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North Pacific krill is widely distributed in the waters around Japan, however its utilization for human consumption has been limited due to its small size. We have investigated the proximate composition, the amino acid, free fatty acid, mineral and protein composition, respectively, as well as the residual proteolytic activity of krill protein, as recovered by acidic- and alkaline-aided isoelectric solubilization/precipitation, in order to facilitate effective utilization of this krill species. Krill protein yield was 31.7 and 73.4 % by acidic and alkaline treatment, respectively. Both treatments resulted in a comparable higher content of essential amino acids than that in whole krill, accompanied with effective removal of insoluble materials. Three major proteolytic active bands with molecular weights of approximately 28, 18 and 16 kDa, as estimated by gelatin-based zymography, were detected. Pronounced residual proteolytic activity was observed in the krill protein after alkaline processing, implying the possible involvement of proteinase(s) in protein degradation during alkaline solubilization. Based on these results, we suggest that inhibition of proteolysis during isoelectric solubilization/precipitation is required for the effective use of North Pacific krill. [ABSTRACT FROM AUTHOR]

Background: It is unclear whether the prevalence of oligoclonal IgG bands (OCBs) in multiple sclerosis (MS) is different between northern and southern regions of Asia., Objective: This study aimed to compare the prevalence of OCBs and positive cerebrospinal fluid (CSF) findings between northern and southern regions of Japan and to investigate the association of these CSF findings with HLA-DRB1 alleles., Methods: The study included 180 MS patients from Hokkaido (northern Japan) and 184 patients from Kyushu (southern Japan). The IgG index was defined as increased if it was >0.658. Presence of CSF OCBs and/or increased IgG index was defined as positive CSF findings., Results: Positive CSF findings and OCB positivity were significantly higher in MS patients from Hokkaido than in those from Kyushu (p < 0.0001 for both). Logistic regression analysis revealed that after adjusting for covariates that can be related to abnormal CSF IgG production, the geographic region (Hokkaido) showed odds ratios (ORs) of 4.08 and 2.57, whereas the HLA-DRB1*04:05 allele showed ORs of 0.36 and 0.30 for positive CSF findings and OCB positivity, respectively., Conclusions: The results indicate that latitude and HLA-DRB1 alleles independently affect the emergence of CSF IgG abnormalities in Japanese patients with MS., (© The Author(s), 2015.)

As hyperphosphorylated paratarg-7 (pP-7) carrier state was shown to be the first molecularly defined autosomal dominantly inherited risk factor for monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma (MM) in a European population, the prevalence of pP-7 carrier state among African-Americans who have a significantly higher incidence of MGUS/MM is of interest. We therefore determined pP-7 carrier state and paraproteins with specificity for P-7 in African-American, European and Japanese patients with MGUS/MM and healthy controls. By isoelectric focusing and ELISA, a paratarg-7-specific paraprotein and the associated pP-7 carrier state was observed in 30/81 (37.0%) African-American, 42/252 (16.7%) European and 7/176 (4.0%) Japanese MGUS/MM patients (p < 0.001). A pP-7 carrier state was found in 11/100 (11.0%) African-American, 8/550 (1.5%) European and 1/278 (0.4%) Japanese healthy controls (p < 0.001), resulting in an odds ratio for MGUS/MM of 4.8 (p < 0.001) among African-American, 13.6 among European (p < 0.001) and 11.5 (p = 0.023) among Japanese carriers of pP-7. We conclude that pP-7 carriers are most prevalent among African-Americans, but a pP-7 carrier state is the strongest molecularly defined single risk factor for MGUS/MM known to date in all three ethnic groups. The high prevalence of pP-7 carriers among African-American patients emphasizes a predominant role of this genetic factor in the pathogenesis of these diseases. The large number of pP7 African-American patients and controls should facilitate the identification of the SNP or mutation underlying the pP-7 carrier state., (© 2014 UICC.)

