Development of a high-level expression system for deuterium-labeled human serum albumin

We have developed an expression system for recombinant human serum albumin (rHSA) using methylotrophic yeast Pichia pastoris Mut(+) transformants together with the multiple cross-over integration of the vector containing human serum albumin (HSA). After 86 h of methanol induction, the secreted rHSA reached levels of approximately 320 mg/l in 100% H(2)O medium and approximately 180 mg/l in 70% D(2)O/30% H(2)O (v/v) medium in a fed-batch fermenter. The structures of the obtained rHSA and plasma-derived HSA (pHSA) were virtually identical as viewed from various physicochemical techniques such as HPLC, SDS gel electrophoresis, and CD. NMR peaks of the partially deuterium (D)-labeled rHSA (DrHSA) were quite sharp compared to those of pHSA due to suppression of the intramolecular nuclear Overhauser effect, promising further structural studies of the whole HSA molecule in the solution state using the recent NMR techniques.     

Three TNFalpha single nucleotide polymorphisms in the Japanese population

BACKGROUND: Tumour necrosis factor-alpha (TNFalpha) is an essential regulator of immune responses and is implicated to relate to several types of disease susceptibilities. Population information on polymorphisms is essential for the study of genetic diseases. AIM: To obtain accurate information about single nucleotide polymorphisms (SNPs) in the TNFalpha gene in the Japanese population. SUBJECTS AND METHODS: The entire TNFalpha gene was screened for SNPs by directly sequencing 48 chromosomes derived from 24 unrelated Japanese individuals. Allele frequencies of each polymorphism were determined and compared with those previously reported in other populations. RESULTS: Three SNPs, -308G/A at nt -308, IVS1 + 125G/A at nt 492 and IVS3 + 104G/A at nt 1359 were observed, of which one (IVS3 + 104G/A at nt 1359) was novel. In addition, allele frequencies of -308G/A were remarkably different from those presented in the NCBI dbSNP, indicating a significant ethnic difference. CONCLUSIONS: The polymorphisms and allele frequencies obtained in this study will be useful for genetic studies of common diseases such as osteoporosis and rheumatoid arthritis in the Japanese population.     

Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in treatment of corneal alkali burns in mice

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 microg/microl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semi-quantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-kappaB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-alpha in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-alpha, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-alpha-/- mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.     

Simple,rapid, and accurate determination of deletion mutations by automated dna sequencing of heteroduplex fragments of the adenomatous polyposis coli (APC) gene generated by PCR amplification

Germline mutations in patients with familial adenomatous polyposis were analyzed by polymerase chain reaction (PCR) amplification of the adenomatous polyposis coli gene. PCR products from heterozygous patients for deletions of this gene formed four distinct bands on polyacrylamide gel electrophoresis. The four fragments were subsequently purified and both strands of each fragment were directly sequenced, using an automated DNA sequencer and the same primers as those for PCR amplification. It was found that the two slower migrating fragments were “bulge” heteroduplexes, while the other two were homoduplexes made up of two wild-type strands and two deletion-mutant strands, respectively. The sites of deletions in the adenomatous polyposis coli gene could be exactly determined in four of the five patients. In an attempt to identify deletion-carriers of familial adenomatous polyposis at the presymptomatic stage, a family study was also carried out, and two children were found to have the same mutations as those of their affected parents. The direct sequencing of heteroduplex fragments generated during PCR amplification is a potentially useful method for detecting mutations of not only the adenomatous polyposis coli gene but also many other genes of genetic diseases. © 1993 Wiley-Liss, Inc.     

Numerical aberrations of chromosome 9 in bladder cancer. A possible prognostic marker for early tumor recurrence

Bilharzial bladder cancer is one of the most common types of malignancy in both men and women in several developing countries including Egypt. It has several unique clinical, epidemiological, and histological characteristics, suggesting that it is an entity distinct from bladder cancer seen in Western countries. Genetic alterations in bilharzial-related bladder cancer have been studied infrequently, especially in the advanced stages of disease, that is, T3 and T4 classifications. The objective of this study was to extend establishing the baseline cytogenetic profile of this type of malignancy to early T1 and T2 classifications. For this purpose, fluorescence in situ hybridization was applied to interphase nuclei of frozen-stored samples with biotinylated repetitive DNA probes specific for all chromosomes to detect numerical chromosome changes in 35 patients presenting with relatively early-stage pT1 and pT2 disease. Eleven cases had squamous cell carcinoma (SCC) and 24 had transitional cell carcinoma. Six of 24 transitional cell carcinomas had diploid chromosome counts with all the probes. Numerical chromosome aberrations were detected in 18 cases (75%). In 12 cases, a loss of chromosome 9 was observed. In three cases, an additional loss of chromosome 17 was detected. One case demonstrated a loss of chromosome 10, whereas another two cases showed a gain of chromosome 7, next to a loss of chromosome 9. Loss of chromosome Y was observed in nine of the 27 male cases studied (33.3%), in which only one case showed an abnormality whereas four cases were detected next to loss of chromosome 9, and one case showed gain of chromosome 7. Five cases showed loss of chromosome 19 whereas gain of chromosome 4 was detected in two cases. Two of 11 samples of SCC had normal diploid chromosome counts with all the probes used. In four of 11 cases (36.4%) underrepresentation of chromosome 9, compared with the other chromosomes, was detected. An additional loss of chromosome 17 and gain of chromosome 7, next to loss of chromosome 9, was detected in three cases. One case showed loss of chromosome 17 as the only numerical aberration. Loss of the Y chromosome was detected in three cases of which one case had gain of chromosome 7 and one case had loss of chromosome 19. No correlation was found between any of the clinicopathologic parameters examined in this study and the presence or absence of any numerical chromosomal aberrations except for the significant association between schistosomal history and loss of Y chromosome (P=0.007).     

Genetic polymorphisms of orosomucoid and alpha-2-HS-glycoprotein in Thai,Sri Lankan and Paraguayan populations

The genetic polymorphism of orosomucoid (ORM) and alpha-2-HS-glycoprotein (AHSG) were studied in Thai, Sri Lankan and Paraguayan populations using isoelectric focusing. Gene frequencies in these populations were compared with those in other populations. Four new ORM variants were detected:ORM1 ^* 15 in Thai,ORM1 ^* 16 in Paraguayan,ORM2 ^* 21 andORM2 ^* 22 in Sri Lankan.     

HISTOPATHOLOGICAL STUDY ON PROGRESSIVE MUSCULAR DYSTROPHY—REPORT OF A CASE WITH AUTOPSY FINDINGS—

A typical case of the D uchenne type of progressive muscular dystrophy with autopsy findings was presented. Changes in the myocardial and smooth muscle of many organs were found, and the skeletal muscles also revealed florid changes.
Histopathological examination of the skeletal muscle was made in detail through light and electron microscopic observation.     

Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Activation of SiO2-supported zirconocene catalysts by common trialkylaluminiums

A modified SiO₂ was prepared by reacting SiO₂ with Cl₂Si(CH₃)₂ in toluene, on which methylaluminoxane (MAO) was supported to obtain a catalyst precursor. The mixture of the precursor and Cp₂ZrCl₂ (Cp: cyclopentadienyl) gave polyethylene in a high yield even by using common trialkylaluminiums as cocatalyst. Surprisingly, the MAO-free catalyst system composed of the modified SiO₂ and Cp₂ZrCl₂ was also found to be activated by common trialkylaluminiums.