Dentilisin Activity Affects the Organization of the Outer Sheath of Treponema denticola

Prolyl-phenylalanine-specific serine protease (dentilisin) is a major extracellular protease produced by Treponema denticola. The gene, prtP, coding for the protease was recently cloned and sequenced (K. Ishihara, T. Miura, H. K. Kuramitsu, and K. Okuda, Infect. Immun. 64:5178–5186, 1996). In order to determine the role of this protease in the physiology and virulence of T. denticola, a dentilisin-deficient mutant, K1, was constructed following electroporation with a prtP-inactivated DNA fragment. No chymotrypsin-like protease activity was detected in the dentilisin-deficient mutant. In addition, the high-molecular-mass oligomeric protein characteristic of the outer sheath of the organism decreased in the mutant. Furthermore, the hydrophobicity of the mutant was decreased, and coaggregation of the mutant with Fusobacterium nucleatum was enhanced compared to that of the wild-type organism. The results obtained with a mouse abscess model system indicated that the virulence of the mutant was attenuated relative to that of the wild-type organism. These results suggest that dentilisin activity plays a major role in the structural organization of the outer sheath of T. denticola. The loss of dentilsin activity and the structural change in the outer sheath affect the pathogenicity of T. denticola.     

Tin-Carbon Cleavage of Organotin Compounds by Pyoverdine from Pseudomonas chlororaphis

The triphenyltin (TPT)-degrading bacterium Pseudomonas chlororaphis CNR15 produces extracellular yellow substances to degrade TPT. Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized. The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from P. chlororaphis ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads. Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I. Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants. F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively. The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production. The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine. On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu²⁺ and Sn⁴⁺, respectively. These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states.     

Effects of shrimp density on transmission of penaeid acute viremia in Penaeus japonicus by cannibalism and the waterborne route.

To investigate the effects of shrimp density on mortalities of Penaeus japonicus in experimental penaeid acute viremia (= white spot syndrome), shrimp injected intramuscularly with penaeid rod-shaped DNA virus (PRDV) were reared at different densities. In Expt 1, challenged (10(-6) dilution of a PRDV preparation) shrimp were reared collectively in a tank or individually in separate chamber units. A significant difference in cumulative mortalities was found between collectively (75.6%) and individually (1.2%) reared groups after 30 d. In Expt 2, effects of density on mortality were clearly shown when challenged (10(-5) dilution) shrimp were reared collectively in tanks at high (260 shrimp m(-2)), middle (135 shrimp m(-2)) and low densities (73 shrimp m(-2)). The cumulative mortalities for 14 d in the high, middle and low density groups were 72, 46 and 18%, respectively. In Expt 3, challenged (10(-5) dilution) shrimp were reared collectively in 3 tanks (Groups A, B and C) at the same high density (260 shrimp m(-2)): Group A, dead shrimp were immediately removed to avoid transmission of the pathogen through cannibalism and the waterborne route; Group B, dead shrimp were removed at scheduled times but were separated from living shrimp by a net partition to avoid cannibalism; and Group C, dead shrimp were removed twice a day at scheduled times. Resulting cumulative mortalities for 20 d in Groups A, B and C were 4, 24 and 64 %, respectively. These results show that the higher mortalities occur in P. japonicus reared at the higher densities in experimental PRDV infection, and this phenomenon is caused mainly by a higher opportunity of horizontal transmission of the virus through cannibalism and the waterborne route.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

Role of isozyme group-specific sequence 4 in the isozyme-specific properties of human aldolase C

To assess which regions of the aldolase C molecule are required for exhibiting isozyme-specific kinetic properties, we have constructed nine chimeric enzymes of human aldolases A and C. Kinetic studies of these chimeric enzymes revealed that aldolase C absolutely required its own isozyme group-specific sequences (IGS), particularly IGS-4, for exhibiting the characteristics of aldolase C which differ significantly from those of isozymes A and B (Kusakabe T, Motoki K, Hori K. Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli. J Biochem (Tokyo) 1994;115:1172–7). Whereas human aldolases A and B required their own isozyme group-specific sequences-1 and -4 (IGS-1 and -4) as the main determinants of isozyme-specific kinetic properties (Motoki K, Kitajima Y, Hori K. Isozyme-specific modules on human aldolase A molecule. J Biol Chem 1993;268:1677–83; Kusakabe T, Motoki K, Sugimoto Y, Takasaki Y, Hori K. Human aldolase B: liver-specific properties of the isoenzyme depend on type B isozyme group-specific sequence. Prot. Eng. 1994;7:1387–93), the present studies indicate that the IGS-1 is principally substitutable between aldolases A and C. The kinetic data also suggests that the connector-2 (amino acid residues 243–306) may modulate the interaction of IGS units with the α/β barrel of the aldolase molecule.     