Objective: Although the concentration of α1-acid glycoprotein (AGP) in serum increases under some conditions, the behavior of the individual genetic variants is not well understood. Therefore, we studied the relative changes in AGP variants pre- and postoperatively in patients with cancer and patients with chronic inflammatory disease states, as well as the distribution of AGP phenotypes in a Japanese population., Methods: Serum samples were taken before and after surgery from 25 female patients with early breast cancer. Serum samples were also obtained from 134 patients with rheumatoid arthritis (RA) and 33 with systemic lupus erythematosus (SLE), and from 103 healthy subjects. The relative concentrations of the individual genetic variants in the serum samples were determined by isoelectric focusing after desialylation with neuraminidase., Results: The postoperative AGP concentrations in patients with early breast cancer were 2-fold higher than before surgery. The relative concentrations of the F1 and S variants were significantly increased, whereas that of the A variant was not changed significantly. The relative concentrations of all the AGP variants in patients with RA and SLE were significantly higher than those in healthy subjects. The distribution of the AGP phenotypes did not differ significantly among the groups examined in this study., Conclusions: The F1/S variants of AGP, but not the A variant, were significantly increased after early breast cancer surgery, but all the variants were increased in patients with chronic inflammatory states such as RA and SLE. The distribution of the AGP phenotypes did not differ significantly among the disease groups studied.

Klebsiella pneumoniae strain KG525, which showed high-level resistance to broad-spectrum cephalosporins, was isolated from the neonatal intensive care unit (NICU) of a Japanese hospital in March 2002. The ceftazidime resistance of strain KG525 was transferable to Escherichia coli CSH-2 by conjugation. Cloning and sequence analysis revealed that production of a novel extended-spectrum class A beta-lactamase (pI 7.0), designated GES-3, which had two amino acid substitutions of M62T and E104K on the basis of the sequence of GES-1, was responsible for resistance in strain KG525 and its transconjugant. The bla(GES-3) gene was located as the first gene cassette in a class 1 integron that also contained an aacA1-orfG fused gene cassette and one unique cassette that has not been described in other class 1 integrons and ended with a truncated 3' conserved segment by insertion of IS26. Another five ceftazidime-resistant K. pneumoniae strains, strains KG914, KG1116, KG545, KG502, and KG827, which were isolated from different neonates during a 1-year period in the same NICU where strain KG525 had been isolated, were also positive for GES-type beta-lactamase genes by PCR. Pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR analyses displayed genetic relatedness among the six K. pneumoniae strains. Southern hybridization analysis with a GES-type beta-lactamase gene-specific probe showed that the locations of bla(GES) were multiple and diverse among the six strains. These findings suggest that within the NICU setting genetically related K. pneumoniae strains carrying the bla(GES) gene were ambushed with genetic rearrangements that caused the multiplication and translocation of the bla(GES) gene.

The cerebrospinal fluid oligodonal IgG bands (OB) are less frequently observed in Japanese multiple sclerosis (MS) patients compared with Caucasian patients. We studied 40 consecutive Japanese MS patients to investigate the differences in the clinical and magnetic resonance imaging (MRI) features of MS between OB-positive patients and OB-negative ones. Among the 40 patients, 22 (55%) patients were OB-positive by either agarose gel electrophoresis (AGE) or isoelectric focusing (IEF), and 18 (45%) patients were OB-negative by both AGE and IEF. There were differences between the two groups only in the clincal forms of MS, but not in terms of gender, onset age, disease duration, or disease severity. In the OB-negative group, nine (50%) of the patients had the optic-spinal form of MS (OS-MS), but only one patient (4.5%) in the OB-positive group had OS-MS. Although most OB-positive patients showed brain MRI lesions typical of MS, 13 (72%) of the OB-negative patients showed no or few brain MRI lesions and the rest of the OB-negative patients showed atypical MS lesions, such as diffuse white matter lesions or large ring-enhanced lesions. Our results suggest that the majority of OB-negative Japanese MS patents show either no or few brain MRI lesions or atypical brain MRI lesions.

In a Japanese paternity test, an alleged father was excluded only by reverse homozygosity of esterase D (ESD) phenotypes (mother, ESD 1; child, ESD 1; alleged father, ESD 2) out of 43 classical and DNA markers investigated. To solve the aberrant inheritance of the ESD phenotypes observed between them, fragments for all eight coding exons amplified by polymerase chain reaction (PCR) were subjected to DNA analysis. The child and alleged father shared a null allele, originating from ESD(*)1. It was characterized by having TGA for the stop codon instead of TCA for serine at codon 63. Thus, the sharing of a rare null gene, ESD(*)Q0(yonago), increased the probability of paternity., (Copyright 2002 Elsevier Science Ireland Ltd.)

Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60 degrees C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD.

