Reliability and validity of a self-administered questionnaire of patient health care behavior and satisfaction

The genes encoding the envelope glycoprotein H (gH) and gB homologues were identified by sequencing genomic clones of human herpesvirus-7 (HHV-7), strain JI. A gB cDNA clone from HHV-7 strain AL was also identified. The deduced primary translation products of the gH and gB genes are a protein of 690 amino acids, with a predicted mass of 80.4 kD, and a protein of 822 amino acids, with a predicted mass of 93.3 kD, respectively. Both the predicted proteins have the characteristics of transmembrane glycoproteins, containing signal and transmembrane sequence motifs and characterized by the presence of 10 (gH) and 11 (gB) potential motifs for N-glycosylation. Comparison of amino acid sequence of HHV-7 gH and gB with the homologous sequences of the other human herpesviruses reveals closest homology with HHV-6 (38.8% identity for gH, 56.2% identity for the gB). In addition, significant sequence similarity was also observed between the gH and gB of HHV-7 and the homologs encoded by human cytomegalovirus (21.6% identity for gH, 37.6% identity for gB). No significant differences existed between the gB sequence of the two different HHV-7 strains analyzed. The products of the HHV-7 gH and gB expressed transiently in eukaryotic cells were specifically recognized by an HHV-7-reactive human serum in immunofluorescence assays.     

Improving critical thinking in nursing practice

In a Japanese population study of the D1S80 locus 24 alleles ranging from allele 16 to allele43 were analysed using PCR-RFLP. As two repeat units were found to contain the restriction cleavage site (CCAGG) for EcoRII, we digested the alleles with EcoRII, separated the digested fragments on polyacrylamide gels and stained with ethidium bromide. Of the 24 alleles 11 band patterns were identified and tentatively labeled E1 to E11. A total of 42 subtypes were detected in a population group of 111 unrelated individuals. All samples of allele 18 were of the E3 type, while about 60% of the allele24 samples were of the E4 type and about 40% were of the E8 type. The third most frequent allele (allele30) contained four types, E4, E8, E5 and E6. No deviations from Hardy-Weinberg equilibrium were observed. Since this method could differentiate those samples which had the same length but different sequences, it is quite useful for paternity testing and individual identification.     

Taste disks are induced in the lingual epithelium of salamanders during metamorphosis

We have established a practical method of complete high-resolution typing for all HLA-A alleles using the polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) technique combined with allele group- and sequence-specific amplification. The second and third exons of the HLA-A gene, in which most allelic variations are observed, were separately amplified by PCRs with 3 and 4 group-specific primer pairs, respectively. Each PCR-amplified product was digested by allele-specific restriction endonucleases and then subjected to electrophoresis on a 10% polyacrylamide gel. In this way, 62 out of 79 HLA-A alleles could be discriminated by the RFLP patterns derived from the genetic polymorphism in the exon 2 and 3 domains. The remaining 17 alleles could be defined unequivocally by either PCR-RFLP analysis after exon 4 amplification or PCR analysis with sequence-specific primers (SSP). By this method, complete HELA-A genotyping for all homozygous and heterozygous combinations can be accomplished, establishing technically simple, economical and practical routine typing of the HLA-A gene, especially for small samples.     

Molecular genetic characterization of new bovine kappa-casein alleles CSN3F and CSN3G and genotyping by PCR-RFLP

In order to characterize the two new kappa-casein variants F and G (CSN3F and CSN3G) recently detected in Ayrshire and Pinzgauer cattle, exon IV of CSN3 from heterozygous animals was amplified by polymerase chain reaction (PCR), cloned and sequenced. The sequencing data revealed single point mutations at nucleotide positions 10530 (G-->A) for CSN3F and 10790 (C-->T) for CSN3G, corresponding to amino acid exchanges in positions 10 (Arg-->His) and 97 (Arg-->Cys) respectively. These mutations alter recognition sites for the restriction enzymes HhaI and MaeII, which were subsequently used to confirm these polymorphisms in cattle carrying CSN3F or CSN3G. A PCR-restriction fragment length polymorphism (RFLP) genotyping procedure for all currently known CSN3 alleles (CSN3A, CSN3B, CSN3C, CSN3E, CSN3F, CSN3G) was developed.     

