Recovery process of arthritis induced by 6-sulfanilamidoindazole (6SAI) in rats

6-Sulfanilamidoindazole (6SAI) is known to induce not only an acute arthritis but also serositis and arteritis which resemble those induced by some vasodilators in rats. In this study, the recovery process of ankle lesions was examined histopathologically for up to 12 weeks of recovery period in rats bearing arthritis induced by administration of 6SAI (500 mg/kg) for 2 weeks. At 2 weeks of 6SAI-treatment, exudative synovitis and exudative/edematous periarthritis with marked formation of granulation tissues and periosteal reactive bone formation were noted in the ankles, but no remarkable neutrophil infiltration was detected in those lesions. The ankle swelling induced by 6SAI diminished by 4 weeks of recovery period, and the elevated plasma fibrinogen levels were normalized by 2 weeks of recovery period. Although fibrosis and newly-formed periosteal bone were still observed after 2 weeks of recovery period, no inflammatory lesion was detected at that point. At 4 or 12 weeks of recovery periods, the ankles showed an almost normal appearance. These results indicate that 6SAI-induced arthritis is reversible in nature and does not develop into chronic phase.     

Crystallization, stability, and enzymatic degradation of poly(L-lactide) thin film

Poly(L-lactide) (PLLA) thin film with 100 nm thickness was crystallized at 160 degreesC for 20 min from the melt obtained at 220 degreesC. Hexagonal crystals with three types of growth (derivative growth lamellae, overgrowth multistacked lamellae, and undergrowth multistacked lamellae) were simultaneously observed by atomic force microscopy (AFM). These phenomena are due to the differences of the formative points of secondary crystal nuclei against the basal lamella. Enzymatic degradation of PLLA thin film revealed two types of amorphous regions. These regions were identified as the free amorphous region around the crystals and the restricted amorphous region between the crystal and glass substrate. In situ observation of thermal behavior of lamellar crystals was performed to understand the correlation between the chain folding and stability of the crystal by using temperature-controlled AFM. The morphology of the sectors with [100] growth plane had changed to a comblike morphology despite the fact that the [110] growth plane remained unchanged, suggesting that the stability of the chain folding and the chain-packing state affected the thermal behavior.     

Apparent lack of trans-dominant negative effects of various vif mutants on the replication of HIV-1

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for virus growth in non-permissive cells such as H9. To elucidate the mechanism of action of the Vif protein, vif mutants, which show trans-dominant negative effects on the replication of HIV-1, would be useful tools. In this study, a new assay system to identify the mutants of this category was established. For this new system, various reporter clones carrying both mutant and authentic vif sequences were generated. By determining the growth ability of the viruses derived from the reporter constructs, the potential negative effect of the mutant vif sequence was readily and sensitively monitored. Ten vif mutant sequences tested were found not to exert the trans-dominant negative effect on the replication of HIV-1.     

Indirect conductimetric assay of antibacterial activities

The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders. Received 02 April 2002/ Accepted in revised form 30 July 2002     

Molecular phylogeny of genus Vigna subgenus Ceratotropis based on rDNA ITS and atpB-rbcL intergenic spacer of cpDNA sequences

The genetic diversity and phylogenetic relationships among species in the genus Vigna subgenus Ceratotropis were investigated using sequence data from the ribosomal DNA ITS and atpB-rbcL intergenic spacer of chloroplast DNA regions. While both sets of sequences were of similar lengths about 700bp the rDNA-ITS was more informative than atpB-rbcL having 170% more polymorphic sites and five times as many parsimony-informative sites. The atpB-rbcL spacer may be appropriate for analysis of taxa above the species level in the genus Vigna. Results of analyzing rDNA-ITS revealed, with low level of statistical bias, separation of the subgenus into three groups that correspond to the three sections Aconitifoliae, Angulares, and Ceratotropis. The ancestral section is Aconitifoliae based on comparison with the outgroup species cowpea, Vigna unguiculata. The V. minima complex, V. minima, V. riukiuensis, and V. nakashimae, has a distinct evolutionary path within section Angulares. Other species in section Angulares are very closely related except V. trinervia. Vigna trinervia has an intermediate position between sections. Sequence data suggests one genome donor to V. reflexo-pilosa came from a lineage within section Angulares close to V. exilis, V. hirtella, and V. umbellata. Data presented supports the view that section Angulares is the most recently diversified section in the subgenus, as inferred by short terminal branch lengths among the species of this section.     

Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD

Enhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes. In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique. The library was constructed by in vitro recombination of the genes for EngB and non-cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous. To obtain thermostable clones without loss of activity, the library was screened by a combination of activity and thermostability screening. We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities. One of the mutants possessed a sevenfold higher thermostability than EngB. The possible mechanisms of thermostabilization are discussed.     

Interactions of endothelin receptor subtypes A and B with Gi, Go, and Gq in reconstituted phospholipid vesicles

To understand the biochemical basis for the functional divergence of the human endothelin receptor subtypes A (ETAR) and B (ETBR), they were expressed, purified from insect Sf9 cells, and reconstituted into phospholipid vesicles with the Go, Gq, and Gi proteins. For each G protein, a unique pattern of reactivity was observed with the different receptor subtypes. Both ETAR and ETBR activated Go to a similar maximal extent, and both subtypes activated Gq with similar EC50 values; however, the ETAR displayed a 2-3-fold higher maximal extent of activation. In contrast, both subtypes activated Gi to a similar maximal extent, but the ETAR displayed a 4-fold higher EC50 value as compared to the ETBR. To test whether these coupling specificities are influenced by C-terminal palmitoylation of the receptor, we mutated a cluster of cysteine residues near the end of the seventh transmembrane helix in both receptors. While the cysteine mutations in the ETBR resulted in a partially palmitoylated receptor, the replacement of these cysteine residues in the ETAR yielded a mostly palmitoyl-deficient receptor and had no effect on Go activation, but caused a reduction in the extents of Gi and Gq stimulation. Together, these studies provide important insights into the specificity of G protein coupling in the endothelin receptors. The ability to discriminate between the different G proteins under various physiological conditions may be a key element in the selection of distinct signal transduction pathways by the two receptor subtypes.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.     

Two enzymatic reaction pathways in the formation of pyropheophorbide a

The demethoxycarbonyl reaction of pheophorbide a in plants and algae was investigated. Two types of enzyme that catalyze alternative reactions in the formation of pyropheophorbide a were found. One enzyme, designated `pheophorbidase (Phedase)', was purified nearly to homogeneity from cotyledons of radish (Raphanus sativus). This enzyme catalyzes the conversion of pheophorbide a to a precursor of pyropheophorbide a, C-13<sup>2</sup>-carboxylpyropheophorbide a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield pyropheophorbide a. The activity of Phedase was inhibited by the reaction product, methanol. The other enzyme, termed `pheophorbide demethoxycarbonylase (PDC)', was highly purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii. This enzyme had produced no intermediate as shown in the Phedase reaction, indicating that it converts pheophorbide a directly into pyropheophorbide a, probably by nucleophilic reaction. Phedase and PDC consisted of both senescence-induced and constitutive enzymes. The molecular weight of both Phedases was 113 000 and of senescence-induced PDC was 170 000. The K _m values against pheophorbide a for both Phedases were 14–15 μM and 283 μM for senescence-induced PDC. The activity of both Phedases was inhibited by the reaction product, methanol, whereas methanol had no specific effect on senescence-induced PDC. Phenylmethylsulfonic fluoride and N-ethylmaleimide inhibited the senescence-induced Phedase and PDC, respectively. Among the 23 species from 15 different families tested, Phedase activity was found in 10 species from three families. PDC activity was not detected in plants lacking Phedase activity, except for Chlamydomonas. Based on these findings, a likely conclusion is that at least two alternative pathways that are catalyzed by two different enzymes, Phedase and PDC, exist for the formation of pyropheophorbide a. This revised version was published online in June 2006 with corrections to the Cover Date.