Regiospecific hydroxylation of 3-(methylaminomethyl) pyridine to 5-(methylaminomethyl) -2 (1H) -pyridinone byArthrobacter ureafaciens

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1H)-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells ofArthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1H)-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.     

Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer

Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4–12 and 7–15, and its disulfide isomer, with disulfides positions 4–15 and 7–12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by ¹H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.     

Social organization in two primitive attine ants,cyphomyrmex rimosus and myrmicocrypta ednaella, with reference to their fungus substrates and food sources

In the Neotropical rainforest of Barro Colorado Island, Panama, social organization and behavior were observed in 2 primitive attine ant species,Myrmicocrypta ednaella andCyphomyrmex rimosus. Both species took nutrients from mycelia on fungus (i.e. mycophagy), and from plant nectar and sap which they collected outside the nest (i.e. phytophagy). They also obtained alimentary liquid by soliciting nestmates (i.e. stomodeal trophallaxis). Queens and larvae were wholly mycophagous, while older workers were much dependent on nectar, sap and alimentary liquid and younger workers were mostly mycophagous but only partly phytophagous.M. ednaella used wood chips as substrate for the fungus garden. Its fungus-growing behavior was similar to those hitherto observed in other primitive attine species. In contrast, the behavior ofC. rimosus was unique in its utilization of crop liquid as a substrate. In the rainforest,C. rimosus workers frequently forage outside the nests to collect nectar and sap, most of which is probably regurgitated for fungus cultivation.     

Demonstration of a Heat-Stable 120-Kilodalton Protein of Rickettsia japonica as a Spotted Fever Group-Common Antigen

Genomic libraries of Rickettsia japonica were cloned into an expression vector λgt11. A clone expressing a protein reactive with antiserum against 120-kilodalton (kDa) proteins, a mixture of heat-modifiable and heat-stable polypeptides, was selected and designated as λRj120-1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with β-galactosidase. The antiserum against 120-kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat-stable 120-kDa polypeptide. The antiserum against the expressed protein reacted with heat-stable 120- to 130-kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group-common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat-stable 120-kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904-909, 1994) and λgt11 lacZ gene amplified the λRj120-1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR-amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat-stable 120-kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.     

Stress chaperone mortalin regulates human melanogenesis

In order to identify the cellular factors involved in human melanogenesis, we carried out shRNA-mediated loss-of-function screening in conjunction with induction of melanogenesis by 1-oleoyl-2-acetyl-glycerol (OAG) in human melanoma cells using biochemical and visual assays. Gene targets of the shRNAs (that caused loss of OAG-induced melanogenesis) and their pathways, as determined by bioinformatics, revealed involvement of proteins that regulate cell stress response, mitochondrial functions, proliferation, and apoptosis. We demonstrate, for the first time, that the mitochondrial stress chaperone mortalin is crucial for melanogenesis. Upregulation of mortalin was closely associated with melanogenesis in in vitro cell-based assays and clinical samples of keloids with hyperpigmentation. Furthermore, its knockdown resulted in compromised melanogenesis. The data proposed mortalin as an important protein that may be targeted to manipulate pigmentation for cosmetic and related disease therapeutics.     

Distinct roles of the two VPS33 proteins in the endolysosomal system in Caenorhabditis elegans

Sec1/Munc‐18 (SM) family proteins are essential regulators in intracellular transport in eukaryotic cells. The SM protein Vps33 functions as a core subunit of two tethering complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) in the endocytic pathway in yeast. Metazoan cells possess two Vps33 proteins, VPS33A and VPS33B, but their precise roles remain unknown. Here, we present a comparative analysis of Caenorhabditis elegans null mutants for these proteins. We found that the vps‐33.1 (VPS33A) mutants exhibited severe defects in both endocytic function and endolysosomal biogenesis in scavenger cells. Furthermore, vps‐33.1 mutations caused endocytosis defects in other tissues, and the loss of maternal and zygotic VPS‐33.1 resulted in embryonic lethality. By contrast, vps‐33.2 mutants were viable but sterile, with terminally arrested spermatocytes. The spermatogenesis phenotype suggests that VPS33.2 is involved in the formation of a sperm‐specific organelle. The endocytosis defect in the vps‐33.1 mutant was not restored by the expression of VPS‐33.2, which indicates that these proteins have nonredundant functions. Together, our data suggest that VPS‐33.1 shares most of the general functions of yeast Vps33 in terms of tethering complexes in the endolysosomal system, whereas VPS‐33.2 has tissue/organelle specific functions in C. elegans.      

Syntheses and biological activities of deoxy-d-allose fatty acid ester analogs

We describe the syntheses of three different deoxy-D-allose analogs [2-deoxy-D-allose (2-DOAll), 1,2-dideoxy-D-allose (1,2-DOAll), and 1,2-didehydro-1,2-dideoxy-D-allose (1,2-DHAll)] and their fatty acid esters via regioselective lipase-catalyzed transesterification. Among them, 2-DOAll and its decanoate (2-DOAll-C10) showed higher inhibitory activity on plant growth, which is similar to D-allose (All) and its decanoate (All-C10). Bioassay results of deoxy-All-C10 on four plant species suggest that the hydroxy group at the C-1 position might be important showing growth inhibitory activity. In addition, co-addition of gibberellin (GA₃) with 1,2-DHAll-C10 and 2-DOAll-C10 recovered plant growth, suggesting that they might mainly inhibit biosynthesis of gibberellin.