Ultraviolet difference spectroscopy of the binary complex of isozyme 4-4 of rat liver glutathione S-transferase with glutathione (GSH) and the enzyme alone or as the binary complex with the oxygen analogue, gamma-L-glutamyl-L-serylglycine (GOH), at neutral pH reveals an absorption band at 239 nm (epsilon = 5200 M-1 cm-1) that is assigned to the thiolate anion (GS-) of the bound tripeptide. Titration of this difference absorption band over the pH range 5-8 indicates that the thiol of enzyme-bound GSH has a pKa = 6.6, which is about 2.4 pK units less than that in aqueous solution and consistent with the kinetically determined pKa previously reported [Chen et al. (1988) Biochemistry 27, 647]. The observed shift in the pKa between enzyme-bound and free GSH suggests that about 3.3 kcal/mol of the intrinsic binding energy of the peptide is utilized to lower the pKa into the physiological pH range. Apparent dissociation constants for both GSH and GOH are comparable and vary by a factor of less than 2 over the same pH range. Site occupancy data and spectral band intensity reveal large extinction coefficients at 239 nm (epsilon = 5200 M-1 cm-1) and 250 nm (epsilon = 1100 M-1 cm-1) that are consistent with the existence of either a glutathione thiolate (E.GS-) or ion-paired thiolate (EH+.GS-) in the active site. The observation that GS- is likely the predominant tripeptide species bound at the active site suggested that the carboxylate analogue of GSH, gamma-L-glutamyl-(D,L-2-aminomalonyl)glycine, should bind more tightly than GSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Several mutations in the surfactant protein C (SP-C) gene (SFTPC) have been reported as causing familial pulmonary fibrosis (FPF). However, the genetic background and clinical features of FPF are still not fully understood.We identified one Japanese kindred, in which at least six individuals over three generations were diagnosed with pulmonary fibrosis. We examined the patients radiologically and histopathologically and sequenced their SFTPC and ABCA3 genes. We also established a cell line stably expressing the mutant gene.All the patients had similar radiological and histopathological characteristics. Their histopathological pattern was that of usual interstitial pneumonia, showing numerous fibroblastic foci even in areas without abnormal radiological findings on chest high-resolution computed tomography. No child had respiratory symptoms in the kindred. Sequencing of SFTPC showed a novel heterozygous mutation, c.298G.A (G100S), in the BRICHOS domain of proSP-C, which co-segregated with the disease. However, in the ABCA3 gene, no mutation was found. In vitro expression of the mutant gene revealed that several endoplasmic reticulum stress-related proteins were strongly expressed.The mutation increases endoplasmic reticulum stress and induces apoptotic cell death compared with wild-type SP-C in alveolar type II cells, supporting the significance of this mutation in the pathogenesis of pulmonary fibrosis.

Appressoria of the phytopathogenic fungus Colletotrichum lagenarium contain melanin, which has been implicated as an important factor in the penetration of host plants. A cDNA clone containing the melanin biosynthetic gene encoding scytalone dehydratase (SCD1) from C. lagenarium was identified by hybridization with a heterologous cDNA probe from Magnaporthe grisea. The cDNA clone was used to identify a cosmid containing SCD1 in a genomic library of C. lagenarium, and the nucleotide sequence was determined for both the cDNA and genomic clones. The SCD1 gene contained one open reading frame composed of 188 codons and two deduced introns of 57 and 67 nucleotides. The deduced amino acid sequence of the N-terminal region of SCD1 showed high similarity to the amino acid sequence of scytalone dehydratase from Cochliobolus miyabeanus. A plasmid containing the SCD1 gene transformed the melanin-deficient mutant 9201Y (Scd ؊) to the wild phenotype but did not complement the conditional scytalone dehydratase-deficient mutant C. lagenarium 8015. Genomic DNA analysis indicated that the SCD1 gene is a single locus in C. lagenarium. Transcripts of the SCD1 gene were detected 2 h after the start of conidial germination.

Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell-cell fusion. Expression of an ezrin dominant negative mutant or knockdown of ezrin, radixin, or moesin with siRNA uniformly decreased transduction titers of HIV-1 vectors having X4-tropic Env. In contrast, transduction titers of R5-tropic Env HIV-1 vectors were decreased only by radixin knockdown: ezrin knockdown had no detectable effects and moesin knockdown rather increased transduction titer. Each of the ERM suppressions had no detectable effects on cell surface expression of CD4, CCR5, and CXCR4 or VSV-Env-mediated HIV-1 vector transductions. Finally, the individual knockdown of ERM mRNAs uniformly decreased efficiency of cell-cell fusion mediated by X4- or R5-tropic Env and HIV-1 infection receptors. These results suggest that (i) the ERM proteins function as positive regulators of infection by X4-tropic HIV-1, (ii) moesin additionally functions as a negative regulator of R5-tropic HIV-1 virus infection at the early step(s) after the membrane fusion, and (iii) receptor protein dynamisms are regulated differently in R5- and X4-tropic HIV-1 infections.