Vaccination against hepatitis B with 4-double doses increases response rates and antibodies titers in HIV-infected adults.

BACKGROUND: Antibody responses to standard regimens of hepatitis B (HBV) vaccination are lower in HIV-infected subjects and the best hepatitis B vaccine schedule in this population is not known. OBJECTIVE: To assess the immunogenicity and to evaluate predictors of serologic response of a modified regimen of a HBV recombinant vaccine in a cohort of HIV-infected subjects. METHODS: HIV-infected subjects received 4 doses (40 µg) of a recombinant HBV vaccine at 0, 1, 2 and 6 months. Demographic information as well as CD4 cell count and plasma viral load were assessed at baseline. Protective and strong responses were defined as an anti-HBs titer ≥10 mIU/mL and ≥100 mIU/mL, respectively and were evaluated one month after the third and the fourth doses. RESULTS: 163 HIV-infected individuals were evaluated 67 (40%) were male and median age was 37 years. Median CD4 cell count was 385 cells/mm(3) and 113 (70%) had undetectable HIV-1 viral load. Protective antibody response was observed in 83 and 91% and a strong antibody response was observed in 62 and 80% of the subjects after 3 and 4 doses, respectively. In a multivariate logistic model undetectable HIV-1 viral load and higher CD4 cell counts were independent predictors of a strong antibody response after 4 doses. Patients with undetectable HIV viral load were almost 3 times more likely to have anti-HBs titers above 100 mIU/mL than those with detectable viral load. CONCLUSIONS: A 4-double-dose regimen of a recombinant HBV vaccine increased response rates and determined higher antibody titers which may translate in prolonged protection against HBV. Inclusion of a fourth dose of HBV vaccine for HIV-infected subjects should be considered in the public health setting.

Prioritising prevention strategies for patients in antiretroviral treatment programmes in resource-limited settings.

Expanded access to antiretroviral therapy (ART) offers opportunities to strengthen HIV prevention in resource-limited settings. We invited 27 ART programmes from urban settings in Africa, Asia and South America to participate in a survey, with the aim to examine what preventive services had been integrated in ART programmes. Twenty-two programmes participated; eight (36%) from South Africa, two from Brazil, two from Zambia and one each from Argentina, India, Thailand, Botswana, Ivory Coast, Malawi, Morocco, Uganda and Zimbabwe and one occupational programme of a brewery company included five countries (Nigeria, Republic of Congo, Democratic Republic of Congo, Rwanda and Burundi). Twenty-one sites (96%) provided health education and social support, and 18 (82%) provided HIV testing and counselling. All sites encouraged disclosure of HIV infection to spouses and partners, but only 11 (50%) had a protocol for partner notification. Twenty-one sites (96%) supplied male condoms, seven (32%) female condoms and 20 (91%) provided prophylactic ART for the prevention of mother-to child transmission. Seven sites (33%) regularly screened for sexually transmitted infections (STI). Twelve sites (55%) were involved in activities aimed at women or adolescents, and 10 sites (46%) in activities aimed at serodiscordant couples. Stigma and discrimination, gender roles and funding constraints were perceived as the main obstacles to effective prevention in ART programmes. We conclude that preventive services in ART programmes in lower income countries focus on health education and the provision of social support and male condoms. Strategies that might be equally or more important in this setting, including partner notification, prompt diagnosis and treatment of STI and reduction of stigma in the community, have not been implemented widely.

Urinary tract infections in renal transplant recipients: virulence traits of uropathogenic Escherichia coli.

BACKGROUND: Urinary tract infection (UTI) is the most common infectious complication after renal transplantation. Most infections are caused by uropathogenic Escherichia coli (UPEC). There are limited data on the prevalence of virulence traits among UPEC isolated from renal transplant recipients. This study compared the phenotypic and genotypic profiles of UPEC strains isolated from recipients with those from control patients. METHODS: E coli isolates that caused UTI in recipients versus nonimmunosuppressed control patients were characterized according to phylogenetic group and the presence of urovirulence genes pap1/pap2; sfa1/sfa2; afa1/afa2; aer1/aer2; and cnf1/cnf2. RESULTS: Thirty-six UPEC isolates from recipients and another 27 from control individuals were included in the study. The proportion of episodes of pyelonephritis in recipients (50%) versus control subjects (41%) was similar (P = .46). However, secondary bacteremia was observed only among recipients (n = 8; P < .001). There was no significant difference in the distribution of phylogenetic groups or the prevalence of analyzed virulence traits between UPEC isolated from the 2 groups. Nevertheless, strains associated with secondary bacteremia in recipients showed a higher prevalence of mannose-resistant hemagglutination (P = .013). CONCLUSION: The phenotypic and genotypic characteristics of UPEC isolated from recipients were similar to those from control patients at a tertiary care center. Secondary bacteremia in recipients was associated with a higher prevalence of mannose-resistant hemagglutination.