The association between apolipoprotein E (ApoE) alleles and schizophrenia has remained controversial. A recent report claiming that ApoE epsilon3 Taiwan Chinese carriers have an increased risk of schizophrenia prompted us to investigate the allele frequency in a large group of Japanese schizophrenic patients. Serum samples were obtained from 314 schizophrenic patients and 188 controls in Japan and examined using isoelectric focusing/immunoblotting. There were no significant differences in ApoE allele frequencies between schizophrenic patients and controls and in the odds ratios for schizophrenia among the epsilon2, epsilon3 and epsilon4 carriers. In contrast to the report from Taiwan, our findings and results of the majority of previous studies suggest no effects of ApoE alleles on the development of schizophrenia., (Copyright 2000 S. Karger AG, Basel.)

Carbohydrate-deficient glycoprotein syndrome type I (CDG1) is an autosomal recessive disorder characterized by severe nervous system involvement and a carbohydrate moiety deficiency in N-linked glycoproteins. Clinical symptoms are psychomotor retardation, stroke-like episodes or hemorrhagic episodes, hepatic dysfunction, polyneuropathy, and cerebellar ataxia. Marked atrophy of the cerebellar hemispheres and pons is recognizable on CT scan or MRI. CDGI has been mapped to human chromosome 16p by linkage studies. Recently, missense mutations in the gene for phosphomannomutase (PMM2) have been detected in Caucasian patients with CDG1. We studied DNA mutations in PMM2 in a Japanese family with CDG1. DNA sequencing of PMM2 in the siblings showed missense mutations of maternal origin in exon 5 and of paternal origin in exon 8. No such mutations were detected in 50 unrelated healthy Japanese. These findings suggest that the PMM2 is responsible for CDG1 in the Japanese as well as in Caucasians, and CDG1 may be the diagnosis in OPCA of neonatal onset, more often than currently thought.

Carbohydrate-deficient glycoprotein syndrome type 1 (CDG1) (MIM: 212065) is an autosomal recessive disorder with psychomotor retardation, strokelike episodes, ataxia, and olivopontocerebellar atrophy (OPCA) of neonatal onset. Recently, DNA substitutions in a gene for phosphomannomutase 2 (PMM2), mapped to 16p13, were identified in patients with CDG1. Biochemical findings in previously reported Japanese patients with CDG1 were slightly different from those of Caucasians, suggesting genetic heterogeneity of CDG1 in Japanese patients. We investigated the DNA sequence of PMM2 in two unrelated Japanese families with CDG1. Missense mutations in exon 5 (Phe144Leu) and exon 8 (Tyr229Ser, Arg238Pro) of the PMM2 gene were present in two families, but they were not present in 72 unrelated healthy Japanese individuals. One of the missense mutations, Phe144Leu in exon 5, was common to two families with CDG1. Our findings confirm that mutations in the PMM2 gene account for at least some Japanese patients with CDG1 similar to that seen in Caucasians and that exons 5 and 8 are hot spots of mutations of CDG1 caused by the PMM2 gene.

Reference preparation for proteins in human serum (RPPHS), also called Certified Reference Material 470 (CRM 470), was prepared by the international Federation of Clinical Chemistry (IFCC) and is intended to serve as a new international plasma protein reference material. It is now being introduced into Japan. RPPHS possesses many excellent properties, including safety, stability, and accuracy in value assignment. Moreover, the physicochemical properties of its proteins are identical to those of fresh serum, giving it immunochemical behavior that is commutable with that of existing reference materials and calibrators in given immunoassays. Reference intervals of 13 serum proteins were determined for the first time using nephelometry and a new working calibrator assigned from RPPHS, which seems certain to play a critical role in the global standardization of specific protein immunoassays.

Katsuwokinase (KK) is a unique fibrinolytic enzyme recently found in skipjack "Shiokara," a Japanese traditional salt-fermented food. A crude enzyme extracted from skipjack Shiokara (Katsuwonus pelamis) showed a very strong fibrinolytic activity above 45 CU/g (fibrin plate method) based on plasmin. KK not only hydrolyzed fibrin but also several synthetic amido substrates, particularly pyro-Glu-Gly-Arg-pNA. The fibrinolytic activity of KK was not affected in the presence of 10% NaCl, was stable in the pH range from 1 to 10 at 37 degrees C for 30 min, and was inhibited by DFP, SBTI, BPTI, and aprotinin but not by epsilon-amino-n-caproic acid and t-4-amino-methylcyclohexane carboxylic acid. The crude enzyme contained at least four kinds of KK, and the major form purified had a pI value of approximately 5.0 and a molecular weight of 35,000. The N-terminal amino acid sequence of 21 residues, I-V-G-G-Y-E-Q-Z-A-H-S-Q-P-H-Q-V-S-L-N-S-G-, had 80% homology with that of trypsin. The fibrinolytic activity of the purified enzyme was approximately 2.6 times greater than that of plasmin by molar ratio, demonstrating its identity as a new and very potent fibrinolytic enzyme.

