Systematic analysis of prophages carried by Porphyromonas gingivalis.

To systematically investigate the prophages carrying in Porphyromonas gingivalis (P. gingivalis) strains, analyze potential antibiotic resistance genes (ARGs) and virulence genes in these prophages. We collected 90 whole genome sequences of P. gingivalis from NCBI and utilized the Prophage Hunter online software to predict prophages; Comprehensive antibiotic research database (CARD) and virulence factors database (VFDB) were adopted to analyze the ARGs and virulence factors (VFs) carried by the prophages. Sixty-nine prophages were identified among 24/90 P. gingivalis strains, including 17 active prophages (18.9%) and 52 ambiguous prophages (57.8%). The proportion of prophages carried by each P. gingivalis genome ranged from 0.5% to 6.7%. A total of 188 antibiotic resistance genes belonging to 25 phenotypes and 46 different families with six mechanisms of antibiotic resistance were identified in the 17 active prophages. Three active prophages encoded 4 virulence genes belonging to type III and type VI secretion systems. The potential hosts of these virulence genes included Escherichia coli, Shigella sonnei, Salmonella typhi, and Klebsiella pneumoniae. In conclusion, 26.7% P. gingivalis strains carry prophages, while the proportion of prophage genes in the P. gingivalis genome is relatively low. In addition, approximately 39.7% of the P. gingivalis prophage genes have ARGs identified, mainly against streptogramin, peptides, and aminoglycosides. Only a few prophages carry virulence genes. Prophages may play an important role in the acquisition, dissemination of antibiotic resistance genes, and pathogenicity evolution in P. gingivalis.

Sequence diversity of SIV(Mne) Nef in vivo and in vitro.

We have compared nef gene sequences isolated by PCR from peripheral blood lymphocyte DNA of macaques which had been inoculated with either biologically or molecularly cloned SIV(Mne). Two samples from each animal obtained either early after infection (week 2-8) or after significant CD4+ depletion (week 21-137) were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two more in all but one. Two of the common exchanges are located about 40 residues apart in the Nef core sequence, but are in proximity on the tertiary structure as judged by computer modelling using the structure of the HIV Nef core protein as a guide. Most recurring in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIVsm/HIV-2 groups. Animals inoculated with virus already containing the "late version" nef gene developed a more aggressive disease. The macaque adapted (MA)nef conferred a threefold higher infectivity to the cloned virus, but had no effects on CD4 downregulation. Propagation of virus with MAnef in tissue culture resulted in the rapid emergence of variants with newly attenuated nef. These findings suggest that the selective pressure on nef in vivo and in vitro are different.

Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein.

Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.

Western impressions of the Hong Kong health care system.

Hong Kong, Taiwan, Singapore, and Malaysia are initiating health care reform to meet the changing demands of populations with improved socioeconomic status and access to modern technologies and who are living longer than in previous generations. Hong Kong, in particular, is facing a unique set of circumstances as its people prepare for the transition in 1997 from a British colony to a Special Administrative Region of China. While spending only 4% of its gross domestic product on health care, it has a large and regulated public hospital system for most inpatient medical care and a separate, loosely regulated private health care system for most outpatient medical care. In 1993 the Secretary for Health and Welfare of Hong Kong initiated a year-long process to debate the pros and cons of 5 fundamental programs for health care reform. After a year of open consultation, options were chosen. We describe the Hong Kong health care system, the fundamental changes that have been adopted, and lessons for reformers in the United States.

Doctor-shopping in Hong Kong: implications for quality of care.

Doctor-shopping is defined as the changing of doctors without professional referral in the same illness episode. Two surveys on samples of patients attending Government Out-Patient Departments (GOPDs) in Hong Kong in 1989 (n = 869) and 1990 (n = 901) estimated the prevalence of shopping at nearly 40%, the main reason being a persistence of symptoms. Doctor-shoppers were likely to be younger with higher expectations of health care and who expressed dissatisfaction about aspects of the present service. In Hong Kong, patients perceive western medicine to be more effective and have high expectations of the effects of western drugs, in particular, in their administration by injection. Patients should be warned about iatrogenic health risks incurred from doctor-shopping; health education programmes are needed to modify unrealistic views about quality care. Health care providers in a mixed care system should promote greater continuity of care between doctors and both the public and private sectors, and identify and resolve problems which may be responsible for discontinuity of care.

