Physiological Studies of Methane and Methanol-Oxidizing Bacteria: Oxidation of C-1 Compounds by Methylococcus capsulatus

Methylococcus capsulatus grows only on methane or methanol as its sole source of carbon and energy. Some amino acids serve as nitrogen sources and are converted to keto acids which accumulate in the culture medium. Cell suspensions oxidize methane, methanol, formaldehyde, and formate to carbon dioxide. Other primary alcohols are oxidized only to the corresponding aldehydes. Oxidation of formate by cell suspensions is more sensitive to inhibition by cyanide than is the oxidation of other one carbon compounds. This is due to the cyanide sensitivity of a soluble nicotinamide adenine dinucleotide-specific formate dehydrogenase. Oxidation of formaldehyde and methanol is catalyzed by a nonspecific primary alcohol dehydrogenase which is activated by ammonium ions and is independent of pyridine nucleotides. Some comparisons are made with a strain of Pseudomonas methanica.     

Role of the environment in the interaction of nonintercalators with Z-DNA

Interaction of the DNA binding nonintercalators Netropsin, Distamycin and the mPD derivative with Z-DNA has been studied. It has been found that environmental factors like the solvent and added cations significantly modulate the interaction of these ligands with Z-DNA. However no definite Z to B transition in presence of these ligands was found in any case, in contrast to previously reported results (Ch. Zimmer, C. Marck and W. Guschlbauer, FEBS Lett. 154, 156-160 (1983)).     

Stereoselective enzymatic hydrolysis of 2-cyclohexyl-and 2-phenyl-1,3-propanediol diacetate in biphasic systems

Summary A key intermediate (S(–) 2-cyclohexyl-1,3-propanediol monoacetate) was made with high optical purity for the total synthesis of a new angiotensin converting enzyme inhibitor, Fosinopril. The stereoselective hydrolysis of 2-cyclohexyl-1,3-propanediol diacetate (I) and 2-phenyl-1,3-propanediol diacetate (II) was carried out with lipases. Among various lipases evaluated, only porcine pancreatic lipase (PPL) and Chromobacterium viscosum lipase demonstrated efficient conversion and gave the desired enantiomer of monoacetate. In aqueous solution, the desired S(–) monoacetate exhibited an optical purity of 65%–80% (30%–60% enantiomeric excess [e.e.]). However, when the same reactions were conducted in a biphasic system, the product S(–) monoacetate exhibited an optical purity of 99%–100% (98%–100% e.e.). The high purity product was achieved with 65 mol% yield at 1% substrate concentration. Among various solvents evaluated in biphasic systems, efficient hydrolysis was achieved in toluene, cyclohexane, and trichloro-trifluoroethane. The crude PPL was partially purified and two lipase fractions (A and B) were identified. Lipases A and B had a molecular mass of 38 000 and 40 000 daltons, respectively, and both were found to catalyze the hydrolysis of I and II to the appropriate monoacetate in a biphasic system. Offprint requests to: R. N. Patel     

Life-history ofNeurospora intermedia in a sugar cane field

The life-history ofNeurospora in nature has remained largely unknown. The present study attempts to remedy this. The following conclusions are based on observation ofNeurospora on fire-scorched sugar cane in agricultural fields, and reconstruction experiments using a colour mutant to inoculate sugar cane burned in the laboratory. The fungus persists in soil as heat- resistant dormant ascospores. These are activated by a chemical(s) released into soil from the burnt substrate. The chief diffusible activator of ascospores is furfural and the germinating ascospores infect the scorched substrate. An invasive mycelium grows progressively upwards inside the juicy sugar cane and produces copious macroconidia externally through fire- induced openings formed in the plant tissue, or by the mechanical rupturing of the plant epidermal tissue by the mass of mycelium. The loose conidia are dispersed by wind and/or foraged by microfauna. It is suggested that the constant production of macroconidia, and their ready dispersal, serve a physiological role: to drain the substrate of minerals and soluble sugars, thereby creating nutritional conditions which stimulate sexual reproduction by the fungus. Sexual reproduction in the sugar- depleted cellulosic substrate occurs after macroconidiation has ceased totally and is favoured by the humid conditions prevailing during the monsoon rains. Profuse micro-conidiophores and protoperithecia are produced simultaneously in the pockets below the loosened epidermal tissue. Presumably protoperithecia are fertilized by microconidia which are possibly transmitted by nematodes active in the dead plant tissue. Mature perithecia release ascospores in situ which are passively liberated in the soil by the disintegration of the plant material and are, apparently, distributed by rain or irrigation water.     

High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.     