Polymorphism of factor H (HF) was investigated in 1060 unrelated Japanese individuals using isoelectric focusing and immunoblotting. Besides 6 different HF types a null type and an unusual type were observed. The family analysis suggested the hereditary occurrence of a new variant allele HF*C. The population data fitted the Hardy-Weinberg equilibrium, assuming that the null allele HF*QO occurs commonly. The allele frequencies were HF*A = 0.407 +/- 0.011, HF*B = 0.491 +/- 0.011, HF*A1 = 0.011 +/- 0.002 and HF*QO = 0.091 +/- 0.006. The HF polymorphism in Japanese was shown to be controlled by the above 4 common alleles.

An antibiotic produced by Alteromonas luteoviolacea strain 9K-V10 was recovered after cold acetone precipitation of culture supernatant fluids or lysates that had been frozen and thawed. The precipitate obtained from cell-free lysates was fractioned by DEAE ion-exchange chromatography. Further purification by gel-filtration chromatography yielded a single peak of antibiotic activity that corresponded to a protein peak with a molecular mass of approximately 100 kDa. After non-denaturing polyacrylamide gel electrophoresis, antibiotic activity co-migrated with a protein band. The isoelectric point of the antibiotic was estimated to be 7.7. Treatment of the concentrated active fraction with proteinase K or heating at 70 degrees C for 10 min resulted in total loss of antibiotic activity. These results show that the antibiotic produced by Alt. luteoviolacea 9K-V10 is of a proteinaceous nature.

We analyzed apolipoprotein E (apo E) genotypes of 104 randomly sampled Japanese subjects by using the polymerase chain reaction-restriction fragment length polymorphism method in which the apo E genomic DNA was amplified between bases 2849 and 3071. We compared the results thus obtained with their corresponding phenotypes assessed by isoelectric focusing (IEF). The overall frequencies of the genotypes were as follows: epsilon 2:2.4%, epsilon 3:86.5%, and epsilon 4:11.1%, which are essentially identical to those previously reported when the IEF method has been used. The phenotypes and genotypes were identical in all but five cases. We could not determine the phenotypes of four cases by IEF because of an atypical IEF pattern presumably caused by sialylation. In one other case the phenotype initially did not correlate with its genotype but eventually did after neuraminidase treatment. These results indicate that apo E polymorphism of most Japanese people can be determined by analyzing the apo E genome between bases 2849 and 3071.

Of the 68 strains of beta-lactamase-producing Moraxella (Branhamella) catarrhalis isolated in Japan that were studied, 62 (91%) produced the BRO-1-type beta-lactamase and 6 (9%) produced the BRO-2 type. There were no strains containing the BRO-3-type beta-lactamase. We compared the susceptibility of BRO-1- and BRO-2-producing strains to various oral beta-lactam antibiotics. We found that the BRO-1-producing strains were less susceptible than the BRO-2-producing strains. Although the BRO-1 and BRO-2 types showed a similar hydrolysis pattern, the specific activity of BRO-1 was 3-fold that of BRO-2. We examined the transfer of the BRO-1 and BRO-2 genes to non-beta-lactamase-producing M. catarrhalis No. 4020 and found that of the 13 donor strains producing BRO-1, 11 (85%) were able to transfer the gene for BRO-1 production by conjugation. Of the 6 donor strains producing BRO-2, 2 (33%) were able to transfer the gene for BRO-2 production by conjugation. For 3 of the 13 (23%) BRO-1-producing strains and 1 of the 6 (17%) BRO-2-producing strains, about 13 Mdalton of plasmids were detected. These plasmid-containing strains were used as donors, and in beta-lactamase-producing transconjugants the same size of plasmids was detected. However, when the total DNA is extracted from strains with the ability to transfer by conjugation, the transformation of the beta-lactamase-producing gene can occur regardless of the presence or absence of plasmids. Furthermore, even if purified plasmids are transformed, beta-lactamase-producing transformants are not obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroxine-binding globulin (TBG) is the major transport protein of thyroid hormones in human serum. In this communication, we present a sequence abnormality of the TBG-gene in a Japanese family manifesting partial TBG deficiency (TBG-PDJ). The propositus was a male with a reduced concentration of TBG (3.2 micrograms/ml). Thyroid function tests suggested that the inheritance of this TBG abnormality was X-linked. The TBG exhibited increased heat-lability compared with the common type TBG (TBG-C). The isoelectric focusing pattern of this TBG molecule was indistinguishable from TBG-C. Genomic DNAs from white blood cells of four members of a TBG-PDJ family were subjected to polymerase chain reaction (PCR), and the products were sequenced. The sequencing of the entire coding exons and exon/intron junctions of TBG allele of the propositus revealed a single nucleotide substitution: CCT (proline) to CTT (leucine) at amino acid 363 of the TBG-C. The heterozygosity as revealed by the direct sequencing of the PCR product correlated with the TBG concentration in serum. The proline to leucine substitution may cause a change in the TBG tertiary structure and result in decreased heat stability, resulting in decreased TBG levels in the affected subjects.