Integration of porin into the outer mitochondrial membrane through the common import machinery located in contact sites.

Although no binding of porin-precursor to trypsin-pretreated rat liver mitochondria was detectable, its integration into these mitochondria was observed to some extent, possibly by the bypass-import system first demonstrated in fungus mitochondria. A topographical study of this bypass-import system demonstrated that import of porin occurs at contact sites between the outer and inner membranes. Furthermore, antibodies to 29 kDa outer membrane protein, a component of the import machinery in contact sites for precursors of ornithine aminotransferase and sulfite oxidase, inhibited the integration of porin, suggesting that the 29 kDa protein is involved in the imports of most mitochondrial precursor proteins.

Transport of prepro-albumin into inverted vesicles prepared from the inner membrane of rat liver mitochondria.

When inverted vesicles prepared from the inner membrane of rat liver mitochondria were incubated with prepro-rat serum albumin, considerable amounts of prepro-albumin and pro-albumin were recovered with the inverted vesicles re-isolated by centrifugation. Pro-albumin was resistant to trypsin, but prepro-albumin was completely digested by trypsin, indicating that prepro-albumin was transported into the vesicles and concomitantly converted to pro-albumin. This transport process required ATP, but not a membrane potential. These results suggest that some export machinery for a protein having an amino acid sequence in its N-terminal portion similar to the signal sequence of secretory protein exists in the inner mitochondrial membrane.

Evidence that rat liver mitochondrial and cytosolic fumarases are synthesized from one species of mRNA by alternative translational initiation at two in-phase AUG codons.

Rat liver contains two isozymes of fumarase, mitochondrial and cytosolic enzymes. Recently, we suggested that the precursors of both isozymes might be synthesized by one species of mRNA [Suzuki, T., Sato, M., Yoshida, T. & Tuboi, S. (1989) J. Biol. Chem. 264, 2581-2586]. To examine this possibility, we have isolated and characterized rat genomic clones for fumarase. The isolated clones covered almost all of the 5' half of the fumarase gene consisting of five exons. The first exon contained the whole 5' non-coding region and the signal peptide of mitochondrial precursor. The second exon encoded 45 amino acid residues of both mature proteins, starting from the N-terminal alanine. By using the boundary region of the first intron and the second exon as an S1-nuclease-analysis probe, we obtained conclusive evidence that rat liver contains no other mRNA specific for the cytosolic isozyme of fumarase. Two transcription-initiation sites were identified by further S1-nuclease-mapping analysis and were shown to be located very close to each other, differing by only four bases in length. Therefore, these sites were considered to be functionally the same. The results obtained by hybrid-selected translation, with a DNA fragment of the 5' non-coding region as a hybridization probe for selecting mRNA, were consistent with the above findings. We found a plausible secondary structure within the 5' non-coding mRNA sequence that may impede initiation and so alter the efficiency of translation. We also discuss the mechanism regulating translational initiation.

Purification of 52 kDa protein: a putative component of the import machinery for the mitochondrial protein-precursor in rat liver.

A protein having a molecular mass of 52 kDa was purified to homogeneity from solubilized mitochondrial membrane proteins by affinity column chromatography using the synthetic presequence of ornithine aminotransferase (OAT) as the ligand. This 52 kDa protein was specifically bound to the affinity column and eluted with 1 mM OAT-presequence, indicating that it recognized the presequence and bound to it specifically. Anti-52 kDa protein Fab fragments specifically inhibited the import of OAT-precursor into mitochondria, showing that the 52 kDa protein plays an essential role in this process. These results suggest that 52 kDa protein is a component of the import machinery of the mitochondrial protein-precursor in the mitochondrial membrane.

Purification and identification of a cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria.

