Gene replacement analysis of the butyrolactone autoregulator receptor (FarA) reveals that FarA acts as a Novel regulator in secondary metabolism of Streptomyces lavendulae FRI-5.

IM-2 [(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl gamma-butanolide] is a gamma-butyrolactone autoregulator which, in Streptomyces lavendulae FRI-5, switches off the production of D-cycloserine but switches on the production of a blue pigment and several nucleoside antibiotics. To clarify the in vivo function of an IM-2-specific receptor (FarA) in the IM-2 signaling cascade of S. lavendulae FRI-5, a farA deletion mutant was constructed by means of homologous recombination. On several solid media, no significant difference in morphology was observed between the wild-type strain and the farA mutant (strain K104), which demonstrated that the IM-2-FarA system does not participate in the morphological control of S. lavendulae FRI-5. In liquid media, the farA mutant overproduced nucleoside antibiotics and produced blue pigment earlier than did the wild-type strain, suggesting that the FarA protein acts primarily as a negative regulator on the biosynthesis of these compounds in the absence of IM-2. However, contrary to the IM-2-dependent suppression of D-cycloserine production in the wild-type strain, overproduction of D-cycloserine was observed in the farA mutant, indicating for the first time that the presence of both IM-2 and intact FarA are necessary for the suppression of D-cycloserine biosynthesis.     

Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus.

A monoclonal antibody (MCI20.6) which inhibited measles virus (MV) binding to host cells was previously used to characterize a 57- to 67-kDa cell surface glycoprotein as a potential MV receptor. In the present work, this glycoprotein (gp57/67) was immunopurified, and N-terminal amino acid sequencing identified it as human membrane cofactor protein (CD46), a member of the regulators of complement activation gene cluster. Transfection of nonpermissive murine cells with a recombinant expression vector containing CD46 cDNA conferred three major properties expected of cells permissive to MV infection. First, expression of CD46 enabled MV to bind to murine cells. Second, the CD46-expressing murine cells were able to undergo cell-cell fusion when both MV hemagglutinin and MV fusion glycoproteins were expressed after infection with a vaccinia virus recombinant encoding both MV glycoproteins. Third, M12.CD46 murine B cells were able to support MV replication, as shown by production of infectious virus and by cell biosynthesis of viral hemagglutinin after metabolic labeling of infected cells with [35S]methionine. These results show that the human CD46 molecule serves as an MV receptor allowing virus-cell binding, fusion, and viral replication and open new perspectives in the study of MV pathogenesis.     

In vivo analysis of the lumenal binding protein (BiP) reveals multiple functions of its ATPase domain

The endoplasmic reticulum (ER) chaperone binding protein (BiP) binds exposed hydrophobic regions of misfolded proteins. Cycles of ATP hydrolysis and nucleotide exchange on the ATPase domain were shown to regulate the function of the ligand-binding domain in vitro. Here we show that ATPase mutants of BiP with defective ATP-hydrolysis (T46G) or ATP-binding (G235D) caused permanent association with a model ligand, but also interfered with the production of secretory, but not cytosolic, proteins in vivo. Furthermore, the negative effect of BiP(T46G) on secretory protein synthesis was rescued by increased levels of wild-type BiP, whereas the G235D mutation was dominant. Unexpectedly, expression of a mutant BiP with impaired ligand binding also interfered with secretory protein production. Although mutant BiP lacking its ATPase domain had no detrimental effect on ER function, expression of an isolated ATPase domain interfered with secretory protein synthesis. Interestingly, the inhibitory effect of the isolated ATPase was alleviated by the T46G mutation and aggravated by the G235D mutation. We propose that in addition to its role in ligand release, the ATPase domain can interact with other components of the protein translocation and folding machinery to influence secretory protein synthesis.     

In vitro cartilage formation: effects of 6-diazo-5-oxo-L-norleucine (DON) on glycosaminoglycan and collagen synthesis.

To determine the relationships between glycosaminoglycan (GAG) synthesis and type-specific collagen synthesis, we have investigated mouse limbs cultured in the presence of antiglutamine DON (6-diazo-5-oxo-l-norleucine). When compared to control limbs, ultrastructural examination of the DON-treated limbs shows that newly formed cartilage lacks matrix granules and the collagen fibrils have an altered morphology. Using [³⁵S]sulfate as a precursor, we have found that DON (5 μg/ml) suppresses chondroitin sulfate synthesis to less than 15% of the control level. We have also examined the collagen synthesized in equivalent limbs labeled with [³H]proline. The α-chain patterns from CM-cellulose chromatography were very similar for control and experimental limbs (α1:α2 ～ 7), suggesting that both (α1)₃- and (α1)₂α2-type molecules were being produced. The (α1)₃ molecules in both cases were identified as type II collagen by fractional salt separation and cyanogen bromide peptide mapping on CM-cellulose columns. We conclude that (1) the synthesis of type II collagen can be dissociated from the production of GAG, and (2) environmental influences can be involved in controlling the fibrillogenesis of collagen.     

