(14C)acetate assimilation by a type I obligate methylotroph, Methylococcus capsulatus.

Methanol and formate oxidation supported the assimilation of [14C]acetate by cell suspensions of Methylococcus capsulatus; oxidation of other primary alcohols, except ethanol, did not. The extent of [1-14C]acetate assimilation supported by methanol oxidation was decreased in the presence of primary alcohols, except ethanol. Potassium cyanide (0.33 mM) completely inhibited the oxidation of formate and its stimulation of [1-14C]acetate assimilation. The amount of [1-14C]acetate assimilation supported by methanol oxidation was significantly inhibited by cyanide.     

Effect of biotin on alkaloid production during submerged cultivation of Claviceps sp. strain SD-58

Addition of biotin to culture medium NL-406 significantly increased alkaloid yield during submerged cultivation of Claviceps sp. strain SD-58. Alkaloid yield was further enhanced by incorporating leucine in biotin-supplemented culture medium. Increased alkaloid production was associated with an increase in the lipid content of cells and in the number of chlamydospores. Biotin deficiency caused a reduction in alkaloid yield and a parallel decrease in lipid content and chlamydospore numbers.     

Lactose-proton symport by purified lac carrier protein

The lac carrier protein of Escherichia coli was purified by an improved procedure and its activity assayed by a rapid filter method. Following reconstitution of the carrier by octyl glucoside dilution, proteoliposomes were concentrated by filtration on a microporous filter. Lactose accumulation by adsorbed or entrapped proteoliposomes is driven by an artificially imposed pH gradient (interior alkaline), by a membrane potential (interior negative), or by a combination of both forces. Activity is almost completely abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone or by the competitive inhibitor thiodigalactoside. Addition of lactose to proteoliposomes under appropriate conditions results in alkalinization of the external medium. This effect is not observed with liposomes devoid of lac carrier or in the presence of proton conducting agents. The results provide a strong indication that the lac gamma gene product is the only protein in the cytoplasmic membrane of Escherichia coli required for lactose-proton symport.     

Expression of cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase in Escherichia coli. Differences in kinetic mechanisms

The cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase (E3) were expressed in Escherichia coli using a lambda PL promoter-driven prokaryotic expression vector. The expressed proteins in total cell extracts were identified by Western blot analysis using anti-pig heart E3 antibody and also by measurement of E3 activity. Most of the expressed human E3 polypeptides (five bands) were found in the insoluble pellet while primarily full-length mature E3 was found in the soluble fraction. About 2% of the total soluble protein was mature human E3 when expressed in wild type E. coli AR120. Since wild type E. coli has its own endogenous E3 activity, the expression of human E3 was performed in a pyruvate dehydrogenase complex-deficient strain of E. coli, JRG1342. The expressed recombinant human E3s in JRG1342 were purified to near homogeneity. The amino-terminal amino acid sequence analysis revealed that the recombinant mature E3 had an expected sequence while the recombinant precursor E3 lost 19 amino acid residues of its 35-amino acid leader sequence presumably due to a proteolytic cleavage. The recombinant mature E3 displayed comparable kinetic properties to those reported for highly purified mammalian E3s. The truncated precursor E3 showed about half of the mature E3 activity. The double-reciprocal plot for the mature E3 in the direction of NAD+ reduction showed parallel lines (ping-pong mechanism) while that for the truncated precursor E3 displayed intersecting lines (sequential mechanism). In the direction of NADH oxidation, the kinetic mechanisms of both E3s were apparently a ping-pong mechanism. These kinetic results showed that the partial 16-amino acid extension in the leader sequence changed the kinetic mechanism of human E3 so that it resembled that of glutathione reductase.     

A complex pattern of H2A phosphorylation in the mouse testis

Phosphorylation of H2A histones in mouse testis was examined using testis tubule cultures labeled with 32PO4. Histones were analyzed by two systems of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography of the gels. Of the 32PO4 detected in histones, 95% was incorporated by certain modified forms of the H2A variants H2A.1 and H2A.X. Phosphorylation sites were mapped to N- and C-terminal regions of the modified variants by SDS gel electrophoresis and autoradiography of peptides generated by cleavage of in vitro-labeled proteins with N-bromosuccinimide. Incorporation rates differed for N- and C-terminal regions from different modified forms, demonstrating a complex pattern of H2A phosphorylation in the mouse testis.     

Multinuclear nuclear magnetic resonance studies of Na cation-stabilized complex formed by d(G-G-T-T-T-T-C-G-G) in solution. Implications for G-tetrad structures.