The occupations of drink drivers: using occupational information to identify targetable characteristics of offenders

The complete DNA sequence of human herpesvirus-7 (HHV-7) strain RK was determined following direct cloning of virion DNA fragments into a sequencing vector. The sequence was compared with the previously published complete sequences of HHV-7 strain JI and human herpesvirus-6 (HHV-6) strain U1102. Despite a very close relationship between the two HHV-7 strains, differences are apparent in regions containing tandem reiterations, particularly in the "telomeric" reiterations located near the termini of the large direct repeat at the genome ends, and in a total of 179 additional positions distributed throughout the genome (i.e., about one nucleotide difference per kbp). This extent of divergence implies that the two strains arose from an ancestral virus several thousands of years ago. Differences that affect coding potential do not cluster in particular protein-coding regions, indicating that specific HHV-7 genes have not been measurably subject to unusual evolutionary pressures since divergence. Reassessments of genetic content indicated that the HHV-7 genome contains 84 different genes, whereas the HHV-6 genome contains 85. All HHV-7 genes but 1 have direct HHV-6 counterparts, and all but 2 HHV-6 genes have HHV-7 homologues. Sequence comparisons between HHV-7 and HHV-6 provided evidence that the protein-coding regions of 11 genes are expressed by splicing.     

Identification of missense mutations and haplotyping of carnitine palmitoyltransferase II gene

Carnitine palmitoyltransferase II(CPTII) deficiency manifests as two different clinical phenotypes: an adult form associated with muscular symptoms and an infantile form presenting with hepatocardiomuscular manifestations. We have investigated three Japanese patients with CPT II deficiency. Molecular analysis revealed two novel missense mutations, a glutamate (174)-to-lyine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPTII cDNA. Transfection experiments demonstrated that the two mutations reduced CPTII catalytic activity. We also identified a novel polymorphism in the CPTII gene, a phenylalanine (352)-to-cysteine substitution (F352C). According to an expression analysis this mutation did not alter CPTII activity. It was present in 21 out of 100 normal alleles in the Japanese population, but was not observed among Caucasians. Genotyping with the F352C polymorphism and the previously reported polymorphisms V368I and M647V allowed normal alleles to be classified into five haplotypes. In all three families, the E174K mutation resided only on F1V1M1 allele, while the F383Y mutation was observed on F2V2M1 allele, suggesting a single origin of each mutation.     

Cerebral cortical phospholipase A2 activity of senescence-accelerated mouse is increased in an age-dependent manner

An immunogenetic examination of 86 cases of stomach cancer established a correlation between predisposition and resistance, on the one hand, and the distribution of allele sets of HLA-genes (classes I and II), on the other. The relationship was found to vary according to sex and age. The most significant relationships with respect to predisposition were identified for HLA-B51 (RR = 19.82) alleles and allele combinations of HLA-DRI-DR7 (RR = 25.52) and HLA-A9-DRI (RR = 33.67). High relative risk of stomach cancer was attributed to the absence of relevant alleles in 91 patients included into the group of comparison. Also, combinations of allele sets were identified in healthy subjects which never occur in stomach cancer patients. The results provide a substantiation for developing an automated system of interpreting HLA-typing data which are instrumental in evaluating the patient's predisposition, resistance and prognosis.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

Typing of Pneumocystis carinii f. sp. hominis by single-strand conformation polymorphism of four genomic regions

To better investigate Pneumocystis carinii f. sp. hominis epidemiology, we have developed a molecular typing method. Because of the limited genetic variability of the P. carinii hominis genome, a multitarget approach was used. Four variable regions of the genome were amplified by PCR, polymorphism in each region was assessed by the single-strand conformation polymorphism (SSCP) technique, and the results for the four regions of each patient were combined. Bronchoalveolar lavage specimens collected from 11 patients were examined. Four patients were probably infected by a single strain, since their specimens yielded simple SSCP patterns (two bands corresponding to one allele). The combinations of these patterns were unique, suggesting that the strains which infected these patients were different. For the other seven patients, complex patterns were found (three or four bands corresponding to two alleles). The presence of more than one allele of a region in a patient is likely to be due to coinfection. Polymorphism was also assessed by sequencing, which revealed variations at nucleotide positions previously reported to vary. About half of the observed alleles had already been reported by laboratories in different countries. Multitarget typing of P. carinii hominis by PCR-SSCP should allow investigation of strain diversity and thus be useful for future epidemiological studies.     

