Noboru Yumoto

Phenotypic diversity of Pseudoalteromonas citrea from different marine habitats and emendation of the description

Four strains of marine, aerobic, agar-decomposing bacteria with one polar flagellum and with DNA G + C contents of 38.9-40.2 mol% were isolated from the Far-Eastern mussels Crenomytilus grayanus and Patinopecten yessoensis. These four strains were identified as Pseudoalteromonas; however, they were phenotypically different from species described previously according to carbon compound utilization tests and the BIOLOG identification system. High agar-decomposing activity was found in two strains, in one of which agarase, alpha-galactosidase, pustulanase and laminarinase had been detected. The level of DNA homology of three of the strains was 70-100%. The fourth isolate was genetically less related to the others (67% DNA relatedness) and phenotypically was more distant from other members of this group; however, all four strains were assigned to a single species genotypically. DNA from the strains isolated from mussels showed 40-45% genetic relatedness with the DNA of Alteromonas atlantica, 8-36% with DNA of Pseudoalteromonas haloplanktis subsp. haloplanktis, Pseudoalteromonas haloplanktis subsp. tetraodonis, Pseudoalteromonas undina, Pseudoalteromonas nigrifaciens and Pseudoalteromonasas carrageenovora, 53% with Pseudoalteromonas elyakovii, 32-48% with marine P. nigrifaciens from mussels and 14-16% with Alteromonas macleodii. The DNA-DNA hybridization data revealed that the levels of relatedness between the strains isolated and the type strains of Pseudoalteromonas citrea and Pseudoalteromonas fuliginea described recently were significant (95-85%). These results were confirmed by serological data employing polyclonal antibodies to cell surface antigens. The strains isolated from mussels were identified as P. citrea. The hybridization data showed that the name P. fuliginea Romanenko et al. 1994 should be recognized as a junior subjective synonym of P. citrea Gauthier 1977. A notable phenotypic diversity of P. citrea which might be a reflection of their ecological habitats is discussed.

Syntheses of optically pure β-hydroxyaspartate derivatives as glutamate transporter blockers

DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a blocker and the transporters, we synthesized the optically pure isomers (L- and D-TBOA) and its erythro-isomers. L-TBOA is the most potent blocker for the human excitatory amino acid transporters (EAAT1-3), while D-TBOA revealed a difference in the pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives) of L-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers.

Effects of threo-β-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5)

d,lthreo‐β‐Benzyloxyaspartate (d,l‐TBOA), an analog of threo‐β‐hydroxyaspartate (THA) possessing a bulky substituent, is a potent non‐transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while lthreo‐β‐methoxyaspartate (l‐TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4‐expressing oocytes, d,l‐TBOA acted as a non‐transportable blocker, while l‐TMOA like d,l‐THA was a competitive substrate. In contrast, d,l‐THA, d,l‐TBOA and l‐TMOA all strongly attenuated the glutamate‐induced currents generated by EAAT5. Among them, l‐TMOA showed the most potent inhibitory action. Moreover, d,l‐THA, d,l‐TBOA and l‐TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that d,l‐TBOA and l‐TMOA, as well as d,l‐THA, not only act as non‐transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate‐gated chloride channel.

Synthesis and application of caged peptides and proteins

Caged compounds have covalently attached groups that are rapidly cleaved upon exposure to UV light. Attachment of photolabile groups makes the molecule inert until photolysis releases it in its bioactive form. When caged compounds are applied to the experimental system in advance, the concentration jump of biologically active substances can be brought about immediately in a limited area upon irradiation with pulsed and focused UV light. Therefore, caged compounds of low molecular weight, which are commercially available, have been used effectively to study the mechanisms of temporal biological phenomena, such as muscle contraction, intracellular signaling, and neurotransmission. Because many proteins and peptides play important roles in these phenomena, their caged derivatives should serve as powerful tools to clarify complex biological systems. To prepare caged proteins and peptides, several groups have improved upon a chemical modification method, as well as developed two new methods: (1) nonsense codon suppression and (2) solid-phase peptide synthesis. In this review, we summarize recent advances made in the design, preparation, and application of caged peptides and proteins.

Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin‐dependent protein kinase II (CaMKII), was studied. Replacement of Arg6, Gln7, or Ala9 by other amino acid residues produced a marked increase in the IC50 value. Leu4 and Val10 were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala3, Glu8, Ala12, and Leu13 by other residues produced no significant increase in the IC50, the substitution of Lys for Ala3 decreased the IC50. An AIP analog (KK LRRQEA DAY), in which Ala3 and Val10 were replaced with Lys and Phe, respectively, showed an IC50 value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.