In vitro activity of 5-(2,4-dimethylbenzyl) pyrrolidin-2-one extracted from marine Streptomyces VITSVK5 spp. against fungal and bacterial human pathogens

BackgroundPharmacological screening and usage of natural products for the treatment of human diseases has had a long history from traditional medicine to modern drugs. The majority of modern drugs are reported to be mostly from natural products.ObjectiveThe aim of the present study was to evaluate the inhibitory activity of 5-(2,4-dimethylbenzyl) pyrrolidin-2-one (DMBPO) extracted from marine Streptomyces VITSVK5 spp. isolated from sediment samples collected at Marakkanam coast of Bay of Bengal, India.MethodsThe lead compound was isolated by bioactive guided extraction and purified by silica gel column chromatography. Structural elucidation of the lead compound was carried out by using UV, FT-IR, ¹H NMR, ¹³C NMR, DEPT and HR-MS spectral data.ResultsSystematic screening of isolates for antimicrobial activity lead to identification of a potential strain, Streptomyces VITSVK5 spp. (GQ848482). Bioactivity guided extraction yielded a compound DMBPO and its inhibitory activity was tested against selected bacterial and fungal strains. DMBPO showed maximal activity against Escherichia coli with a MIC value of 187 μg/ml, followed by Klebsiella pneumoniae (MIC of 220 μg/ml and 10.3 mm zone of inhibition), Staphylococcus aureus (MIC of >1000 μg/ml and 4.4 mm zone of inhibition) and Bacillus subtilis (MIC of 850 μg/ml and 2.6 mm zone of inhibition). Furthermore, DMBPO was found to be a potent inhibitor of opportunistic fungal pathogens too. It showed a maximum activity against Aspergillus niger with a MIC value of 1 μg/ml and 28 mm zone of inhibition.ConclusionThe result of this study indicates that DMBPO possess antibiotic activity to selected bacterial and fungal pathogens and exhibited better activity against fungi than bacteria.     

In vitro phosphorylation of the adipocyte lipid-binding protein (p15) by the insulin receptor. Effects of fatty acid on receptor kinase and substrate phosphorylation.

Phosphorylation of the adipocyte lipid-binding protein (ALBP) isolated from 3T3-L1 cells has been studied in vitro utilizing the wheat germ agglutinin-purified 3T3-L1 adipocyte insulin receptor and the soluble kinase domain of the human insulin receptor. Following insulin-stimulated, ATP-dependent autophosphorylation of the wheat germ agglutinin-purified receptor beta-subunit, ALBP was phosphorylated exclusively on tyrosine 19 in the sequence Glu-Asn-Phe-Asp-Asp-Tyr19, analogous to the substrate phosphorylation consensus sequence observed for several tyrosyl kinases. The concentration of insulin necessary for half-maximal receptor autophosphorylation (KIR0.5) was identical to that necessary for half-maximal ALBP phosphorylation (KALBP0.5), 10 nM. Kinetic analysis indicated that stimulation of ALBP phosphorylation by insulin was attributable to a 5-fold increase in the Vmax (to 0.33 fmol/min/fmol insulin-binding sites) while the Km for ALBP was largely unaffected. By utilizing the soluble kinase domain of the human receptor beta-subunit, the presence of oleate bound to ALBP increased the kcat/Km greater than 3-fold. Oleate dramatically inhibited autophosphorylation of the 38-kDa fragment of the soluble receptor kinase in a concentration dependent fashion (I0.5 approximately 4 microM). The 48-kDa kinase exhibited much less sensitivity to the effects of oleate (I0.5 approximately 190 microM). The inhibition of autophosphorylation of the 48-kDa soluble kinase by oleate was reversed by adding saturating levels of ALBP. These results demonstrate that in vitro the murine adipocyte lipid-binding protein is phosphorylated on tyrosine 19 in an insulin-stimulated fashion by the insulin receptor and that the presence of a bound fatty acid on ALBP increases the affinity of insulin receptor for ALBP. Inhibition of insulin receptor kinase activity by unbound fatty acids suggests that the end products of the lipogenic pathway may feedback inhibit the tyrosyl kinase and that fatty acid-binding proteins have the potential to modulate such interaction.     

Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis.

The nuclease resistance of a short, thermostable mini-hairpin, d(GCGAAGC), and other related hairpins was examined. Hairpins possessing a purine-rich (GAA) or (GAAA) loop appeared to be more resistant against nucleases than those with a pyrimidine-rich loop or single-stranded oligomers. Among 8 kinds of oligodeoxyribonucleotides examined, the fragment most resistant against nucleases was a hairpin with the sequence of d(CGCGAAGCG). This hairpin was then utilized for the stabilization of mRNA in an in vitro translation system; the 3'-terminal region of an mRNA was hybridized with an oligodeoxyribonucleotide including the sequence complementary to the 3'-terminus of the mRNA tagged with the nuclease-resistant d(CGCGAAGCG) hairpin sequence. By using this method, dihydrofolate reductase (DHFR) mRNA was stabilized against nucleases contaminating a cell-free translation system of E.coli, with a consequent increase in protein synthesis efficiency of 200%.     

Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro.

Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.