Multiplex amplified product-length polymorphism analysis for rapid detection of human mitochondrial DNA variations.

A number of mutations in coding and noncoding regions of mitochondrial DNA (mtDNA) have previously been studied. In the present study, we simultaneously typed six mutation sites in the coding region by use of amplified product-length polymorphism (APLP) analysis. The mtDNA variations of 2471 individuals from 20 populations of Japanese, Korean, Chinese, and German were examined and classified into 18 haplotypes. Two of these haplotypes, B1 (estimated ancestral haplotype) and C1, were distributed among all populations tested. However, the haplotypes A1, A2, B2, B3, and C2 were mostly restricted to the Mongoloid populations, whereas haplotypes B5 and C5 appeared almost exclusively in the German population. Phylogenetic analysis by the neighbor-joining method revealed that the Japanese populations were more closely related to each other than to the other East Asian populations surveyed. The multiplex APLP method is suitable for large-scale screening studies of mtDNA variability because it is both rapid and economical.

Rat glioma cell death induced by cationic liposome-mediated transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir treatment.

BACKGROUND AND OBJECTIVES: We studied antitumor effects and cell death induced by cationic liposome-mediated gene transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir treatment in cultured rat T9 glioma cells and in experimental gliomas produced from this cell line. METHODS: To transfer genes we used small unilamellar cationic liposomes containing N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride. Video-enhanced contrast differential interference contrast microscopy was used for morphologic observations of cultured cells. RESULTS: When we treated the cells or implanted gliomas with the liposomes and ganciclovir, a strong effect was seen against tumor cells, and survival of tumor-implanted rats was increased. Morphologically, cell death observed after HSV-tk gene/liposome and ganciclovir treatment in the cultured glioma cells included both apoptosis and necrosis. CONCLUSIONS: Introduction of the HSV-tk gene in a DNA-liposome complex followed by ganciclovir treatment induced both apoptosis and necrosis, which together resulted in a potent antitumor effect.

A histological study on the mechanism of epidermal nuclear elongation in electrical and burn injuries.

Epidermal nuclear elongation is one of the most important signs for the diagnosis of electrical injury. In this study, we investigated the mechanism responsible for this phenomenon by comparing the findings from burn injuries and those from contusions. Electrical and burn injuries were made in the dorsal skin of rats using energy ranging from 100 to 790 joules for electrical injury, and 170-690 joules for burn injury. Contusions were also made by compressing the skin with a vice. In electrical and burn injuries, the dermis under the epidermal elongated nuclei was homogeneous and without empty spaces between collagen bundles and the number of dermal fibroblasts per 0.01 mm2 below the damaged epidermis decreased significantly (P < 0.05). The incidence of this change correlated with the depth of denatured dermal collagen fibres and in both types of injuries, dermal cells had no nuclear antigenicity for ubiquitin. The width of the injured epidermis with nuclear elongation decreased significantly (P < 0.05) and the elongated nuclei were parallel to the basal membrane. In electrical injury however, nuclear elongation occurred more frequently near the external root sheath. Nuclear elongation of fibroblasts and external root sheath cells was also found, but those of sebaceous gland cells were not detected. Epidermal elongated nuclei were also found in contusions. The evidence strongly suggests that epidermal nuclear elongation in electrical and burn injuries is due to dermal expansion by heat.

Production of a single-chain variable fragment antibody recognizing type III mutant epidermal growth factor receptor.

The type III deletion mutant of the epidermal growth factor receptor (EGFR) is a potential target in diagnostic and therapeutic approaches for those glioblastomas characterized by its expression. We previously raised a mouse monoclonal antibody, 3C10 (IgG2b) specifically recognizing this mutant EGFR. In this study, a single-chain variable fragment (scFv) antibody was produced. Partial determination of its N-terminal amino acid sequence and preparation of adequate primers for variable heavy chain (V(H)) and variable light chain (V(L)) genes were performed to allow cloning by means of reverse transcriptase-polymerase chain reaction. The genes cloned were assembled with a linker, (Gly4Ser)3, and ligated into a bacterial expression vector to express the scFv as cytoplasmic inclusion bodies. After appropriate refolding, the antibody activity of the V(H)-V(L) scFv was examined in an enzyme-linked immunosorbent assay. 3C10 scFv showed a selective reactivity with the mutant peptide, similarly to the parental 3C10 antibody. A mouse transfectant expressing the type III mutant EGFR and a glioblastoma with type III deletion-mutant EGFR were positively stained by immunofluorescence. By Biacore analysis, the affinity (K(A)) of the parental 3C10 for the mutant peptide was 9.7 x 10(7) M(-1), while that of 3C10 scFv was 2.45 - 2.48 x 10(7) M(-1), being approximately 4-fold weaker. The results together suggested that the scFv antibody retained the appropriate structure to recognize a conformational epitope of the mutant receptor, similarly to the parental antibody.

Efficacy of post operative adjuvant therapy with human interferon beta, MCNU and radiation (IMR) for malignant glioma: comparison among three protocols.

