Cloning of the Genes Concerned in Phenylalanine Biosynthesis in Corynebacterium glutamicum and its Application to Breeding of a Phenylalanine Producing Strain

The chorismate mutase and prephenate dehydratase genes of phenylalanine producing Corynebacterium glutamicum K38, which is resistant to p-fluorophenylalanine and m-fluorophenylalanine, were cloned into plasmid pCE53 in C. glutamicum KY9456, which lacks chorismate mutase and prephenate dehydratase. One of the resultant plasmids, pCmB4, contained a 9.4kb BamHI DNA fragment inserted into the unique BamHl site of pCE53. Plasmid pCmB4 complemented a phenylalanine and tyrosine double auxotroph of C. glutamicum KY9456. Introduction of pCmB4 into C. glutamicum RRL5 resulted in an about ten times increase in chorismate mutase activity. C. glutamicum K38 carrying the plasmid accumulated 19.0mg/ml of phenylalanine (50% increase over the yield of K38).     

Essential Role of Aspartokinase in L-Threonine Production by Escherichia coli W Mutants

We previously constructed an l-threonine-producing strain of E. coli W, KY8280, which is an Ile⁺ revertant of KY8279 which requires l-methionine, a,£-diaminopimelic acid and l-isoleucine [H. Kase et al., Agric. Biol. Chem., 35, 2089 (1971)]. From KY8280, another l-threonine-hyperproducing strain, KY8366, was obtained as an α-amino-β-hydroxy valeric acid (AHV, a threonine analog)-resistant mutant. Enzymatic analysis revealed that KY8280 constitutively expressed 8-fold higher l-threonine-sensitive aspartokinase I activity than KY8279. In addition, KY8366 constitutively expressed 13-fold higher l-lysine-sensitive aspartokinase III activity than KY8280. Such elevated levels of aspartokinases may contribute to the hyperproduction of l-threonine by these mutant strains. KY8366 produced 28 mg/ml of l-threonine in a culture medium fed with 12% glucose.     

Crucial Residue Involved in L-Lactate Recognition by Human Monocarboxylate Transporter 4 (hMCT4)

Background

Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4.

Experimental approach

Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4.

Results

In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization.

Conclusions

Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.     

Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

Organization of the Mitochondrial Genome of a Deep-Sea Fish, Gonostoma gracile (Teleostei: Stomiiformes): First Example of Transfer RNA Gene Rearrangements in Bony Fishes

We determined the complete nucleotide sequence of the mitochondrial genome (except for a portion of the putative control region) for a deep-sea fish, Gonostoma gracile. The entire mitochondrial genome was purified by gene amplification using long polymerase chain reaction (long PCR), and the products were subsequently used as templates for PCR with 30 sets of newly designed, fish-universal primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products showed that the genome contained the same 37 mitochondrial structural genes as found in other vertebrates (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes), with the order of all rRNA and protein-coding genes, and 19 tRNA genes being identical to that in typical vertebrates. The gene order of the three tRNAs (tRNA^Glu, tRNA^Thr, and tRNA^Pro) relative to cytochrome b, however, differed from that determined in other vertebrates. Two steps of tandem duplication of gene regions, each followed by deletions of genes, can be invoked as mechanisms generating such rearrangements of tRNAs. This is the first example of tRNA gene rearrangements in a bony fish mitochondrial genome. Received August 5, 1998; accepted February 19, 1999.     

