Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction. The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit. From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex. Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit. The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).     

Hepatitis in used syringes: the limits of sensitivity of techniques to detect hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, and antibodies to HBV core and HCV antigens

Hepatitis virus infections are common among injecting drug users. Syringes containing hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA were identified by polymerase chain reaction (PCR); syringes containing antibodies to HBV core antigen and HCV were identified by EIA. Syringe use was simulated to determine the sensitivity of these assays. The mean limits for PCR were 0.082 microliter of blood for HBV and 0.185 microliter for HCV; the mean limits for EIA were 0.185 microliter for HBV and 0.023 microliter for HCV. HBV PCR testing of 681 syringes returned to the needle exchange program in New Haven, Connecticut, revealed a decline from 7.8% HBV-positive at the program's outset to 2.6%. HCV antibodies were found in 12.1% of 207 syringes tested. Syringe testing can help estimate the prevalence and incidence of hepatitis virus infections when standard seroepidemiologic analyses cannot be applied.     

Evaluation of human immunodeficiency virus (HIV) type 1 RNA levels in cerebrospinal fluid and viral resistance to zidovudine in children with HIV encephalopathy

The amount of human immunodeficiency virus (HIV) type 1 RNA and the presence of a codon 215 mutation indicative of zidovudine resistance were evaluated in cerebrospinal fluid (CSF) and plasma obtained from HIV-1-infected children. The level of HIV-1 RNA in CSF was highest in children with severe encephalopathy (n = 25; median, 430 copies/mL; range, 0-2.2 x 10(5) copies/mL) followed by the moderately encephalopathic (n = 7; median, 330; range, 0-1130) and nonencephalopathic groups (n = 9; median, 0; range, 0-566) (P = .007). There was no correlation between CSF and plasma HIV-1 RNA levels. Five of 7 children with the codon 215 mutation in CSF had a progression of encephalopathy, while all 8 children with wild type codon 215 had improved or stable disease during zidovudine treatment (P = .007). These findings suggest that increased viral replication and emergence of drug-resistant HIV-1 variants within the central nervous system may play a role in progression of HIV encephalopathy.     

Identification of immunoreactive epitopes in proteins coded by gag, env, and pol genes of the type I human T-lymphotropic virus (HTLV-I) using synthetic peptides

Reactivity of 26 synthetic peptides that comprise 12 to 26 amino acid residues corresponding to segments of the gag p19, env gp46, and pol proteins of human T-lymphotropic virus type I toward 31 positive sera was studied using enzyme-linked immunosorbent assay. Specific reactivity with high titers of antibodies (presented in reciprocal dilution values) was detected for the synthetic peptides corresponding to fragments 110-130 and 100-130 (titers up to 4050) of p19, 174-197 (up to 800), 186-201 (up to 4050), 191-215 (up to 1350), 242-257 (up to 800), and 272-292 (up to 450) of gp46. Immunoreactivity of seven peptides, fragments of pol-proteins, was weak. New linear epitopes in the regions 145-158, 272-277, and 292-300 of gp46 were detected. In addition, location of the known linear epitopes in p19 and gp46 was refined on the basis of comparative study of overlapping peptides from these proteins.     

Localization of the human phosphotyrosine phosphatase-related genes (h-PRL-1) to chromosome bands 1p35-p34, 17q12-q21, 11q24-q25 and 12q24

The h-PRL-1 gene codes for a new phosphotyrosine phosphatase that may play an important role in the control of basic cellular processes such as cell growth and proliferation. Using the cDNA of the h-PRL-1 gene as a probe, we examined a somatic mouse and hamster x human hybrid panel and found that chromosomes 1, 17 and 11 harbor sequences homologous to h-PRL-1. By in situ hybridization of metaphase spreads, subchromosomal localizations were determined at bands 1p35-p34, 17q12-q21 and 11q24-q25; in addition, a faint signal was detected at 12q24. The chromosomal assignment of the genes homologous to h-PRL-1 will help the investigation of its possible involvement in human diseases involving genetic alteration at these chromosomal regions.     