Two CPT2 mutations in three Japanese patients with carnitine palmitoyltransferase II deficiency: functional analysis and association with polymorphic haplotypes and two clinical phenotypes

Carnitine palmitoyltransferase II (CPT II) deficiency manifests as two different clinical phenotypes: a muscular form and a hepatic form. We have investigated three nonconsanguineous Japanese patients with CPT II deficiency. Molecular analysis revealed two missense mutations, a glutamate (174)-to-lysine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPT II cDNA. Transfection experiments in COS-1 cells demonstrated that the two mutations markedly decreased the catalytic activity of mutant CPT II. Case 1 (hepatic form) was homozygous for the F383Y mutation, whereas case 3 (muscular form) was homozygous for the E174K mutation. Case 2 and her brother, who were compound heterozygotes for E174K and F383Y, exhibited the hepatic phenotype. We also identified a novel polymorphism in the CPT2 gene, a phenylalanine (352)-to-cysteine substitution (F352C), which did not alter CPT II activity in transfected cells. It was present in 21 out of 100 normal alleles in the Japanese population, but absent in Caucasian populations. Genotyping with the F352C polymorphism and the two previously reported polymorphisms, V368I and M647V, allowed normal Japanese alleles to be classified into five haplotypes. In all three families with CPT II deficiency, the E174K mutation resided only on the F1V1M1 allele, whereas the F383Y mutation was observed on the F2V2M1 allele, suggesting a single origin for each mutation.     

Penetration of oligonucleotides into mouse organism through mucosa and skin

The increased concordance rate of nickel sensitivity in monozygotic compared to dizygotic twins indicates a genetic causal component. We have previously described an association in nickel-sensitive subjects with an HLA-DQA restriction fragment length polymorphism (RFLP) (4.5-kb TaqI band, DQA1*0501). The purpose of the present study was to investigate if our previous finding could be confirmed in an independent study, and also to investigate the distribution of HLA class II alleles in chromium- and cobalt-sensitive individuals. Using TaqI- or MspI-digested DNA and DQA, DQB, DRB, DPA and DPB cDNA probes alleles were defined by RFLP analysis. The association with the DQA1*0501 allele was not confirmed in the new group of 37 nickel-sensitive subjects (compared to 150 new controls), nor when the two groups of patients were combined. The distribution of HLA class II alleles and DR-DQ haplotypes were similar in the pooled group of 70 nickel-sensitive subjects and the combined control groups (n = 250). No significant changes in the distribution of HLA class II allele among the chromium- (n = 26) and/or cobalt- (n = 38) sensitive individuals were found. Our results indicate that it is unlikely that the tendency to develop metal sensitivity is associated with alleles of the HLA class II region.     

Prospective study of human herpesvirus 6, human herpesvirus 7, and cytomegalovirus infections in human immunodeficiency virus-positive patients

Blood samples from human immunodeficiency virus (HIV)-positive patients were monitored for cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and HHV-7 by PCR. We detected CMV in 17% of the patients, HHV-6 in 6%, and HHV-7 in 3%. The viral loads of CMV were significantly higher than those of HHV-6 (P = 0.007) or HHV-7 (P = 0.01). Detection of CMV and HHV-6 was associated with low and high CD4 counts, respectively.     

Effect of cysteine substitutions on dimerization and interfragment linkage of human cytomegalovirus glycoprotein B (gp UL55)

Experiments were carried out to analyze the function of cysteine residues at amino acid positions 506 (cI), 550 (cII), 573 (cIII), and 610 (cIV), in dimerization and/or disulfide linkage of human cytomegalovirus (HCMV) glycoprotein B (gB). Single c-codons or pairs were substituted in the gB sequence of constructs which were used for transfection and selection of stable transfectants. Analysis of gB expression products revealed that single substitutions of cIII or cIV, but neither single nor double substitutions of cI or/and cII prevented gB dimerization. All substituted gB derivatives were, however, no longer processed by proteolytic cleavage. After deletion of the membrane anchor domain, correct proteolytic processing was again observed for anchorless gB forms. Substitutions of cI or cI/cII in secretory gB appeared to interfere with disulfide linkage between gB cleavage fragments. In the case of anchorless gB with substitutions of cII, cIII, or cIII/cIV, however, extracellular gB forms were not recovered. Using the Sindbis expression system recovery of all anchorless gB forms with cysteine substitutions was achieved. Analysis verified involvement of cI/II substitutions in intrachain disulfide linkage between cleavage fragments of HCMV gB.     