In order to develop ultimate adjuvant therapy for malignant gliomas, we analysed 77 patients with malignant gliomas (29 anaplastic astrocytomas (AAs) and 48 glioblastoma multiformes (GMs)) treated by three protocols of IMR therapy (human interferon-beta (HuIFN-beta), MCNU and radiation). In protocol 1 (n = 45: AA = 13, GM = 32), 1 x 10(6) IU of HuIFN-beta was administrated intravenously once a day for 7 days. On day 2, MCNU was administrated at a dose of 2 mg/kg b.w. intravenously and from day 3, radiation was started in five weekly fractions of 2 Gy for 6 weeks. Total dose was 60 Gy. Protocol 2 (n = 19: AA = 11, GM = 8) was comparable with protocol 1 except HuIFN-beta was administrated twice a day at a dose of 1 x 10(6) IU each. Protocol 3 (n = 13: AA = 5, GM = 8) differed from protocol 2 only in a high dose-hyperfractionated radiation which was given twice a day at a dose of 1.5 Gy each and for a total dose of 66 Gy. Antitumor effects were evaluated by survival and response rate determined by decrease of tumor size. Significant improvement was obtained in patients with AAs by protocol 2 and 3. Response rates of patients with AAs and GMs were 46.2% and 50% in protocol 1, 63.6% and 50% in protocol 2, and 80% and 50% in protocol 3, respectively. One and two year survival rates in AAs were 46.4% and 34.8% in protocol 1, both 75% in protocol 2, and both 100% in protocol 3. Survival rates in GMs were not different among them. Except of radiation necrosis, which was observed in 38.5% of the patients under protocol 3, there was no significant difference in the adverse effects among the three protocols. In the present study, the efficacy of IMR therapy for patients with malignant gliomas, especially for AAs, was cofirmed. We conclude that twice a day administrations of HuIFN-beta in combination with a high dose-hyperfractionated radiation provide increased efficacy in IMR therapy.

Suppression of experimental myasthenia gravis by monoclonal antibodies against MHC peptide region involved in presentation of a pathogenic T-cell epitope.

We have prepared monoclonal antibodies (mAbs) against an antigen-binding region of I-A, region 62-76 of I-Abeta(b), which is involved in the T-cell participation in the pathogenesis of EAMG. The mAbs reacted with its parent molecules and inhibited the proliferation of disease-related T-cells. Passive transfer of these mAbs suppressed the occurrence of clinical EAMG, which was accompanied by decreased T-cell and Ab responses to tAChR. The results indicated that blocking the function of disease-related MHC by targeting a disease-associated region on MHC molecules could be an effective, straightforward and feasible strategy for immunointervention in MG.

An application of PCR-single strand conformation polymorphism to MN genotyping.

A PCR-based genotyping of MN blood group system was investigated for DNA samples taken from a population of 409 northern Japanese. DNA fragment (257bp) including exon 2 of glycophorin A (GPA) gene, in which encodes the determinants of MN antigens, was specifically amplified. On the analysis of PCR-single-strand conformation polymorphism (PCR-SSCP) for M alleles, band patterns of M(G) and M(T) were easily discriminated each other. For N alleles, three band patterns were observed, and we tentatively named these alleles as N(1), N(2) and N(V). The N(1) allele appeared predominantly and N(2) had two base substitutions at 1st (C-->A) and 56th (C-->T) in exon 2 of N(1). The other N(V), which was detected from a pair of a mother and her child, possessed a single base substitution at 23rd (A-->G) in intron 2. The allele frequencies of M(G), M(T), N(1) and N(2) were 0.4450, 0.0978, 0.4303 and 0.0269, respectively. The polymorphism information content and the probability of paternity exclusion by this MN genotyping were estimated to be 0.5252 and 0.3219, respectively.

Detection of ABH blood group antigens from minute human hairs using polyvinylidene difluoride membrane with elution--ELISA method.

A sensitive and simple method for ABH blood grouping of human hairs using a polyvinylidene difluoride (PVDF) membrane was described. The hairs sensitized with monoclonal antibodies were applied onto the membrane and heated at 60 degrees C on a hot plate. When the membrane was examined with ELISA using a peroxidase-conjugated anti-IgM antibody, group-specific streaky stains were perceived on the membrane, because the eluates directly bound to the membrane. The present method could be available to the detection of minute antigens of short hairs.

ITIH1*Q0iwate, a null allele of inter-alpha-trypsin inhibitor H1 caused by deletion/frameshift mutation.

The molecular characterization of the first example of null allele in the inter-alpha-trypsin inhibitor H1 (ITIH1) system, ITIH1*Q0iwate, encountered as apparent inverse homozygosity of ITIH1 phenotypes between mother and child in a paternity case, is described. Single-strand conformation polymorphism analysis and subsequent sequencing showed that deletion of a single nucleotide in the codon for Lys87 results in a frameshift causing a terminator codon downstream of the deletion. This leads to premature termination of ITIH1 protein translation at amino acid 128, resulting in a truncated protein.

An improved method for MN genotyping by the polymerase chain reaction.