A functionally inactive plasminogen (PLG) variant designated as PLG M5 is polymorphic in the Japanese population and has a feature common to PLG with type-I mutation that has a codon 601 missense mutation in exon 15 (GCT for Ala-->ACT for Thr). This study was conducted to clarify whether the type-I mutation of PLG is present in PLG M5 and polymorphic in the Japanese population. Direct sequencing of the amplified DNA from the PLG gene in a heterozygote for PLG M5 revealed that the sequence of the exon 15 in the gene for PLG M5 is identical with that in the PLG gene with type-I mutation. In addition, the amplified DNA from the PLG gene in 12 heterozygotes for PLG M5 reacted with the probe for the type-I mutation in dot blot hybridization with an allele-specific oligonucleotide probe. The heterozygote for PLG with type-I mutation was found in 2.2% of 360 unrelated healthy subjects. These data indicate that the type-I mutation of PLG is present in PLG M5 and polymorphic in the Japanese population. The data also suggest that the PLG M5 is identical with PLG Tochigi and Kagoshima.

The polymorphism of FXIIIB was investigated in 555 unrelated Japanese individuals using isoelectric focusing and immunoblotting. Five common phenotypes and a rare variant type FXIIIB 15-3 were observed. The allele frequencies were FXIIIB*1 = 0.3063, FXIIIB*2 = 0.0162, FXIIIB*3 = 0.6766 and FXIIIB*15 = 0.0009. Phenotyping was also possible from bloodstains stored at 37 degrees C for up to 4 months and from bloodstains stored at room temperature and at 4 degrees C for over 6 months. The FXIIIB system can provide a new powerful genetic marker for the medicolegal grouping of bloodstains.

We describe 3 children (from two families) with a multisystemic disorder characterized by mental retardation, nonprogressive ataxia, polyneuropathy, hepatopathy during infancy and growth retardation. Due to the clinical similarities to a recently recognized disorder associated with carbohydrate-deficient transferrin, we examined serum transferrin by means of isoelectric focusing, and found increases in disialo transferrin and asialotransferrin. Removal of sialic acid with neuraminidase revealed the same transferrin phenotypes as in their parents. Similarly, carbohydrate-deficient fractions of serum alpha 1-antitrypsin were also detected. Therefore, the diagnosis was made of the recently identified carbohydrate-deficient glycoprotein syndrome. This is a genetic disorder with distinctive clinical features and multiple carbohydrate-deficient glycoproteins. These seem to be the first reported Japanese patients with this syndrome.

Polymorphism of the complement component factor I (IF) was investigated in 645 unrelated Japanese individuals using isoelectric focusing and immunoblotting. The allele frequencies were IF*A = 0.1031 and IF*B = 0.8969. The population data fitted the Hardy-Weinberg law. Phenotyping was also possible from bloodstains stored at 37 degrees C for up to 1 week, at room temperature for up to 2 weeks and at 4 degrees C for up to 5 weeks. The IF system can be a useful supplement for the medicolegal identification of bloodstains, but seems useless in Caucasoids.