Phylogenetic relationship of the populations within and around Japan using 105 short tandem repeat polymorphic loci

We have analyzed 105 autosomal polymorphic short tandem repeat (STR) loci for nine East and South-eastern Asian populations (two Japanese, five Han Chinese, Thai, and Burmese populations) and a Caucasian population using a multiplex PCR typing system. All the STR loci are genomewide tetranucleotide repeat markers of which the total number of observed alleles and the observed heterozygosity were 756 and 0.743, respectively, for Japanese populations. Phylogenetic analysis for these allele frequency data suggested that the Japanese populations are more closely related with southern Chinese populations than central and/or northern ones. STRUCTURE program analysis revealed the almost clearly divided and accountable population structure at K=2–6, that the two Japanese populations always formed one group separated from the other populations and never belong to different groups at K≥3. Furthermore, our new allele frequency data for 91 loci were analyzed with those for 52 worldwide populations published by previous studies. Phylogenetic and multidimensional scaling (MDS) analyses indicated that Asian populations with large population size (six Han Chinese, three Japanese, two Southeast Asia) formed one distinct cluster and are closer to each other than other ethnic minorities in east and Southeast Asia. This pattern may be the caviar of comparing populations with greatly differing population sizes when STR loci were analyzed.     

Leptodiscaceans (Noctilucales, Dinophyceae) from the Pacific Ocean: First records of Petalodinium and Leptodiscus beyond the Mediterranean Sea

Records of dinoflagellates of the family Leptodiscaceae (Noctilucales) from the Kuroshio Current, Philippine, Celebes, Sulu, South China Seas and the western and central Equatorial Pacific Ocean are described. Scaphodinium mirabile was the most common leptodiscacean. Two specimens that differed from the type species of Scaphodinium were found: one specimen showed a highly bifurcate proximal extremity and another showed two dissimilar proboscides from the distal extremity. Another unidentified leptodiscacean showed an arrowhead-shaped contour with the margins folded. Six specimens of Petalodinium porcelio were found, being the first record beyond the Mediterranean-Black Seas. Six specimens were tentatively assigned to the genus Leptodiscus, being the first record beyond the western Mediterranean Sea. The folded specimens that ranged from 90 to 120 μm in diameter and with a prominent flagellum were tentatively considered to be young specimens of Leptodiscus. The abundance of the leptodiscaceans is underestimated in the world's oceans.     

Linear alpha-human atrial natriuretic peptide analogs display receptor binding activity and inhibit alpha-hANP-induced cGMP accumulation

We have synthesized a series of [Cys(R)7,23]alpha-hANP analogs, in which the two Cys residues were modified with various alkyl groups(R); i.e., R=Acm, Pe, Qe, Cam, Me, Ae, Bzl, Cm, Ocam and sulfo. The Acm-, Cam-, and Me-analogs exhibited binding activity as potent as alpha-hANP in rat vascular smooth muscle cells (VSMC). Binding activity of the analogs decreased progressively as the bulkiness of the R group increased. None of the analogs caused accumulation of cGMP in VSMC and vasorelaxant activity in rat aorta. Acm-, Cam- and Me-analogs substantially antagonized alpha-hANP-induced cGMP accumulation, but did not antagonize vasorelaxation induced by alpha-hANP in vitro.     

Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats

It is widely recognized that activated hepatic stellate cells (HSC) play a pivotal role in development of liver fibrosis. A platelet-derived growth factor (PDGF) is the most potent mitogen for HSC. The aim of this study was to examine the effect of imatinib mesylate (STI-571, Gleevec), a clinically used PDGF receptor (PDGFR) tyrosine kinase inhibitor, on development of experimental liver fibrosis. The rat model of pig serum-induced hepatic fibrosis was used to assess the effect of daily oral administration of STI-571 on the indexes of fibrosis. STI-571 markedly attenuated development of liver fibrosis and hepatic hydroxyproline and serum fibrosis markers. The number of alpha-smooth muscle actin-positive cells and mRNA expression of alpha2-(I)-procollagen, tissue inhibitor of metalloproteinases-1, and transforming growth factor-beta were also significantly suppressed by STI-571. Our in vitro study showed that STI-571 markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner. STI-571 also significantly attenuated PDGF-BB-induced phosphorylation of PDGFR-beta, MEK1/2, and Akt in activated HSC. Because STI-571 is widely used in clinical practice, it may provide an effective new strategy for antifibrosis therapy.