A cytosolic factor required for import of the precursor of mitochondrial protein into mitochondria was purified to homogeneity from a rabbit reticulocyte lysate by affinity column chromatography using a synthetic peptide containing the presequence of ornithine amino-transferase as a ligand. The molecular mass of the purified protein was estimated as 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The import of precursors of ornithine aminotransferase and sulfite oxidase into mitochondria was inhibited by anti-28-kDa protein IgG raised in guinea pigs. This antibody also blocked the binding of these precursors to mitochondria. These results suggest that the 28-kDa protein is an essential component of the import machinery in the cytosol and that anti-28-kDa protein IgG blocked the binding of the precursor of ornithine aminotransferase to mitochondria, but not the penetration step. Therefore, the 28-kDa protein may be a factor that should be named the "targeting factor" for import of mitochondrial protein.

Purification of the putative import-receptor for the precursor of the mitochondrial protein.

The receptor protein for the mitochondrial protein precursor synthesized in the cytosol was extensively purified from the mitochondrial membrane fraction by affinity column chromatography using a synthetic peptide containing the extrapeptide of ornithine aminotransferase as a ligand. The purified fraction contained two major proteins with molecular masses of 52 and 29 kDa. Of these proteins, only the 29 kDa protein bound to the extrapeptide of ornithine aminotransferase. Furthermore, anti-29 kDa protein Fab fragments inhibited the import of pre-ornithine aminotransferase into mitochondria, suggesting that the 29 kDa protein plays an essential role in the process of import of the mitochondrial protein precursor.

Presence of the cytosolic factor stimulating the import of precursor of mitochondrial proteins in rabbit reticulocytes and rat liver cells.

Previously we purified a cytosolic factor that stimulates the import of the extrapeptide (the synthetic peptide of the presequence of ornithine aminotransferase) into the mitochondrial matrix (Ono, H., and Tuboi, S., 1988, J. Biol. Chem. 263, 3188-3193). In this work this cytosolic factor was shown also to stimulate the import of the precursors of ornithine aminotransferase, a large subunit of succinate dehydrogenase, and sulfite oxidase. The amounts of these precursors bound to the outer mitochondrial membrane were increased by this cytosolic factor, suggesting that the cytosolic factor participates in the recognition step in the import process of the precursor protein. When the cytosolic factor was applied to an ATP-agarose column, the import-stimulating activity was recovered entirely in the unadsorbed fraction. Immunochemical studies showed that in these conditions the 70-kDa heat shock-related protein (Hsp 70) was present exclusively in the fraction adsorbed to the ATP-agarose column. The cytosolic factor is thus different from the 70-kDa heat shock-related protein, which was identified as a factor required for the import of mitochondrial proteins in yeast. The cytosolic factor was also detected in the cytosol of rat liver cells, and a considerable amount of this factor was recovered from rat liver mitochondria by washing them with high salt buffer, suggesting that the cytosolic factor has affinity to the outer mitochondrial membrane and binds to its receptor on the membrane. From these results, we conclude that the cytosolic factor forms a complex with the precursor of mitochondrial protein and then this complex binds to the outer mitochondrial membrane, probably via the receptor of the cytosolic factor.

Specific pattern of ruptured annulus fibrosus in lumbar degenerative spondylolisthesis.

In order to study the antepriori etiologic factors of degenerative spondylolisthesis, the discograms and CTD were analyzed and the rate of disc slipping and disc indices wer evaluated in 30 cases with degenerative spondylolisthesis. (1) The characteristic S-shaped image which extended from anteroinferior to posterosuperior up to the posterior margin of a vertebral body was observed in 89.7% of slipped discs in lateral discograms. CTD revealed that this image represented a circular splitting in the external and intermediate annulus fibrosus. (2) Discographic degeneration of the discs adjacent to a slipped disc was relatively mild, and their disc indices were not significantly different from those of controls. (3) A negative correlation with r = -0.434 was found between the slipping rate and the disc index. From these results, it was postulated that the site and direction of the circular splitting in laminae of the annulus fibrosus, and the direction of the load applied to an intervertebral disc are important etiologic factors of degenerative spondylolisthesis.

Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single mRNA with two alternative in-phase AUG initiation sites.