Polyacrylamide gel isoelectric focusing (PAGIEF) of neuraminidase treated EDTA plasma samples at pH 7-9 with 1.0 M urea followed by semi-dry horizontal electroblotting with enzyme immunoassay was done for the detection of factor I (IF) allotypes in 604 unrelated Japanese blood donors living in Tokyo. In these samples three common types, IF A, IF AB, and IF B, and a new variant were observed. A new variant which had major bands in a more acidic region than IF A was designated IF A1. Family data was in accordance with the Mendelian inheritance. The allele frequencies were estimated as IF*B = 0.8924, IF*A = 0.1068 and IF*A1 = 0.0008, respectively. The IF protein level of various allotypes was measured using the single radial immunodiffusion technique. The mean IF protein level for IF B was slightly higher than that for IF A (P less than 0.001) and that for IF A1B (P less than 0.05).

Allele frequencies for human transferrin (TF) subtypes were determined using serum samples from Japanese subjects living in Fukui prefecture, Japan, and compared with other Japanese populations using isoelectric focusing (IEF) and immunoblotting. The application of IEF revealed considerable heterogeneity in the TF system, enhancing its potential value for anthropologic and genetic studies. So far, TF subtypes of about 27,000 Japanese individuals from 35 population groups have been analyzed to evaluate the degree of genetic variation at the TF locus. Possible geographic and biologic factors are discussed.

Serum samples from 270 healthy blood donors of Iwate prefecture, northern Japan, were examined for polymorphism of factor I (IF) by using polyacrylamide gel isoelectric focusing followed by semidry horizontal electroblotting with enzyme immunoassay. In 270 individuals four different patterns were observed, and these were controlled by two common alleles, IF*A and IF*B, and one rare allele, IF*A2. Allele frequencies were estimated to be 0.1019, 0.8963 and 0.0018 for IF*A, IF*B and IF*A2, respectively. The data of IF allele frequencies thus far reported in Japan excluding Okinawa Island were compared, and a statistically significant (p less than 0.01) geographical cline was detected for IF*A and IF*B alleles.

The allotypes of C6, C7, factor B (BF) and factor I (IF) of the human complement system were studied in 11 Japanese patients with pemphigus (5 with pemphigus vulgaris and 6 with pemphigus foliaceus) and 17 with bullous pemphigoid (BP) to investigate the genetic background of these diseases. The allotypes were detected by using isoelectric focusing and immunoblotting. The frequency for IF*A allele in the pemphigus patients was significantly higher (p = 0.009) than that in healthy controls (n = 60). A significant association of IF A allotype with pemphigus was also observed (p = 0.027), with a relative risk of 6.3. There was no association between the C6, C7, BF or IF allotypes and BP. These data suggest that IF A allotype may be an etiological genetic factor in the development of pemphigus.

Genetic polymorphism of glutamate oxaloacetate transaminase (GOT1) was demonstrated in human erythrocytes by isoelectric focusing in thin layer polyacrylamide gels and a sensitive and positive detection method. Using this technique, five phenotypes, GOT1 1, 2, 2-1, 3-1 and 3-2 were determined and the estimated gene frequencies of GOT1*1, GOT1*2 and GOT1*3 in the Japanese population were 0.9740, 0.0173 and 0.0087, respectively.

The polymorphism of C1R was investigated in 570 unrelated Japanese individuals using isoelectric focusing and immunoblotting. A total of 11 different C1R phenotypes including a new pattern designated C1R 11-1 were observed. The allele frequencies were C1R*1 = 0.4561, C1R*2 = 0.3377, C1R*5 = 0.1956, C1R*8 = 0.0088 and C1R*R (C1R*9 and C1R*11) = 0.0018. The population data fitted the Hardy-Weinberg equilibrium. The C1R polymorphism in Japanese was shown to be controlled by 3 common alleles, C1R*1, C1R*2 and C1R*5, as compared to Caucasians where only the former 2 are present commonly. This complement system can be a useful genetic marker for anthropological studies.

Two complement C7 protein polymorphisms exist. One is determined by isoelectric focussing whereas the other is determined by the reaction pattern of a monoclonal C7-allospecific antibody in an ELISA assay. C7 concentrations quantitated by an ELISA based on polyclonal C7-specific IgG and data from functional haemolytic assays were compared with the allotypes of both C7 protein polymorphisms. Testing a randomly selected Japanese population revealed that not only C7*3 but also C7*N is a hypomorphic allele. However, no significant disease association of C7*N or C7*M was found in studies of hospitalized patients.