Molecular dynamics simulations of oligosaccharides and their conformation in the crystal structure of lectin-carbohydrate complex: importance of the torsion angle {psi} for the orientation of {alpha}1,6-arm

The conformation of the heptasaccharide Man-1,6-(Man-1,3)(Xyl-ß1,2)-Man-ß,4-GlcNAc₂-ß1,4-(L-Fuc-1,3)-GlcNAc₁,the carbohydrate moiety of Erythrina corallodendron lectin (EcorL),the hexasaccharide Man-1,6-(Man-1,3) (GlcNAc-ß1,4)-Man-ß1,4-GlcNAc-ß1,4-GlcNAcand their disaccharide fragments have been studied by moleculardynamics (MD) simulations for 1000 ps with different initialconformations. In the isolated heptasaccharide, the most frequentlyaccessed conformation during MD has a value of 180° aroundMan-1,6-Man linkage. This conformation is stabilized by theformation of a hydrogen bond between the carbonyl oxygen ofGlcNAc₂ with the O3/O4 hydroxyls of the 1,6-linked mannose residue.The conformation of the heptasaccharide found in the crystalstructure of the EcorL-lactose complex (Shaanan et al., Science,254, 862, 1991), that has a value of 76° around Man-1,6-Manlinkage, is accessed, although less frequently, during MD ofthe isolated oligosaccharide. The ,, = 58°,–134°,–60°conformation around Man-₁,6-Man fragment observed in the crystalstructure of the Lathyrus ochnrs lectin complexed with a biantennaryoctasaccharide (Table I in Homans,S.W., Glycobiology, 3, 551,1993) has also been accessed in the present MD simulations.These values for the 1,6-linkage, which are observed in theprotein-carbohydrate crystal structures and are accessed inthe MD simulations, though occasionally, have not been predictedfrom NMR studies. Furthermore, these different values of leadto significantly different orientations of the 1,6-arm for thesame value of . This contrasts with the earlier predictionsthat only different values of can bring about significant changesin the orientation of the ₁,6-arm. The MD simulations also showthat the effects of bisecting GlcNAc or ß1,2-xyloseare very similar on the ₁,3-arm and slightly different on the₁,6-arm. bisecting GlcNAc carbohydrates glycoprotein lectinsaccharide complex     

Molecular dynamics simulations of high-mannose oligosaccharides

Conformations of several high-mannose-type oligosaccharidesthat are generated during the biosynthetic degradation of Man₉GlcNAc₂to Man₅GlcNAc₂ have been studied by molecular dynamics (MD).Simulations were performed on NCI-FCRDC's Cray Y-MP 8D/8128supercomputer using Biosym's CVFF force field for 1000 Ps withdifferent initial conformations. The conformations of the two1,3- and the two 1,6-linkages in each oligomannose were different,suggesting that deriving oligosaccharide conformations basedon the conformational preferences of the constituent disaccharidefragments will not always yield correct results. Unlike otheroligomannoses, Man₉GlcNAc₂ appears to take more than one distinctconformation around the core 1,6-linkage. These various conformationsmay play an important role in determining the processing pathways.Using the data on the preferred conformations of these oligomannosesand the available experimental results, possible pathways forprocessing Man₉GlcNAc₂ to Man₅GlcNAc₂ by 1,2-linkage-specificmannosidases have been proposed. Conformational analysis ofMan₅GlcNAc₂ indicates that the addition of ß1,2-GlcNActo the 1,3-linked core mannose, besides serving as a prerequisitefor mannosidase II action as suggested earlier, may also preventthe removal of 1,3-mannose. The MD simulations also suggestthat the processing of the precursor oligosaccharide duringAsn-linked complex and hybrid glycan biosynthesis proceeds ina well-defined pathway involving more than one 1,2-linkage-specificmannosidase. Knowledge of the conformation of the processingintermediates obtained from the present study can be used todesign highly specific substrate analogues to inhibit a particularmannosidase, thereby blocking one processing pathway withoutinterfering with the others. carbohydrates conformation glycosidase inhibitors mannosidase oligosaccharide processing     

Behavioral responses of the whitebacked planthopperSogatella furcifera (Homoptera: Delphacidae) on rice plants whose odors have been masked

In a two-choice test, moreS. furcifera females settled more often on exposed plants than on parafilm-masked ones, regardless of the susceptibility of rice varieties. This indicates that rice volatiles play an important role in the insect's short-range orientation to its host. The fact that more insects settled on exposed resistant Rathu Heenati (RHT) than to masked susceptible Taichung Native 1 (TN1) suggests that there must be certain common volatiles released by both varieties. Few females landed on masked plants of either RHT or TN1. This implies that the insect could not recognize at a distance that a plant was resistant or susceptible without olfactory stimuli.S. furcifera excreted less honeydew on masked plants than on exposed ones for both varieties and more on masked TN1 than on exposed RHT. The electronic monitoring of feeding behavior demonstrates that the insect made more frequent probes and had shorter phloem ingestion durations on exposed RHT than on exposed TN1 and on masked RHT than on masked TN1. Moreover, the insect had longer phloem ingestion durations on masked TN1 than on exposed RHT. These results suggest that volatile chemicals given off by resistant RHT plants have a negative effect on feeding.     

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid–liquid phase separation of tau

Liquid–liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.