An exact test for the association between the disease and alleles at highly polymorphic loci with particular interest in the haplotype analysis

The association analysis between the disease and genetic alleles is one of the simple methods for localizing the susceptibility locus in the genes. For revealing the association, several statistical tests have been proposed without discussing explicitly the alternative hypotheses. We therefore specify two types of alternative hypotheses (i.e., there is only one susceptibility allele in the locus, and there is an extension or shortening of alleles associated with the disease) and derive exact tests for the respective hypotheses. We also propose to combine these two tests when the prior knowledge is not sufficient enough to specify one of these two hypotheses. In particular, these ideas are extended to the haplotype analysis of three-way association between the disease and bivariate allele frequencies at two closely linked loci. As a by-product, a factorization of the probability distribution of the three-way cell frequencies under the null hypothesis of no three-way interaction is obtained.     

Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate

The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. AspATs from many species were classified into aminotransferase subgroups Ia and Ib. The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved. The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them. (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site. However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate. (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate. The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT.     

Biological characteristics of cultured cells derived from various types of human brain tumors

We placed in culture brain tumors from 45 cases (7 cases of astrocytoma, 2 from oligodendrogliomas, 2 glioblastomas, 2 ependymomas, 13 meningiomas, 6 pituitary adenomas, 5 neurinomas, a malignant lymphoma, a choroid plexus papilloma, and 6 metastatic tumors) and succeeded in making a primary culture from 33, and maintained 17 in vitro over a considerable period of time (greater than three months). In the early period of the primary cultures, the astrocytoma cells had cytoplasmic processes which contacted each other, the oligodendroglioma cells were small and spindle-shaped, the glioblastoma cells were neoplastic with pleopmorphic features and possessed cytoplasmic processes, the ependymoma cells formed a rosette-like cell arrangement, the meningioma cells were spindle- or round-shaped cells and characterized as forming psammoma bodies, the pituitary adenoma cells were round- or oval-shaped cells and produced growth hormone (GH), adenocorticoid tropic hormone (ACTH), prolactin, or other hypophyseal hormones, the choroid plexus papilloma cells were round-or polygonal and showed a papillary cell arrangement, the neurinoma cells were spindle- or fibrous-shaped cells, and the malignant lymphoma cells were round and formed cell aggregates floating in the culture medium.     

Establishment and characterization of human uterine cervical epidermoid carcinoma cell line HHUS containing HPV 59 DNA

The cell line designated HHUS was established from human uterine cervical keratinizing squamous cell carcinoma. The HHUS cell line was subcultivated more than 70 times within 3 years. The cultured cells, polygonal or spindle, with neoplastic and pleomorphic features, appeared epithelial in shape, with a pavement-like arrangement and grew in multi-layers without contact inhibition. The chromosome number was varied from 40 to 88, and the modal number was stable in diploid range. The cultured cells produced keratinizing squamous cell carcinomas by heterotransplantation into the subcutis of nude mice. The HHUS cells were characterized as producing large amounts of SCC, in vitro and possessing HPV-59 DNA genomes.     

Changes in the thermal dissipation and the electron flow in the water-water cycle in rice grown under conditions of physiologically low temperature

Effects of low temperature on chlorophyll (Chl) fluorescence, gas exchange rate, the amounts of xanthophyll cycle pigments (Xp) and the activities of several antioxidant enzymes were examined in the 8th leaf of two rice (Oryza sativa L.) cultivars (japonica and indica types) and rbcS antisense rice. All plants were grown hydroponically at 25/20 degrees C (day/night), and then exposed to 20/17 degrees C (day/night) after full expansion of the 8th leaf, or exposed to either 20/17 degrees C or 15/13 degrees C (day/night) during the expansion of the 8th leaf. All plants exposed to low temperatures showed a decrease in CO(2) assimilation rate without photoinhibition, and increases in the fraction of thermal dissipation in PSII, and in the electron flux through the water-water cycle (WWC) were observed. Although the increase of thermal dissipation was associated with increases in the ratio of carotenoids to Chl, the ratio of Xp to carotenoids and the de-epoxidation state of Xp, the increase of the electron flux of WWC was not accompanied by an increase in the activities of antioxidant enzymes. Such photoprotective responses did not differ between during and after full expansion of the leaf, and did not differ among the three genotypes. Quantitative analyses on the dissipation of excess light energy showed that thermal dissipation makes a larger contribution than WWC. Thus, although low temperature led to a decrease in CO(2) assimilation, rice potentially coped with the excess light energy by increasing the thermal dissipation and the electron flux of WWC under low temperature irrespective of leaf development and genotypes.     