Cultivation characteristics ofIsaria japonica

Cultivation characteristics of fruit-body (synnema) formation ofIsaria japonica were examined using liquid and solid media in order to produce fruit-bodies on a large scale. Mycelia grew well at 18–28°C on PDA medium with an initial pH of 7.0. The formation of fruit-bodies ofI. japonica was induced by lowering temperature to below 20°C in PD liquid medium. In sawdust-rice bran basal medium mixed with pupal powder prepared from silkworms (Bombyx mori), the fresh weight of fruit-bodies increased with increasing content of pupal powder. The highest yields of fruit-bodies were obtained in carbon-rich barley grain medium supplemented with pupal powder. The fruit-bodies grown under CO₂ concentrations of 1,000 μl/L had coral-like, many-branched synnemata with numerous conidiospores, whereas those formed under high concentrations (9,000 μl/L) of CO₂ had unbranched and longer synnemata. High concentrations of CO₂ remarkably inhibited conidiospore formation on synnemata. Continuous high-intensity illumination at 2.93 W·m⁻² inhibited the elongation of synnemata, and low-intensity illumination at 0.088 W·m⁻² slightly inhibited the branching of synnemata. Fruit-bodies were produced on the pupa metamorphosed from living larvae ofAgrotis fucosa placed on the surface of a culture ofI. japonica incubated in sawdust-rice bran medium.     

Enhancement of interferon-β production with sphingomyelin from fermented milk

A fermented milk, Kefir, contains an active substance which enhances IFN- secretion of a human osteosarcoma line MG-63 treated with a chemical inducer, poly I: poly C. The active substance in the fermented milk was identified to be sphingomyelin (SpM) by a combined use of a fast atom bombardment mass spectrometry (FAB-MS) and a fast atom bombardment tandem mass spectrometry (FAB-MS/MS). SpM from fermented milk (F-SpM) was a mixture of four molecular species of SpMs having C₂₁-, C₂₂-, C₂₃- and C₂₄-fatty acids. F-SpM enhanced the IFN secretion 14 times, SpMs from other sources also enhanced moderately (2–3 times). Sphingosine and lysosphingomyelin also enhanced the activity but ceramide and cerebroside did not.Abbreviations IFN- interferon- - SpM sphingomyelin - Lyso-SpM lysosphingomyelin - SpS sphingosine - FAB-MS fast atom bombardment mass spectrometry - FAB-MS/MS fast atom bombardment tandem mass spectrometry     

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.)

Summary The number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.     

Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells

In order to enhance recombinant protein productivity in animal cells, we developed the oncogene activated production (OAP) system. The OAP system is based on the premise that oncogenes are able to enhance promoter activity. To this end, we constructed reported plasmids by fusing various promoters to the human interleukin-6 (hIL-6) cDNA, and the effector plasmids by inserting individual oncogenes, for example c-myc, c-fos, v-jun, v-myb and c-Ha-ras, downstream from the human cytomegalovirus immediate early (CMV) promoter. Results of transient expression experiments with BHK-21 cells suggest that the CMV promoter is the most potent promoter examined and that theras product is able to transactivate the -actin, CMV and SR promoters. Recombinant BHK-21 cells producing hIL-6 under the control of the CMV promoter were contransfected with theras oncogene and dihydrofolate reductase gene, then selected with 50 nM methotrexate to coamplify theras oncogene. We were able to rapidly establish a stable and highly productive clone which exhibited a 35-times higher production rate as compared to the control value.     

Chromosomal Assignments of Novel Genes Expressed in HL60 Granulocytes

By collecting 3'-directed cDNA sequences called gene signatures(GSs) on a large scale, it is possible to make an expressionprofile of genes in a particular tissue, as well as discoveringa number of novel genes. A total of 305 novel GSs collectedfrom granulocytoid cells derived from HL60, a human promyelocyticleukemia cell line, by exposure to dimethyl sulfoxide were radiolabeledand used for Southern blot analyses to determine the copy numberof the corresponding genes. Of these, 198 GSs identified asrepresenting single-copy genes were then used as probes forhybridization analyses using a monochromosomal hybrid cell DNApanel. Sixty-nine of them were assigned to individual chromosomes.These results demonstrate that the chromosomal distributionof the GSs seems not to be proportional to the cytogenetic lengthof each chromosome.