Two cis-acting elements in negative RNA strand of Hepatitis C virus involved in synthesis of positive RNA strand in vitro

Sequences at the 3'-ends of both positive and negative strands of Hepatitis C virus (HCV) RNA harbor cis-acting elements required for RNA replication. However, little is known about the properties of the negative RNA strand as a template for the synthesis of positive RNA strand. In this study, a purified recombinant HCV RNA-dependent RNA polymerase (RdRp) was used to investigate the synthesis of positive RNA strand using the 3'-terminal region of negative RNA strand ((-)3'T RNA) as template. A mutagenesis analysis was performed to evaluate the role of the 3'-proximal stem-loop and the first 3'-cytidylate (3'C) of the negative RNA strand in the synthesis of the positive RNA strand. A negative RNA strand of wild type (wt) HCV as template was able to direct the synthesis of a full-length positive RNA strand. Deletion of the 3'-proximal stem-loop resulted in an approximately 90% decrease in RNA synthesis. Disruption of the 3'-proximal stem-loop structure by nucleotide substitutions led to a 70-80% decrease in RNA synthesis. However, the restoration of the stem-loop by compensatory mutations in the stem region restored also the RNA synthesis. Likewise, the deletion or substitution of the first 3'C by guanylate (G) led to a 90% decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in a 60-80% decrease in the RNA synthesis only. These findings demonstrate that the 3'-proximal stem-loop and the first 3'C of the negative RNA strand of HCV are two cis-acting elements involved in the synthesis of the positive RNA strand.     

Diverse locations of amino acids in HLA-DR beta chains involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0101), DR(alpha, beta 1*1101), and DR(alpha,beta 3*0202) molecules.

In a previous study, we used transfectants expressing hybrid HLA-DR(beta 1*0403)/DR(beta 1*0701) chains to map sequences involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0403) or DR(alpha, beta 1*0701) molecules. Amino acids 1-40 of the beta 1 domain were found to make the major contributions to most of the antibody binding epitopes studied. To begin to localize sequences that contribute to polymorphic antibody epitopes on DR(alpha,beta 1*0101), DR(alpha,beta 1*1101) and DR(alpha,beta 3*0202) molecules, we used indirect immunofluorescence and flow cytometry to assess the binding of mAb to transfectants expressing hybrid DR(beta 1*0101)/DR(beta 1*1101) or DR(beta 1*1101)/DR(beta 3*0202) chains that divide the DR beta chain into three segments: amino acids 1-40, 41-97, and the beta 2 domain. The results indicate that amino acids 41-97 of the beta 1 domain on DR(beta 1*0101), DR(beta 1*1101), or DR(beta 3*0202) are critical in most of the epitopes, including those recognized by human antibodies MP4 and MP12, and mouse mAb GS88.2, I-LR1, 21r5, and 7.3.19.1, whereas amino acids 1-40 of DR(beta 1*1101) are critical in the epitope recognized by the MCS-7 mAb, and both segments 1-40 and 41-97 of DR(beta 1*1101) are important in the epitopes recognized by the I-LR2 and UL-52 mAbs. Based on these data and comparison of DR beta allelic protein sequences, the residues that may play critical roles in these antibody binding epitopes are predicted.     

HLA-A*02 allele frequencies and haplotypic associations in Koreans

We have investigated the frequencies of HLA-A*02 alleles and their haplotypic associations with HLA-B and -DRB1 loci in 439 healthy unrelated Koreans, including 214 parents from 107 families. All of the 227 samples (51.7%) typed as A2 by serology were analyzed for A*02 alleles using polymerase chain reaction (PCR)-low ionic strength-single-strand conformation polymorphism (LIS-SSCP) method. A total of six different A*02 alleles were detected (A*02 allele frequency 29.6%): A*0201/9 (16.6%), *0203 (0.5%), *0206 (9.3%), *0207 (3.0%), and one each case of *0210 and *02 undetermined type. Two characteristic haplotypes showing the strongest linkage disequilibrium were A*0203-B38-DRB]*1502 and A*0207-B46-DRB1*0803. Besides these strong associations, significant two-locus associations (P<0.001) were observed for A*0201 with B61, DRB1*0901 and DRB1*1401, and for A*0206 with B48 and B61. HLA haplotypes carrying HLA-A2 showed a variable distribution of A*02 alleles, and all of the eight most common A2-B-DR haplotypes occurring at frequencies of > or =1% were variably associated with two different A*02 alleles. These results demonstrate that substantial heterogeneity is present in the distribution of HLA-A*02 alleles and related haplotypes in Koreans.     