A novel method of human MN blood group genotyping is reported using the polymerase chain reaction. Genotyping is based on two base substitutions characteristic of M and N alleles in the 2nd exon of the glycophorin A gene. Using a newly designed primer trio, PCR products for M (255 bp) and N (270 bp) alleles are rapidly and simultaneously detected by a single PCR procedure and subsequent polyacrylamide gel electrophoresis. This method enables MN genotyping from not only minute but also degraded DNA samples.

Five new alleles of plasma Zn-alpha 2-glycoprotein variants phenotyped by isoelectric focusing and immunoblotting in twelve populations.

Human Zn-alpha 2-glycoprotein (ZAG) in plasma samples from twelve populations was tested by immunoblotting after polyacrylamide gel isoelectric focusing. Eleven ZAG phenotypes produced by one common and nine rare alleles, including five new ones (ZAG*6-ZAG*10), were detected. Additionally, an application of separator IEF with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) was found to be useful for discriminating the rare ZAG 7 band.

Sweat protein components tested by SDS-polyacrylamide gel electrophoresis followed by immunoblotting.

Sweat samples were collected in a sauna from 74 healthy volunteers (72 men and 2 women) and concentrated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the individual samples revealed, in general, five main proteins and four PAS positive components. In pooled sweat, a method of SDS-PAGE followed by immunoblotting with specific antisera or antibodies against 24 human serum components was applied, and three out of the five main proteins showed the same molecular weights and antigenicities corresponding to serum albumin (67,000 Da), Zn-alpha 2-glycoprotein (42,000 Da) and lysozyme (14,000 Da). Moreover, orosomucoid, transferrin, IgG and IgA were demonstrated in the pooled sweat. Although alpha 1-antitrypsin was probably in the pooled sweat, other serum components could not be detected. On the pooled and individual sweat samples, anti-carcinoembryonic antigen (CEA) formed three bands at 42,000, 19,000 and 18,000 Da, but the antibody did not react with normal serum. It might be considered from these molecular weights that those sweat components are CEA-related antigens.

Distribution of human Zn-alpha-2-glycoprotein types in Chinese and Korean populations.

The genetic variation in Zn-alpha 2-glycoprotein (ZAG) was investigated by polyacrylamide gel isoelectric focusing and immunoblotting. The samples comprised 590 Chinese from 2 localities (Shenyang: n = 390; Kaohsiung: n = 200) and 873 Koreans from 2 localities (Seoul: n = 523; Cheju: n = 350). The allele frequencies were: ZAG*1 = 0.9962, ZAG*5 = 0.0038 in Shenyang; ZAG*1 = 1.0000 in Kaohsiung; ZAG*1 = 0.9971, ZAG*3 = 0.0029 in Seoul, and ZAG*1 = 0.9929, ZAG*3 = 0.0071 in Cheju.

Four variants of human plasma Zn-alpha 2-glycoprotein (ZAG) in the Japanese population.

Zn-alpha 2-glycoprotein (ZAG) of plasma from the general Japanese adult population (n = 1224) was studied by polyacrylamide gel isoelectric focusing (IEF) followed by immunoblotting with specific antiserum to ZAG. Most of the plasmas showed a common band pattern, while 16 samples showed four other patterns. These ZAG band patterns were easily differentiated by desialyzing the samples prior to IEF. The asialo form of ZAG commonly showed a single band. The 16 plasma samples presenting double bands were classified into four types containing the common single band. The differences in ZAG phenotypes may be suggested to be due to amino acid substitutions of the ZAG molecule. The statistical frequencies of five alleles, which we proposed to designate ZAG*1, ZAG*2, ZAG*3, ZAG*4, and ZAG*5, were 0.9935, 0.0025, 0.0016, 0.0004, and 0.0020, respectively. The genetic transmission of the rare alleles ZAG*3 and ZAG*4 was confirmed by two family studies.

Glutamate-pyruvate transaminase (GPT) variant found in a disputed paternity case.

GPT variants, GPT2C-1 and GPT2A-2C, were found in samples from a mother and her child, respectively, in a disputed paternity case. The phenotype GPT 2C-1 was determined by polyacrylamide gel electrophoresis performed at alkaline pH and acidic pH; this is probably the first GPT*2C-1 detected in Japanese. It was considered that the GPT 2C allele in this family was inherited in a manner of autosomal codominant transmission.

A case of a newborn infant with Hb M Iwate.

Hemoglobin of a newborn infant who was suspected to hereditarily have Hb M Iwate was examined. The infant hemolysate was separated into five fractions by column chromatography on Amberlite CG-50, and two of these fractions showed absorption spectra corresponding with that of Hb M Iwate. Five bands were found after the isoelectric focusing of the hemolysate, and two of these bands were brown. The two Hb M fractions obtained by column chromatography was focused to the positions of the brown bands. One of these Hbs M corresponded with Hb M Iwate (alpha M2 beta 2) from an adult carrier of this trait, but the other was not found in adult hemolysates. The latter species of Hb M was shown to be composed of the abnormal alpha chain and the normal gamma chain (alpha M2 gamma 2) by chain analysis, and was assumed to be specific for infants. A quantitative estimation of the hemoglobins in the infant hemolysate showed that there was no difference between the relative quantities of the fetal and adult forms of Hb M Iwate.