There has been much recent interest in the self-association of short deoxyguanosine-rich motifs within single-stranded DNAs to generate monovalent cation modulated four-stranded helical segments called G-quadruplexes stabilized by hydrogen-bonded G-tetrad alignments. We have addressed structural aspects of this novel alignment and report on multinuclear 1H, 31P and 13C nuclear magnetic resonance studies on the d(G2T4CG2) deoxynonanucleotide with Na cation as counterion in aqueous solution at low temperature. This sequence forms stable structures even though it cannot align by Watson-Crick hydrogen bond formation (see the paper on d(G2T5G2) describing optical and calorimetric measurements by Jin, R., Breslauer, K. J., Jones, R. A. & Gaffney, B. L. (1990), Science, 250, 543-546). The four narrow exchangeable protons detected between 11.5 and 12.0 parts per million (p.p.m.), which are common to the d(G2T4CG2) deoxynonanucleotide and the d(G2TCG2) deoxyhexanucleotide sequences, are assigned to deoxyguanosine imino protons hydrogen-bonded to carbonyl acceptor groups. These narrow imino protons are not detected for d(IGN5IG) and d(I2N5G2), where two deoxyguanosine residues are replaced by two deoxyinosine residues in the deoxynonanucleotide sequences. This implies that the 2-amino protons of deoxyguanosine must also participate in hydrogen bond formation and stabilize the structured conformation of d(G2T4CG2) in Na cation-containing solution. We have completely assigned the base and sugar H1', H2',2', H3', and H4' protons of the d(G2T4CG2) oligomer following analysis of two-dimensional nuclear Overhauser enhancement spectroscopy and two-dimensional correlated spectroscopy data sets in 0.1 M-NaCl, 10 mM-sodium phosphate, 2H2O solution at 0 degree C. The relative magnitude of the nuclear Overhauser enhancements (NOEs) between the base H8 and its own sugar H1' protons of individual deoxyguanosine residues establishes that G1 and G8 adopt syn orientations while G2 and G9 adopt anti orientations about the glycosidic bond in the d(G1-G2-T3-T4-T5-T6-C7-G8-G9) sequence in both Na and K cation-containing aqueous solution. Consequently, any structure proposed for the tetramolecular complex of d(G2T4CG2) must exhibit alternating G(syn) and G(anti) glycosidic torsion angles within each strand. The directionality and magnitude of the observed NOEs are consistent with the G(syn)-G(anti) steps adopting right-handed helical conformations in solution. We also note that the H8 protons of G1 and G8 (7.35 to 7.45 p.p.m.) in a syn alignment are shifted significantly upfield from the H8 protons of G2 and G9 (8.0 to 8.3 p.p.m.) in an anti alignment.(ABSTRACT TRUNCATED AT 400 WORDS)     

The asparaginyl-tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316.

Escherichia coli strain N4316 is a mutant that exhibits temperature-sensitive growth at 43 degrees C and temperature-sensitive translation in vivo and in vitro. Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA. Previous work has shown that a protein, called 'rescue', isolated from the parental strain partly corrects the defective translation in vitro. Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild-type parental strain; the purified protein is a homodimer of 54 kDa. The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature-sensitive growth phenotype of the mutant at the non-permissive temperatures. The cloned gene was sequenced, mapped to the 20.9-min region of the E. coli chromosome and shown to code for a 466-amino-acid protein with a molecular mass of 52 kDa. Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl-tRNA synthetase. Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase. A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read-through of nonsense codons and protein copy number is discussed.     

Novel leukocyte agonists are released by endothelial cells exposed to peroxide.

Reactive oxygen species do not activate isolated neutrophils, yet in vivo, such oxidants promote their adhesion to, and subsequent migration through, the vascular wall. We show human endothelial cells exposed to t-butylhydroperoxide shed large, sealed membrane vesicles that contained potent neutrophil agonists. This activity migrated on TLC like platelet-activating factor (PAF). Since neutrophils have a receptor for this phospholipid, which recognizes its unique characteristics including the short sn-2 acetyl residue, we examined the effect of PAF receptor antagonists and PAF acetylhydrolase on this activity. Structurally unrelated PAF receptor antagonists blocked neutrophil stimulation by vesicular phospholipids, and digestion with PAF acetylhydrolase, which is specific for short sn-2 residues, destroyed this activity. However, metabolic labeling, inhibition of synthesis, phospholipase A1 digestion, and high performance liquid chromatographic studies demonstrated that the vesicles did not contain PAF. Instead, the bioactivity migrated on high performance liquid chromatography like the phospholipids generated by oxidative fragmentation of synthetic arachidonoyl phosphatidylcholine that we have shown previously (Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104-11110) to stimulate neutrophils through their receptor for PAF. Thus, peroxide treatment of endothelial cells fragments cellular phosphatidylcholines, forming novel PAF-like phospholipids, and induces the shedding of membrane vesicles that contain these bioactive phospholipids.     

Molecular analysis of the Smith-Magenis syndrome: a possible contiguous-gene syndrome associated with del(17)(p11.2).

We undertook clinical evaluation (32 cases) and molecular evaluation (31 cases) of unrelated patients affected with Smith-Magenis syndrome (SMS) associated with an interstitial deletion of band p11.2 of chromosome 17. Patients were evaluated both clinically and electrophysiologically for peripheral neuropathy, since markers showing close linkage to one form of Charcot-Marie-Tooth disease (CMT1A) map to this chromosomal region. The common clinical findings were broad flat midface with brachycephaly, broad nasal bridge, brachydactyly, speech delay, and hoarse, deep voice. Fifty-five percent of the patients showed clinical signs (e.g., decreased or absent deep tendon reflexes, pes planus or pes cavus, decreased sensitivity to pain, and decreased leg muscle mass) suggestive of peripheral neuropathy. However, unlike patients with CMT1A, these patients demonstrated normal nerve conduction velocities. Self-destructive behaviors, primarily onychotillomania and polyembolokoilamania, were observed in 67% of the patients, and significant symptoms of sleep disturbance were observed in 62%. The absence of REM sleep was demonstrated by polysomnography in two patients. Southern analysis indicated that most patients were deleted for five 17p11.2 markers--FG1 (D17S446), 1516 (D17S258), pYNM67-R5 (D17S29), pA10-41 (D17S71), and pS6.1-HB2 (D17S445)--thus defining a region which appears to be critical to SMS. The deletion was determined to be of paternal origin in nine patients and of maternal origin in six patients. The apparent random parental origin of deletion documented in 15 patients suggests that genomic imprinting does not play a role in the expression of the SMS clinical phenotype. Our findings suggest that SMS is likely a contiguous-gene deletion syndrome which comprises characteristic clinical features, developmental delay, clinical signs of peripheral neuropathy, abnormal sleep function, and specific behavioral anomalies.