Isolation and sequence analysis of the human syntaxin-encoding gene.

STX-related genes (encoding syntaxin) have been identified in rat and yeast; however, no human STX gene has been isolated thus far. Here, we describe the nucleotide sequence of the first human STX gene isolated from a placental library. It encodes a 297-amino-acid (aa) protein and is 89% identical to the aa sequence of rat STX 4a.

Aralkylation of 2'-deoxyguanosine: medium effects on sites of reaction with 7-(bromomethyl)benz[a]anthracene.

Product distributions were determined for reactions between 2'-deoxyguanosine, or its anion, and 7-(bromomethyl)benz[a]anthracene in either acetone/H2O (1:1) or 2,2,2-trifluoroethanol at 50 or 70 degrees C. The exocyclic amino-substituted product, N2-(benz[a]anthracen-7-ylmethyl)-2'-deoxyguanosine, was always the major nucleoside product formed in these reactions, although its yield was higher in reactions involving 2'-deoxyguanosine anion than the neutral nucleoside. Reaction with the anion also led to formation of the 1-substituted 2'-deoxyguanosine and a guanidinoimidazole nucleoside resulting from reaction of 2'-deoxyguanosine at carbon 5. Reactions of 2'-deoxyguanosine anion in 2,2,2-trifluoroethanol are shown to produce significant amounts of the N2-substituted product, which is difficult to prepare by other routes.

Molecular analysis of O6-substituted guanine-induced mutagenesis of ras oncogenes.

We have designed an Ha-ras/thymidine kinase (TK) cassette that permits the incorporation of chemically synthesized adducts within specific domains of the rat Ha-ras protooncogene. This cassette has been used to evaluate the mutagenicity of O6-substituted guanine residues, including O6-methylguanine and O6-benzylguanine, incorporated within the 12th codon of this locus. Mutations were monitored by the ability of these modified Ha-ras DNAs to transform Rat4 TK-cells. Our results indicate that both types of O6-substituted guanines are substantially mutagenic, although the methyl substituent induced a 2-fold higher percentage of transformed Rat4 TK+ colonies than its bulkier benzyl analogue. Interestingly, the mutagenicity of both O6-substituted guanines was found to be independent of their relative position within codon 12, therefore suggesting that the specific activation of Ha-ras oncogenes by GGA----GAA mutations in tumors induced by methylating carcinogens might be due to differences in the accessibility of these guanine residues to the carcinogen rather than to a differential rate of repair. Molecular analysis of the mutations induced by these O6-substituted guanines indicated that O6-methylguanine exclusively induced G----A transitions. In contrast, O6-benzylguanine produced G----C and G----T transversions in addition to G----A transitions. These results suggest that O6-methylguanine and its bulkier analogue O6-benzylguanine may induce mutagenesis by different mechanisms.

Synthesis and properties of H-ras DNA sequences containing O6-substituted 2'-deoxyguanosine residues at the first, second, or both positions of codon 12.

Nine 16-base oligodeoxyribonucleotides having the sequence of codons 9 through the first base of codon 14 of the rodent H-ras gene, i.e., 5'-d(GTGGGCGCTG*G*AGGCG)-3', have been synthesized containing either an O6-methyl- (G* = m6G), O6-ethyl- (G* = e6G), or the newly described O6-benzyl-2'-deoxyguanosine residue (G* = b6G) at position 10 and/or 11 from the 5'-end. The conversion of the protected O6-substituted 2'-deoxyguanosine derivatives to the corresponding 3'-[O-(2-cyanoethyl) diisopropylphosphoramidites] and their incorporation into oligodeoxyribonucleotides were conveniently accomplished by using an "in situ" activation approach and automated phosphite triester synthetic methods. These oligomers were characterized by enzymatic digestion to their component nucleosides and were shown to be free of detectable contamination by known nucleoside impurities that can be produced during these syntheses. The melting behavior and circular dichroism spectra are described for duplexes of the nine O6-substituted 2'-deoxyguanosine containing oligomers paired with the complementary strand 5'-d(CGCCTCCAGCGCCCAC)-3', and these data have been compared with those for the "wild-type" unsubstituted duplex.