radiologic manifestation was a round opacity type "p" and a 1/1 to 1/3 profusion. From a functional respiratory perspective, a mild reduction of FVC, DLco (SB) and pO2 similar to that described in coal miners' pneumoconiosis was observed. It seems that "stone workers" had a higher incidence of suffering severe silicosis than stone "tearing" workers. Surprisingly, in the analysis of inhaled dust of such an activity which is performed in the open air the rates of dust and SiO2 are much higher than those found in coal workers. We believe that this is the first time that these measurements are performed, and published, in a group of stone workers.