By use of anticytosolic fumarase antibody, a cDNA clone was isolated from a rat liver cDNA library in the expression vector lambda gt11 and in the pBR 322 vector. A clone with an insert of about 1.7 kbp was isolated. Nucleotide sequence analysis of the insert revealed that the cDNA contained a noncoding region composed of 25 nucleotides in the 5' terminus, the coding region composed of 1,521 nucleotides, and the 3' nontranslated region composed of 43 nucleotides followed by a poly(A)+ tail. The open reading frame encoded a polypeptide of 507 amino acid residues (predicted Mr = 54,462), which contained an additional sequence composed of 41 amino acid residues on the N-terminus of the mitochondrial mature fumarase (the presequence). Thus, this reading frame was concluded to encode the precursor of mitochondrial fumarase. The amino acid sequence predicted from the nucleotide sequence contained all the amino acid sequences of 12 proteolytic polypeptides obtained by digestion of purified mitochondrial fumarase with V8 protease. The total amino acid sequence of the mitochondrial fumarase also contained all the sequences of 14 proteolytic peptides prepared from the cytosolic fumarase, indicating that the amino acid sequences of these two isozymes are identical. Furthermore, the results obtained by hybrid-selected translation, Northern blot and primer-extension analyses using appropriate cDNA segments prepared from fumarase cDNA (1.7 kbp) as the probe or primer suggested a possibility that both precursors of the mitochondrial and cytosolic fumarases were synthesized with one species of mRNA having base sequence coding presequence of the mitochondrial fumarase by unknown post-transcriptional mechanism(s). Rat liver cells may contain a specific RNA(18S) modulating the translational activity of mRNA for fumarase. This RNA(s), which was contained in poly(A)- fraction, was partially purified by high-performance gel filtration. The partially purified RNA(s) suppressed the translational activity of the cytosolic fumarase, whereas the translational activity of the mitochondrial one was accelerated by this RNA(s).

Immunocytochemical localization of creatine kinase M in canine myocardial cells: most creatine kinase M is distributed in the A-band.

The localization of creatine kinase (CK) M in canine myocardium was immunocytochemically studied by a direct immunoperoxidase method. Specific antiserum against CK-M was produced in rabbits immunized with canine CK-MM. An anti-CK-M Fab'-horseradish peroxidase conjugate was prepared by the maleimide method. Frozen sections prepared from fixed canine myocardium were stained with the conjugate and observed by light and electron microscopy. In light microscopy of longitudinal sections, CK-M showed a cross-striated pattern consisting of distinct broad and narrow brown bands. Immunoelectron microscopy revealed that the regions of the broad and narrow brown bands corresponded to the A-band and the Z-line, respectively. Most CK-M in the A-band was associated with the thick fibers, and a small amount of CK-M was found in the M-line. These findings suggest that ATP regeneration from the ADP produced by myosin ATPase is related to the participation of this CK associated with the thick fibers rather than that of the M-line-bound CK. Creatine kinase M was also found in the sarcolemmal membrane, the membranes of the sarcoplasmic reticulum, and the mitochondrial outer and inner membranes. This report provides new information for understanding the physiological role of the phosphorylcreatine shuttle in the myocardial energy transport system.

Evidence for intra-mitochondrial degradation of the extrapeptide of ornithine aminotransferase.

When rat liver mitochondria that had imported a synthetic extrapeptide of ornithine aminotransferase (composed of 34 amino acids) were incubated at 25 degrees C, the extrapeptide in their matrix was degraded inside the mitochondria. The degradation of the extrapeptide did not depend on energy either inside or outside the mitochondria. The degrading activity was found exclusively in the mitochondrial soluble fraction and only inhibited by N-ethylmaleimide of eight protease-inhibitors tested. These observations show that the extrapeptide cleaved from the precursor of the mitochondrial protein in the mitochondria is degraded by some ATP-independent proteases inside the mitochondria.

Primary structure of rat brain protein carboxyl methyltransferase deduced from cDNA sequence.

Two cDNA clones for protein carboxyl methyltransferase were isolated from a rat brain cDNA library in lambda gt 11 with synthetic oligonucleotides as probes. The two clones differ in size, but the nucleotide sequence including the whole coding region of the shorter cDNA is completely identical with the corresponding sequence of the longer cDNA. The open reading frame encodes a polypeptide of 227 amino acid residues, with a molecular weight of 24,626. This molecular weight is comparable to those reported for other protein carboxyl methyltransferases from several animals, which were determined by gel filtration chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene.