Tobacco farmers and diversification: opportunities and barriers

PCR/SSOP typing methods were used to analyze the HLA Class II DRB1, DQA1, DQB1 and DPB1 loci of samples from three African American populations of Colombia. Forty samples from the Cauca (Pacific), and twenty samples each from the Choco (North Pacific Coast) and the Providencia (Caribbean island) populations, were collected and the Class II loci analyzed under the auspices of the Expedicion Humana. Despite the limited number of samples analyzed, the African Colombian populations exhibit a very high degree of class II polymorphism. A great diversity of DRB1 alleles was found, with representatives from all serological classes, including 19 DRB1 alleles in the Providencia, 16 in the Cauca and 14 in the Choco groups. In addition, a novel DQB1*02 allele (*0203) was found in two individuals from the Cauca population of the Pacific Coast. The sequence of the DQB1*0203 allele, associated with DR3, differs from DQB1*0201 by only one nucleotide substitution (C-->A) in the second position of codon 57, resulting in an Ala to Asp change. The addition of DQB1*0203 brings the total number of DQB1 alleles identified to date to 26. HLA class II diversity is much greater in these African Colombian populations than that seen in nearby Amerindian populations. Analysis of regional Colombian African American HLA population genetics is discussed with respect to the Colombian Amerindian HLA genetics described in an accompanying paper.     

A HinfI polymorphism in the 5' flanking region of the human VNTR locus D1S80

A restriction fragment length polymorphism (RFLP) characterized by the presence (HinfI+) or absence (HinfI-) of a HinfI site has been found in the 5' flanking region of the VNTR locus D1S80. RFLP-allele frequencies were determined from 82 unrelated individuals: HinfI+ = 0.49, HinfI- = 0.51. The RFLP/VNTR haplotype frequencies show an absolute association between the HinfI+ allele and the VNTR allele of 18 repeat units and an extreme association between the HinfI- allele and the VNTR allele of 24 repeat units. The remaining VNTR alleles associate more randomly with the 2 flanking HinfI alleles.     

The effect of alpha-tocopherol and pentoxyfilline on ischemia-reperfusion induced liver injury in rats

BACKGROUND/AIM: In the present study our purpose was to investigate the effect of pentoxyfilline, that plays a role in microcirculation and tissue oxygenation, alone and in combination with an antioxidant vitamin E on tissue damage in the rat liver induced by ischemia-reperfusion. METHODOLOGY: Thirty-one albino rats were divided into four groups. Rats in group I (n= 7), group II (n= 8) and group III (n= 8) were given, respectively, pentoxyfilline (25 mg/kg), pentoxyfilline and vitamin E in combination (25 mg/kg and 50 mg/kg, respectively) and equal volume of saline solution intraperitoneally for 7 days. Rats in group IV (n= 8) served as controls and received no treatment. On day 7 ischemia was induced by cross-clamping the hepatic artery, portal vein and left branch of the biliary duct for 30 minutes. Malondialdehyde (MDA) and catalase activity were assessed in tissue sample, and the level of ALT was measured in serum obtained after reperfusion for 30 minutes. Histological examination of tissue sample was also carried out. RESULTS: There was no significant difference in ALT level between three study groups. Group I and group II had significant lower MDA and catalase levels than those of group III. The results of histopathologic examination in group I and group II were better than that of group III. CONCLUSION: Our findings suggested that the treatment of pentoxyfilline alone and in combination with vitamin E decreased liver damage induced by ischemia-reperfusion and that the effect of latter was more effective but the difference between the two treatment patterns was not statistically significant.     

Molecular genetic analysis of the ABO blood group system: 4. Another type of O allele

We have encountered an allele which seems to be another type of O allele at the human histo-blood group ABO locus. We have determined the nucleotide sequence of this allele over the coding region in the last two coding exons. This allele does not possess the single-nucleotide deletion found common among all the O alleles previously analyzed. Compared with A1 allele, this allele has three nucleotide substitutions resulting in two amino acid substitutions. The introduction of these amino acid substitutions into the A1 transferase expression construct apparently abolished the enzymatic activity of A1 transferase.