Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Gap Junctions Mediate Glucose Transport Between GLUT1-Positive and -Negative Cells in the Spiral Limbus of the Rat Cochlea

To elucidate the role of the spiral limbus in glucose transport in the cochlea, we analyzed the expression and localization of GLUT1, connexin26, connexin30, and occludin in the spiral limbus of the rat cochlea. GLUT1 and occludin were detected in blood vessels. GLUT1, connexin26, connexin30, and occludin were also expressed in fibrocytes just basal to the supralimbal lining cells. Connexin26 and connexin30 were present among not only these GLUT1-positive fibrocytes but also GLUT1-negative fibrocytes. In vivo glucose imaging using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG, MW 342) together with Evans Blue Albumin (EBA, MW 68,000) showed that 6-NBDG was rapidly distributed throughout the spiral limbus, whereas EBA was localized only in the vessels. Moreover, the gap junctional uncoupler heptanol inhibited the distribution of 6-NBDG. These findings suggest that gap junctions play an important role in glucose transport in the spiral limbus, i.e., that gap junctions mediate glucose transport from GLUT1-positive fibrocytes to GLUT1-negative fibrocytes in the spiral limbus.     

Analysis of two distinct B cell activation pathways mediated by a monoclonal T helper cell. I. MHC-restricted activation of B cells by an IL 2-dependent pathway

The present study was carried out to determine whether the MHC-restricted and MHC-unrestricted B cell activation pathways mediated by a single cloned Th cell are separable, and whether these two pathways are mediated by distinct mechanisms. It was demonstrated that the two B cell activating functions of a single cloned Th cell could be separated by their sensitivity to irradiation. It was shown that MHC-restricted B cell activation is mediated by a radiosensitive Th cell function, whereas MHC-unrestricted B cell activation is mediated by a radioresistant function of the same Th cell. In addition, it was shown that recombinant IL 2 can restore or replace the radiosensitive component of MHC-restricted cognate helper function.     

Evolutionary trends of the hairy-faceSaguinus in terms of the dental and cranial morphology

Shape variations in the dentition and the cranium were analyzed for sevenSaguinus forms of the hairy-face tamarin by applying the factor analysis method. The results obtained for the dental and cranial measurements were almost consistent with each other. The magnitude of the difference in shape factors between theS. nigricollis group and theS. midas group is appreciably larger than that between the former group and theS. mystax group. If the ancestral geographic centre of origin is postulated as being within the region which is inhabited by the livingS. nigricollis group, the morphological distances between any pairs of groups correlate well with the geographic distances between them. Concerning the dental and cranial morphologies, the physical changes in the three species group probably took place in two directions; that is, from theS. nigricollis group to theS. mystax group, and from theS. nigricollis group to theS. midas group. The forms belonging to each species group are more closely related to each other, with the exception ofS. imperator in theS. mystax group. The uniqueness ofS. imperator was clearly demonstrated by factor analysis and distance analysis. In theS. mystax group, although still hypothetical,S. imperator may have been related only through the basic ancestral stock toS. labiatus andS. mystax.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

Comparison of growth properties of carrot hairy root in various bioreactors

Summary Growth properties of carrot hairy root cells in various bioreactors were investigated. A turbine-blade reactor and an immobilized rotating drum reactor were found to be advantageous for the hairy root culture because of a high oxygen transfer coefficient (k in L a). After 30 days of culture, 10 g/l of dry hairy root cells were obtained in both bioreactors and maximum growth rates (V _m ) were found to be 0.63 and 0.61 g/l per day for the turbine-blade reactor and immobilized rotating drum reactor, respectively. Specific growth rates () at various cultivation times were observed to be linearly proportional to X/k _l a for both bioreactor configurations where X is the cell concentration. The estimated specific oxygen uptake rate of 0.34 mmol O₂/g dry cells per hour compares fairly well with an experimental value of 0.3.     

A cladistic analysis of interspecific relationships ofSaguinus

The dental and cranial morphologies of all species ofSaguinus, S. oedipus, S. geoffroyi, S. leucopus, S. nigricollis, S. fuscicollis, S. labiatus, S. mystax, S. imperator, S. bicolor, andS. midas are examined. The following hypotheses are developed by cladistic methodology, using only synapomorphic characters to assess the interspecific relationships ofSaguinus.Saguinus are divided into two main groups; one consists ofS. oedipus, S. geoffroyi, andS. leucopus, and the other includesS. inustus, S. nigricollis, S. fuscicollis, S. labiatus, S. mystax, S. imperator, S. bicolor, andS. midas. In the former group,S. oedipus is more closely related toS. geoffroyi than either is toS. leucopus. In the latter group,S. labiatus, S. mystax, andS. imperator are classified into one group, andS. bicolor andS. midas form one monophyletic group.     

Effects of antipyretics and anticancer drug on human cells at high temperature condition

The effects of antifebriles and anticancer drug on human vascular endothelial cells (HVE) and several cultured human cells were investigated. The HVE were isolated from umbilical cord veins by enzyme treatment and cultured successively in aerated synthetic medium, RPMI-1640, with 20% preclostrum new born calf serum. The presence of factor VIII antigen in the HVE was determined by enzyme-labeled antibody method. Cell count and protein amount were examined at regular intervals. At 3 hour-expose, sulpyrine was more toxic to the cultured cells than aspirin at 37 degrees C. The cytotoxicity of sulpyrine was markedly enhanced at 40 degrees C than at 37 degrees C. However, there was no enhancement in the cytotoxicity of aspirin at 40 degrees C. Cultured HVE and normal human fetal lung (HAIN-55) cells at 37 degrees C were sensitive to sulpyrine, and their sensitivity of the cells to the drug were markedly enhanced when they were incubated at 41 degrees C. In contrast, sensitivity of malignant human cells (HeLa cells) to sulpyrine was not found at 37 degrees C, however sensitivity of the cells to the drug was manifested at 41 degrees C of incubation. There was no effect of 5-fluorouracil (FU) on the growth of HVE and HAIN-55 cells at 41 degrees C, while HeLa cells showed high susceptibility to FU at the same temperature. The results showed the possibility that normal human cells may be sensitive to antifebrile drugs but not to anticancer drug at ordinary and high temperature, whereas malignant human cell may be susceptible to both antifebrile drugs and anticancer drug at high temperature.     

Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.