Cytokine mRNA expression in unilateral ischemic-reperfused rat lung with salt solution supplemented with low-endotoxin or standard bovine serum albumin

Our aim was to determine whether cytokine mRNA expression is induced by experimental manipulation including artificial perfusate or ischemia-reperfusion (I/R) in an isolated, perfused rat lung model. Constant pulmonary flow [Krebs-Henseleit solution supplemented with low-endotoxin (LE) or standard (ST) bovine serum albumin 4%, 0.04 ml/g body wt] and ventilation were maintained throughout. Right and left pulmonary arteries were isolated, and the left pulmonary artery was occluded for 60 min and then reperfused for 30 min. Analysis of tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-10, and IFN-gamma mRNA expression by RT-PCR and evaluation of vascular permeability by bronchoalveolar lavage (BAL) fluid albumin content were conducted separately in right and left lung. Both LE and ST groups (each 12 rats) showed increases in vascular permeability by I/R (BAL fluid albumin content: 5.53 +/- 1.55 vs. 15.63 +/- 8.87 and 4.76 +/- 2.71 vs. 16.72 +/- 4.85 mg.ml BAL fluid-1.g lung dry wt-1, mean +/- SD; right vs. left lung in LE and ST groups, P < 0.05 between right and left). Cytokine mRNA expression was significantly higher in the I/R lung than in the control lung in the LE group, whereas it was higher in the control lung in the ST group (P < 0.05). mRNAs of not only proinflammatory but also anti-inflammatory cytokines were expressed in I/R lung, which are expected to aggravate I/R injury. The reversed pattern of cytokine mRNA expression in the ST group was possibly due to the longer perfusion of control lung with perfusate containing endotoxin, which caused no lung damage without I/R.     

Characterization of histidinol phosphate aminotransferase from Escherichia coli

Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6. The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn(157) to Ala increased the pK(a) to 9.2. Replacement of Arg(335) by Leu increased the pK(a) to 8.6. On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.     

The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro

To elucidate the function of protein disulfide isomerase (PDI), we screened for PDI-binding proteins in a bovine liver extract using affinity column chromatography. One of the binding proteins was identified by SDS-PAGE and N-terminal amino acid sequence analysis to be cyclophilin B (Cyp B). Use of the BIACORE system revealed that purified bovine Cyp B bound specifically to bovine PDI with a K(D) value of 1.19 x 10(-5) M. Interestingly, the binding affinity between PDI and Cyp B was strengthened by preincubation of the Cyp B with cyclosporin A (CsA), yielding a K(D) value of 3.67 x 10(-6) M. Although the interaction between PDI and Cyp B affected neither the isomerase activity of PDI nor the peptidyl-prolyl cis-trans isomerase activity of Cyp B, Cyp B increased the chaperone activity of PDI. However, the complex of Cyp B and CsA completely inhibited the chaperone activity of PDI. Thus, PDI and Cyp B appear to cooperate with each other to regulate the functional expression of proteins in vivo.     

Establishment and characterization of a lymphoepithelial-like carcinoma cell line (HUUCLEC) derived from the human uterine cervix

A cell line designated HUUCLEC was established from a human uterine cervical lymphoepithelial carcinoma obtained from a 61-year-old Japanese woman. The cell line has grown slowly without interruption and serial passages were successively carried out 60 times within 3 years. The cultured cells were spindle or round in shape, showing anaplastic and pleomorphic features, a pavement cell arrangement and multilayering without contact inhibition. The population doubling time of the HUUCLEC line was 72 hours while the chromosomal number varied widely and showed aneuploidy. The modal chromosomal number was stable at the triploid range and marker chromosomes were present; the Ebstein-Barr virus was absent in the cultured cells.