A procedure which can detect subtype-specific minor bands of factor B (BF) by polyacrylamide gel isoelectric focusing is presented. After zymosan-mediated fragmentation of BF in serum via alternative pathway for complement activation, serum samples are subjected to isoelectric focusing in a narrow pH range (4.2-4.9). The Ba fragments are detected by using immunoblotting. In addition to the previously reported minor bands with subtypic specificities, heterogeneities are observed in other minor band group, where a single minor band corresponds exclusively to a subtype in a regular combination with the previously announced subtypic patterns. A one-to-one correspondence of a single band to each subtype provides an unambiguous determination for three subtypic phenotypes deduced from the two divided BF*F alleles, BF*FA and BF*FB. An autosomal codominant heredity is confirmed through segregation analysis. A population survey reveals that four common alleles, BF*S, BF*FA, BF*FB, BF*Fb1, occur in a Japanese population and the former three alleles, except BF*Fb1, occur in a Cambodian population. The presence or absence of a single anodal minor band was found to be the only difference after neuraminidase treatment of FA and FB, implying that an amino acid substitution responsible for the FA-FB subtypic difference is involved in an additional acquisition in FA of an oligosaccharide unit with a charged sialic acid.

Specific antibodies against the human haptoglobin (HP) alpha chain were raised and used for immunoblotting after isoelectric focusing to determine Hp alpha subtypes in a reproducible and simplified manner. By eliminating the staining of the HP beta chain, HP alpha subtypes were visualized more precisely and simply than by previously reported methods. Subtypes of a total of 1211 sera, obtained from two different populations in Japan, were examined by the new method. Four HP alpha subtypes (HP 1S-1S,2FS-1S, 2FS-2FS and 2FS-2SS) and five subtypes combined with a new variant (HP 2FS-1V1, 2V1-1S, 2FS-2V1, 2FS-2V2 and 2FS-2V3) were observed. HP 1V1 belonged to HP alpha 1, and HP 2V1, 2V2 and 2V3 belonged to HP alpha 2, and their alleles were designated HP A*1V1, HP A*2V1, HP A*2V2 and HP A*2V3, respectively. The allele frequencies were calculated to be as follows: HP A*1S, 0.2597; HP A*1V1, 0.0004; HP A*2FS, 0.7333; HP A*2SS, 0.0008; HP A*2V1, 0.0045; HP A*2V2, 0.0008; and HP A*2V3, 0.0004. The allele frequency of HP A*2SS, which is common in the European population, is less frequent than HP A*2V1 in the Japanese population.

Three new variants of acidic proline-rich proteins (At, Au, Aw) were found in human parotid saliva by isoelectric focusing and basic gel electrophoresis. Electrophoretic comparison of the purified proteins and their tryptic peptides suggested minor charge and size differences from other acidic PRPs. Genetic and biochemical studies indicate that the At and Aw proteins are allelic products of the PRH1 locus. Gene frequencies of the At productive allele (PRH1(6)) in Japanese, Chinese, and Malays were 0.008, 0.012, and 0.004, respectively. The Au protein was also found in Japanese (2 in 746 samples), Chinese (1 in 215 samples), and Malays (1 in 220 samples), however, the Aw protein was found only in one Japanese (n = 746). These three proteins were not found in 106 Indian subjects.

Genetic polymorphism of transferrin (TF) was revealed in human urine by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels. Using this technique more than 300 urine samples were examined, and correct TF typing from a small volume of urine (approx. 0.5 ml) was achieved, in comparison with the results of direct grouping for plasma. Three common phenotypes, TF C1, C2-1 and C2, were differentiated. In addition, the rare types TF C1D, C2D, and C1B were observed. The frequencies of the TF alleles in our samples were found to be: TF*C1 = 0.7265, TF*C2 = 0.2624, TF*D = 0.0083 and TF*B = 0.0028.

Orosomucoid (ORM) of plasma from 287 Japanese was typed by polyacrylamide gel isoelectric focusing followed by immunoprinting with specific antiserum to ORM. Two new variants were observed and they were designated ORM2 16 and ORM2 17.

Polyacrylamide gel isoelectric focusing (PAGIEF) of EDTA plasma and neuraminidase-treated plasma samples at pH 3.5-9.5 containing 8.0 M urea followed by an electroblotting with enzyme immunoassay was applied for the detection of factor H (HF) phenotypes in 536 unrelated Japanese blood donors living in Tokyo. In the major cathodal components, phenotypes of HF were classified into three common and five rare patterns, and these were considered to be controlled by two common and two rare alleles. The data suggest that the HF*Q0 allele also exists in the Japanese population. Family studies confirm the hypothesis that the HF polymorphism is controlled by autosomal codominant Mendelian inheritance.