The experience of Japan as a clue to the etiology of breast and ovarian cancers: relationship between death from both malignancies and dietary practices

Although breast and ovarian cancers are rare in Japan compared with other developed countries, the death rates for both are increasing. In Japan, dramatic lifestyle changes occurred after World War II. Over the past 50 years (1947-1997), the age-standardized death rates of breast and ovarian cancers increased about 2- and 4-fold, respectively, and the respective intake of milk, meat, and eggs increased 20-, 10-, and 7-fold. The increase in the annual death rates from breast and ovarian cancers might be due to the lifestyle changes (increased consumption of animal-derived food) that occurred after 1945. Among the food, milk and dairy products should receive particular attention since they contain considerable amounts of estrogens.     

Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical,biochemical, and molecular genetic aspects

We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the biotin-dependent mitochondrial carboxylases in lymphocytes remained below 50% of the mean control values. Not only urinary 3-hydroxyisovaleric acid excretion has been persistently elevated, but also plasma and, with even higher concentrations, cerebrospinal fluid 3-hydroxyisovaleric acid have not normalized. The unusual and insufficient response of this patient to biotin treatment can be explained by the effect of the combination of the common HLCS allele IVS10 +5 g>a on one chromosome and a truncating mutation on the other. This case illustrates mechanisms involved in the genotype-phenotype correlation that unequivocally exists in HCS deficiency.     

Prevalence of HIV in Mayotte

Mayotte is a little French island, located in the Indian ocean, between Madagascar and Mozambic. Officially, the population goes up to 150,000 inhabitants, but in fact, it probably comes up to about 200,000 people, because of a very numerous illegal immigration, especially coming from Anjouan, the nearest Comorian island. Up to now there are no data about HIV in Mayotte. There is only one adult medical unit (except intensive care). All patients detected as seropositive for HIV are sent for treatment in this unit. The only case of systematically proposed HIV serology is pregnancy, and this since 1994. 70% to 80% of women accept it. There are annually 6000 deliveries in Mayotte. When a seropositivity is detected, screening is systematically proposed to other members of the family. Epidemiology does not seem to worsen, even if detection is increasing: 8675 tests done in 1998, 9142 in 1999, 12,085 in 2000. All cases of seropositivity attended to at present time in our unit have been registered and studied, apart from patients who died before this study, and those who did not consult for more than two years. There were 8 HIV positive people who died since 1990 in Mayotte, most of them in 1991, 1992. There was no death in the last years, nor sanitary evacuation, except one. 50 people are registered: 20 males, 30 females. Average age is 34. 39 Comorian people, 10 metropolitans, and one from another origin. Contamination was heterosexual in 43 cases, intravenous drug user in 1 case, homosexual in 2 cases, professional in 1 case, unknown in 3 cases. There are 4 double contamination Ag Hbs/HIV, and 2 HCV/HIV. 39 subjects are at A stage, 5 B, 6 C. The beginning of the epidemic in Mayotte took place probably around 1990, among militaries and prostitutes. Now, transmission keeps going on, in most cases, heterosexual, as it occurs in the nearby Africa. Polygamy, official or officious, has a leading role. Females who were detected during pregnancy have obtained zidovudine, or have been able to pursue previous therapy. Medical care and therapy are free in Mayotte, but sometimes there are delays in supplying medicines or in returning results. Epidemic has not increased up to now; even though screening has. There are very few pediatric cases: only 4 cases were notified in Mayotte until now: one who died very soon, one who has gone to live in Reunion island, and two who are still in Mayotte: one is 12 years old, and another one 5 years old.