Localization of human transcription factor TEF-4 and TEF-5 (TEAD2, TEAD3) genes to chromosomes 19q13.3 and 6p21.2 using fluorescence in situ hybridization and radiation hybrid analysis

The tetrapeptide Ala-lle-Gly-Met bound to a Wang resin via the methionine residue was studied by NMR under MAS conditions and compared to the same peptide in solution. The bound peptide exhibits average linewidths superior to those observed for the peptide in solution. The origin of the residual NMR linewidth observed for the bound form was investigated. The dynamics of the peptide is shown to be only marginally responsible for the increased linewidth; the major cause of the line broadening appears to be nonaveraged magnetic susceptibility differences.     

Evaluation of third-generation assays for detection of anti-hepatitis C virus (HCV) antibodies and comparison with presence of HCV RNA in blood donors reactive to c100-3 antigen

We tested serum samples from blood donors by using first-, second-, and third-generation enzyme immunoassays or recombinant immunoblot assays. Second- and third-generation assays gave comparable results. Circulating hepatitis C virus RNA was found in a high proportion of reactive samples. A lack of reactivity or low-level reactivity predicted the absence of hepatitis C virus RNA in 100% of the cases.     

Comparison of NucliSens and Amplicor monitor assays for quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma of persons with HIV-1 subtype A infection in Abidjan, C?te d'Ivoire

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, C?te d'Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (n = 67 [96%]; mean RNA levels, 5.2 log10 HIV-1 RNA copies/ml) than the NucliSens assay (n = 56 [80%]; 4.3 log10 HIV-1 RNA copies/ml) or the standard HIV Monitor assay (n = 44 [63%]; mean RNA levels, 3.8 log10 HIV-1 RNA copies/ml) (all P values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (r = 0.76; P < 0.001) and the standard HIV Monitor assay (r = 0.57; P < 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (r = 0.60; P < 0. 001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (r = -0.47 for the NucliSens assay, -0.45 for the standard HIV Monitor assay, and -0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.     

Deletion mapping in breast tumor cell lines points to two distinct tumor-suppressor genes in the 1p32-pter region, one of deleted regions (1p36.2) being located within the consensus region of LOH in neuroblastoma

The nucleotide sequence of the cry11Bb1 gene from Bacillus thuringiensis subsp. medellin was determined. The corresponding protein has a deduced molecular mass of 88.2 kDa, and is 60.9% and 83% identical to the proteins Cry11Aa1 and Cry11Ba1, respectively. The Cry11Bb1 protein contains five repetitive blocks of 16 amino acids at the C terminal part. It is highly toxic to first instar laboratory reared Aedes aegypti, Anopheles albimanus and Culex quinquefasciatus larvae.     

Mutation of residues in the C3dg region of human complement component C3 corresponding to a proposed binding site for complement receptor type 2 (CR2, CD21) does not abolish binding of iC3b or C3dg to CR2

Most evidence points toward there being a shared binding site in complement receptor type 2 (CR2, CD21) for the complement ligand C3dg and the EBV surface envelope glycoprotein gp350/220. Indeed, synthetic peptide studies have suggested that the CR2-binding sites in human C3dg and EBV gp350/220 share a similar sequence motif. The proposed CR2-binding sequence in C3dg is EDPGKQLYNVEA (residues 1199-1210 of mature C3), whereas that in EBV gp350/220 is EDPGFFNVEI (residues identical to C3dg are underlined). To further examine the role of amino acids 1199-1210 in the binding of the C3 fragments iC3b and C3dg to CR2, the following alanine-substitution variants of human C3 were tested in two independent CR2-binding assays: ED1199,1200AA; KQ1203,1204AA; L1205A; Y1206A; NV1207,1208AA; E1209A; and ED-KQ-NV1199,1200-1203,1204-1207,1208AA-AA-AA. Also engineered and tested was a chimeric C3 molecule in which the 1199-1210 sequence (PVPGGYQLTLEA) from the non-CR2-binding trout C3 molecule was grafted onto a human C3 background. Recombinant C3 proteins were expressed transiently in COS-1 cells, deposited as C3b on C3 convertase-bearing sheep erythrocytes and finally converted to cell-bound iC3b or C3dg using factors H and I. Binding of EAC423bi and EAC423dg to CR2 on Raji cells or EAC423dg to soluble CR2 was assessed. In most cases, the substitutions had little effect on CR2-binding activity and even in the case of the most highly substituted variants, the decrease in CR2-binding activity was less than twofold. Thus, contrary to the results anticipated from synthetic peptide studies, the single and multiple substitutions to the C3 sequence tested failed to corroborate a role for the 1199-1210 sequence in the C3dg-CR2 interaction.