A new polymorphic acidic proline-rich protein (As) was found in human parotid saliva by SDS and basic polyacrylamide gel electrophoresis. The phenotypic relationships and family studies support the hypothesis that the As protein is another allelic product of the PRH1 locus. The As protein could not be discriminated from the parotid isoelectric focusing (PIf) protein by isoelectric focusing gel electrophoresis due to similar migration of the two proteins. In order to determine salivary PRH1 phenotypes it is necessary to use SDS or basic gel electrophoresis in addition to the isoelectric focusing gel electrophoresis. The As protein was not found in Caucasians. The allele frequencies of the PRH1 locus in Japanese were PRH1 (double-band protein) = 0.035, PRH1(2) (acidic protein) = 0.193, PRH1(4) (PIf) = 0.751, and PRH1(5) (As) = 0.021.

A new separator isoelectric focusing method for typing of orosomucoid (ORM) was developed. This method provided a superior resolution of ORM patterns: two close bands of ORM1*5.2 products were clearly separated. A total of 364 subjects from Okinawa (Japan) were classified into 21 ORM phenotypes determined by 6 ORM1 and 7 ORM2 alleles including a polymorphic silent allele, ORM2*QO, and 2 new rare variants, ORM2*18 and ORM2*19. These phenotypes were also explained by 12 ORM haplotypes, half of which were polymorphic.

The polymorphism of BF was investigated in 765 unrelated Japanese individuals by isoelectric focusing and immunoblotting. Besides five common subtypes three rare variants were observed. The allele frequencies were: BF*S = 0.8078, BF*FA = 0.1797, BF*FB = 0.0105, BF*Var. = 0.0020. The above method was successfully applied to subtyping BF in stored bloodstains. The determination limits were: at 4 degrees C 8 weeks, at room temperature 2 weeks and at 37 degrees C only 2 days after storage. The BF subtyping is of practical use in medicolegal individualization of unknown bloodstains.

A fast-moving abnormal hemoglobin was detected in a heterozygous condition from a patient with benign goiter. The hemoglobin has a slight but significant high oxygen affinity. The patient does not show any hematologic abnormality. Structural analysis indicated that a lysine residue normally present at alpha 11 was substituted by an asparagine residue. This substitution which has not been described previously was discovered coincidentally by another laboratory (1). The new variant has been named Hb Albany-Suma to denote the origin of the variant in both the United States and Japan.

Genetic polymorphism of the seventh component of complement (C7) was studied by the methods of agarose gel isoelectric focusing and immunoblotting. Serum or plasma samples were treated with neuraminidase prior to isoelectric focusing. A cathodal variant, named C7 5, was recognized in the treated samples. Family study indicates that the C7 5 component is genetically determined. The allele frequencies calculated from 183 healthy Japanese individuals were C7*1 = 0.809, C7*2 = 0.104, C7*4 = 0.038, and C7*5 = 0.049. An association between C7 and C6 alleles was not found.

The polymorphism of C7 was investigated in neuraminidase-treated sera from 513 unrelated Japanese individuals using isoelectric focusing followed by an electroimmunoblotting technique. Besides the common phenotypes 5 rare variants including 2 types of new variants were detected. The family analysis suggested the hereditary occurrence of a new allele C7*8. The allele frequencies were: C7*1 = 0.8314, C7*2 = 0.0926, C7*3 = 0.0380, C7*4 = 0.0331, C7*6 = 0.0010, and C7*8 = 0.0039.

The characterization of haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) is described. This new mutation is unique since it only induces modification of the stability of the molecule. In vitro precipitation of haemoglobin Saki upon heat or in the presence of chemicals is compared to the stability of haemoglobin A and haemoglobin S.

Using isoelectric focusing (IEF) in thin-layer polyacrylamide gel, the polymorphism of the serum alpha-1-antitrypsin (Pi system) was investigated in 335 healthy unrelated Japanese individuals living in Miyagi prefecture, the northern part of Japan. Six common and five rare variant phenotypes were identified in our population samples, and the estimated allele frequencies for the genes PiM1, PiM2 and PiM3 were 0.718, 0.238 and 0.044, respectively. Family studies (n = 46) showed an autosomal codominant inheritance, and no exclusion was found in 23 mother-child pairs.