By use of anti-cytosolic fumarase antibody, a cDNA clone was isolated from a rat liver cDNA library in the expression vector lambda gt11 and in the pBR 322 vector. A clone with an insert of about 1.7 kilobases was isolated. Nucleotide sequence analysis of the insert revealed that the cDNA contained a 5'-noncoding region of 25 nucleotides, the coding region of 1,521 nucleotides, and a 3'-nontranslated region of 43 nucleotides followed by a poly(A)+ tail. The open reading frame encoded a polypeptide of 507-amino acid residues (predicted Mr = 54,462), which contained an additional sequence of 41-amino acid residues on the NH2 terminus of the mitochondrial mature fumarase (the presequence). Thus, this reading frame was concluded to encode the precursor of mitochondrial fumarase. The amino acid sequence predicted from the nucleotide sequence contained all the amino acid sequences of 12 proteolytic polypeptides produced by digestion of purified mitochondrial fumarase with V8 protease. The total amino acid sequence of the mitochondrial fumarase also contained all the sequences of 14 proteolytic peptides obtained from the cytosolic fumarase, indicating that the amino acid sequences of these two isozymes are identical. Furthermore, the results obtained by hybrid-selected translation, Northern and Southern blot, and primer-extension analyses using appropriate cDNA segments prepared with fumarase cDNA (1.7 kilobases) as a probe or primer suggested that the mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene and a possibility that the precursors of both these fumarases were synthesized by one species of mRNA having a base sequence coding the presequence of the mitochondrial fumarase by some unknown post-transcriptional mechanism(s).

The cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria.

The mechanism of import of proteins into mitochondria was studied by using the peptide of the presequence of ornithine aminotransferase (the extrapeptide), which was chemically synthesized and is composed of 34 amino acids. When the extrapeptide was incubated with isolated mitochondria in the presence of a rabbit reticulocyte lysate at 25 degrees C, it was imported into the mitochondrial matrix, and the import depended on the inner membrane potential, but not added ATP. The import of several precursors of mitochondrial proteins was competitively inhibited by the presence of excess extrapeptide in the reaction system, indicating that the extrapeptide and mitochondrial proteins were imported by the same machinery. Import of the extrapeptide was significantly stimulated by addition of a rabbit reticulocyte lysate, and a component of the lysate (the cytosolic factor) stimulating import of the extrapeptide was purified about 20,000 times by successive column chromatography on DEAE-cellulose and aminopentyl-Sepharose 4B. The binding of the extrapeptide to liposomes composed of egg lecithin and partially purified receptor of the precursor of mitochondrial protein (Ono, H., and Tuboi, S., (1985) Biochem. Int. 10, 351-357) required the cytosolic factor when the concentration of the peptide was less than 1.5 X 10(-8) M, suggesting that the physiological binding of the precursors of mitochondrial proteins to the receptor is dependent on the cytosolic factor. The extrapeptide and the cytosolic factor were shown to form a complex. From these results, the mechanism of binding of the extrapeptide to the receptor of the mitochondrial outer membrane is suggested to be as follows: the peptide (the precursor of mitochondrial protein) and the cytosolic factor form a complex, and then the complex is recognized by and bound to the receptor.

[Multiple primary malignant tumors of rhabdomyosarcoma in an upper arm and breast cancer].

Multiple primary malignant tumors of rhabdomyosarcoma in a upper arm and cancer of breast are extremely rare. In September 1970, a female, aged 86 years, underwent the first resection operation performed on a pleomorphic rhabdomyosarcoma of the right upper arm. She underwent radiation therapy (8000 rads at the operative field) postoperatively. In August 1980, a simple mastectomy was performed for a cancer of the right breast. In December 1984, fourteen years after her first operation, a recurrence was detected. The tumor recurred five times during the next two years and was resected each time. The patient died of cachexia sixteen years after the first operation.

Integration of porin synthesized in vitro into outer mitochondrial membranes.

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).

Tubulin and high molecular weight microtubule-associated proteins as endogenous substrates for protein carboxymethyltransferase in brain.

The endogenous substrate for protein carboxymethyltransferase in brain was examined. Several polypeptides were methylated when brain slices were incubated with L-methionine or when subcellular fractions of brain, such as the cytosolic fraction, were incubated with S-adenosyl L-methionine. Two methyl-accepting proteins in the cytoplasm were identified as tubulin and high molecular weight microtubule-associated proteins (300 kDa), which are components of microtubules. Tubulin behaved as a 43 kDa protein in acidic polyacrylamide gel electrophoresis, but as a 55 kDa protein in SDS-polyacrylamide gel electrophoresis. The methyl moiety transferred to these proteins from L-methionine was labile at alkaline pH. The high molecular weight microtubule-associated proteins showed higher methyl-accepting activity than tubulin or ovalbumin, which was used as a standard substrate: about 20 mmol of high molecular weight microtubule-associated proteins, 2 mmol of tubulin and 10 mmol of ovalbumin were methylated per mol of each protein in 30 min under the experimental conditions used.