Change in Substrate Preference of Streptomyces Aminopeptidase through Modification of the Environment around the Substrate Binding Site

We attempted to alter the substrate preference of aminopeptidase from Streptomyces septatus TH-2 (SSAP). Because Asp198 and Phe221 of SSAP are located in the substrate binding site, we screened 2,000 mutant enzymes with D198X/F221X mutations. By carrying out this examination, we obtained two enzymes; one specifically hydrolyzed an arginyl derivative, and the other specifically hydrolyzed a cystinyl derivative (65- and 12.5-fold higher k_cat values for hydrolysis of p-nitroanilide derivatives than those of the wild type, respectively).     

Biochemical characterization of a novel metalloendopeptidase from Streptomyces aureofaciens TH-3 with post-proline hydrolysis activity

Streptomyces aureofaciens TH-3 secretes a protease termed ‘kibilysin’, for which we showed unique substrate specificity and preference for Tyr, Pro, and Leu at the P₁ position using fluorescence energy transfer substrate (FRETS) combinatorial libraries. Using (7-methoxycoumarin-4-yl) acetyl-Lys-Pro-Leu-Gly-Leu-d-2,3-diamino propionic acid (2,4-dinitrophenyl)-Ala-Arg-NH₂, we confirmed that kibilysin digests the substrate between Pro and Leu. Its gene was cloned and sequenced. The primary structure of the enzyme showed 40, 66, and 61% identity, respectively, with those of thermolysin from Bacillus thermoproteolyticus, and metalloendopeptidases from Streptomyces cinamoneus TH-2 and S. griseus. Its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, which is a common motif of the peptidase family M4. Moreover, we succeeded in over-expression of kibilysin using Streptomyces lividans.     

Prognostic value of CD208-positive cell infiltration in gastric cancer

Background and aim

A new marker, CD208, was recently explored as a mature interdigitating dendritic cell (DC), and the correlation between the infiltration of CD208-positive cells and clinical factors has been reported in various types of cancers. In this study, we tried to clarify the clinical implication of CD208-positive cell infiltration in gastric cancer immunohistochemically.

Patients and methods

A total of 128 gastric cancer patients who underwent a curative operation were enrolled. DCs in tumor nests were identified with two DC markers, CD208 and S-100 protein (S100), by immunohistochemistry. The correlation between clinicopathological features and the CD208- or S100-positive cell infiltration degree was analyzed.

Results

Infiltration of S100-positive cells did not correlate with the degree of CD208-positive cell infiltration. Patients with high CD208-positive cell infiltration in the peritumor had a poorer surgical outcome than those with low CD208 infiltration (p < 0.05). Multivariate analysis revealed that CD208-positive cell infiltration was not an independent prognostic factor.

Conclusion

We showed that intratumoral CD208-positive cells, as mature DCs, had an inverse correlation to patients’ postoperative outcome in gastric cancer, unlike a conventional DC marker. Evaluation of CD208-positive cell infiltration with S100-positive cell infiltration in gastric cancer is useful to predict antitumor immunological conditions in gastric cancer.     

Isolation and characterization of homocholine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strains A9 and B9b

Soil isolates, identified as Pseudomonas sp. strain A9 and Pseudomonas sp. strain B9b (based on the phenotypic features and phylogenetic analysis) were found to degrade homocholine aerobically. Morphological characterization using the optical microscope under light and phase contrast conditions showed that cells of strain A9 formed short rods measuring approximately 0.5–1 × 1.5–2.0 μm in size while those of B9b formed long rods of 0.5–1 × 2.5–3.0 μm during the early growth phase on both nutrient broth and basal-homocholine (basal-HC) media. Strain A9 was able to grow on basal-HC medium at a wide range of temperatures (4–41°C) whereas strain B9b was not able to grow at either 4 or 41°C. Comparative 16S rRNA sequencing studies indicated that strain A9 fell into the Pseudomonas putida subclade whereas strain B9b located in Pseudomonas fulva subclade. Washed cells of strains A9 and B9b degraded homocholine completely within 6 h with concomitant formation of several metabolites. Analysis of the metabolites by capillary electrophoresis, fast atom bombardment–mass spectrometry, and gas chromatography–mass spectrometry, showed trimethylamine (TMA) as the major metabolite beside β-alanine betaine and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strains is through successive oxidation of the alcohol group (–OH) to aldehyde (–CHO) and acid (–COOH), and thereafter the cleavage of β-alanine betaine C–N bonds yielding trimethylamine and an alkyl chain.     

Neuronal differentiation of neuro 2a cells by inhibitors of cell cycle progression, trichostatin A and butyrolactone I

Trichostatin A (TSA, 17 nM), a specific and reversible inhibitor of histone deacetylase induced neurite network formation at and after 4 days. The networks were preserved for at least 3 weeks in the presence of TSA. Butyrolactone I (BLI, 23.6 microM), an inhibitor of cdc2 and cdk2 kinases, also induced neurite extension. Both compounds enhanced the acetylcholinesterase activity of the cells. Cell cycle progression of the cells was blocked by TSA (17 nM) at G1 phase alone. Furthermore, the level of histone hyperacetylation and p21(WAF1) expression in TSA-treated cells increased transiently. These findings suggest that the induction of the neuronal differentiation in Neuro 2a cells by these agents requires the cell cycle arrest at G1 phase, which is caused by inhibition of cycline dependent kinase, a target molecule of BLI and p21(WAF1).     

Gene cloning and overproduction of an aminopeptidase from Streptomyces septatus TH-2, and comparison with a calcium-activated enzyme from Streptomyces griseus

An aminopeptidase secreted from Streptomyces septatus TH-2 (SSAP) was identified as a heat stable enzyme, and the Ssap gene was cloned and sequenced. The primary structure of SSAP showed 71% identity with that of a Streptomyces griseus aminopeptidase (SGAP), however, it lacked a unique calcium binding site. The recombinant SSAP was overexpressed in the culture supernatant of Escherichia coli harboring pET-KmS2. A comparison of recombinant SSAP and SGAP showed that both enzymes are different in terms of modulation by calcium and substrate specificity. The activity of SSAP was not modulated by calcium, while SGAP is a calcium-activated enzyme. SSAP catalyzed the hydrolysis of L-Lys-pNA efficiently whereas the reaction rate for L-Lys-pNA hydrolysis of SGAP was significantly low. Furthermore, in SGAP, the presence of Ca2+ decreased the reaction rate of L-Lys-pNA hydrolysis. SSAP also had different pKas s of reaction from that of SGAP, although almost all the residues which compose the active site were conserved in both enzymes. This result indicates that SSAP has a different environment of substrate binding and active sites from those of SGAP.     

Unique Distribution of <Emphasis Type="Italic">GAL</Emphasis> Genes on Chromosome XI in the Yeast <Emphasis Type="Italic">Saccharomyces naganishii</Emphasis>

A region of DNA extending from GAL7 to GAL1 was cloned in the yeast Saccharomyces naganishii. Sequence analysis revealed that GAL7 and GAL1 are separated by approximately 2 kbp and share a common promoter region. Although GAL7, GAL10, and GAL1 are clustered in this order in previously studied hemiascomycetous yeasts, GAL10 was not found between GAL7 and GAL1 in S. naganishii. Southern blotting of S. naganishii chromosomal DNA showed that both the GAL7–GAL1 region and GAL10 are located on chromosome XI, but that GAL10 is located more than 10 kbp away from GAL7–GAL1. Thus, S. naganishii and S. cerevisiae, while related phylogenetically, do not share the same orientation with respect to GAL genes. These data are highly relevant to studies of chromosomal evolution in yeast.     

Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.