A caged sperm‐activating peptide that has a photocleavable protecting group on the backbone amide

A backbone‐caged sperm‐activating peptide (caged speract) that has a 2‐nitrobenzyl group at a backbone amide and a vastly reduced affinity for its receptor (IC50=950 nM) was synthesized. UV irradiation of caged speract photocleaves the 2‐nitrobenzyl group (τ 1/2=26 μs), restoring its affinity (IC50=0.67 nM) and ability to increase sperm intracellular pH and Ca2+, as intact speract. Backbone caging of the biological activity was more efficient than side chain caging, which adds a nitrobenzyl group on the peptide side chain. The backbone caging strategy described can be used as a general procedure to cage biologically active peptides, which have no side chain for introduction of a caging group.

ATRA-regulated Asb-2 gene induced in differentiation of HL-60 leukemia cells

Suppressors of cytokine signaling (SOCS) proteins possess common structures, a SOCS box at the C‐terminus and a SH2 domain at their center. These suppressors are inducible in response to cytokines and act as negative regulators of cytokine signaling. The ASB proteins also contain the SOCS box and the ankyrin repeat sequence at the N‐terminus, but do not have the SH2 domain. Although Socs genes are directly induced by several cytokines, no Asb gene inducers have been identified. In this study, we screened the specific genes expressed in the course of differentiation of HL‐60 cells, and demonstrated that ASB‐2, one of the ASB proteins, was rapidly induced by all‐trans retinoic acid (ATRA). Typical retinoid receptors (RARs) or retinoid X receptors (RXRs) binding element (RARE/RXRE) were presented in the promoter of the Asb‐2 gene. We showed that RARα, one of the RARs, binds to the RARE/RXRE in the Asb‐2 promoter. In addition, we demonstrated by luciferase reporter assay that this element was a functional RARE/RXRE. These findings indicate that ASB‐2 is directly induced by ATRA and may act as a significant regulator, underlying such physiological processes as cell differentiation.

Pseudoalteromonas ruthenica sp. nov., isolated from marine invertebrates

On the basis of phenotypic and genotypic characteristics and analysis of 165 rRNA sequences, a novel species belonging to the genus Pseudoalteromonas is described. Two pale-orange-pigmented strains, KMM 300T and KMM 290, isolated respectively from a mussel, Crenomytilus grayanus, and a scallop, Patinopecten yessoensis, are marine, gram-negative, aerobic, rod-shaped bacteria that produce a number of antimicrobial compounds. The strains are able to degrade gelatin, elastin, starch, DNA and Tween 80. Chitin and agar are not degraded. The isolates from marine invertebrates grew at NaCl concentrations of 1-9% and a temperature range of 10-35 degrees C and did not utilize most of the wide range of carbohydrates tested, with the exception of D-glucose, cellobiose and sucrose. The DNA G+C content was 48.4-48.9 mol%. The level of DNA homology of the two strains was 98%. DNA from the strains isolated from marine invertebrates showed 5-15% genetic relatedness to the DNA of other type strains of the genus Pseudoalteromonas. 16S rRNA analysis indicated a clear affiliation of the novel bacteria to other species of the genus. The strains are assigned to a novel species, Pseudomonas ruthenica sp. nov., with the type strain KMM 300T (= LMG 19699T = CIP 106857T).

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm.

Abstract Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.

Amino acid intrinsic α‐helical propensities III: Positional dependence at several positions of C terminus

In this study, we have analyzed experimentally the helical intrinsic propensities of noncharged and nonaromatic residues at different C‐terminal positions (C1, C2, C3) of an Ala‐based peptide. The effect was found to be complex, resulting in extra stabilization or destabilization, depending on guest amino acid and position under consideration. Polar (Ser, Thr, Cys, Asn, and Gln) amino acids and Gly were found to have significantly larger helical propensities at several C‐terminal positions compared with the α‐helix center (−1.0 kcal/mole in some cases). Some of the nonpolar residues, especially β‐branched ones (Val and Ile) are significantly more favorable at position C3 (−0.3 to −0.4 kcal/mole), although having minor differences at other C‐terminal positions compared with the α‐helix center. Leu has moderate (−0.1 to −0.2 kcal/mole) stabilization effects at position C2 and C3, whereas being relatively neutral at C1. Finally, Met was found to be unfavorable at C1 and C2 ( +0.2 kcal/mole) and favorable at C3 (−0.2 kcal/mole). Thus, significant differences found between the intrinsic helical propensities at the C‐terminal positions and those in the α‐helix center must be accounted for in helix/coil transition theories and in protein design.