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PubMed1996

ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype.

The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels.

Amino Acid Sequence↗
PubMed2006

The mitomycin C (MMC)-binding protein from MMC-producing microorganisms protects from the lethal effect of bleomycin: crystallographic analysis to elucidate the binding mode of the antibiotic to the protein.

Antibiotic-producing microorganisms must be protected from the lethal effect of their own antibiotic. We have previously determined the X-ray crystal structure of the bleomycin (Bm)-binding protein, designated BLMA, as a self-resistance determinant from Bm-producing Streptomyces verticillus, which suggests that the binding of the first Bm to one of two pockets formed in the BLMA homodimer induces the cooperative binding of the second Bm to the other pocket. In the present study, we noticed that the X-ray crystallographic structure of a self-resistance determinant from a mitomycin C-producing microorganism, designated MRDP, reveals similarity to the folding pattern on the BLMA, although no sequence homology exists. To clarify the hypothesis that MRDP may function as a resistance determinant to Bm, we characterized and determined the crystal structure of MRDP complexed with the Cu(II)-bound form of BmA(2) grouped into the Bm family of antibiotics. The biochemical and structural studies for Bm binding provide evidence that the first Bm binds anti-cooperatively to a pocket of MRDP with binding affinity of the nanomolar order, whereas the second Bm binds to the other pocket, which has binding affinity of the micromolar order. The invisibility of the second Bm in the structure agrees with the observation that Escherichia coli-expressing MRDP displays lower resistance to Bm than that expressing BLMA. The structure of MRDP, which is complexed with the Cu(II)-bound BmA(2), revealed that the gamma-aminopropyldimethylsulphonium moiety of the antibiotic is sandwiched between the peripheral residues of the binding pocket and that its positively charged sulphonium head is accommodated completely in the negatively charged region of the MRDP pocket. Furthermore, the Cu(II)-bound BmA(2) has a very compact structure, in which the bithiazole ring of BmA(2) is folded back to the metal-binding domain.

Bacterial Proteins↗
PubMed2006

In vivo and in vitro regulation of type I IFN synthesis by synergistic effects of CD40 and type II IFN.

During cognate interaction with CD40 ligand (CD154)-expressing T cells, Ag-presenting accessory cells are activated for increased cytokine synthetic and costimulatory function. We examined whether CD40 modulates in vivo innate immune function over time, hypothesizing that distinct cytokine responses evolve to delayed microbial exposure. C3H/HeN mice pretreated with activating anti-CD40 Ab (FGK45) produced 10-fold more serum IFN-gamma and IL-12 p70 to delayed, but not synchronous, challenge with LPS. A novel finding was that LPS-induced IFN-alpha increased by 20-fold in mice pretreated for 24 h, but not 6 h or less, with anti-CD40. Anti-CD40-pretreated C57BL/6 RAG-2(-/-) mice similarly increased IFN-alpha responses to delayed LPS challenge, confirming mediation by innate immunity. Type I IFNR- and IFN-gamma-deficient mice treated with anti-CD40 failed to expand serum IFN-alpha responses to LPS challenge. Combined pretreatment with anti-CD40 and anti-IFN-gamma mAb showed that IFN-gamma produced after anti-CD40 pretreatment, but before LPS challenge, was necessary for IFN-alpha synthetic enhancement. Anti-CD40 also increased polyinosinic-polycytidylic acid (poly(I:C))-inducible IFN-alpha by 5-fold in an IFN-gamma-dependent fashion, but did not significantly increase IFN-alpha production to CpG or Pam(3)Cys challenges. Poly(IC)-stimulated splenocytes from anti-CD40-pretreated mice produced 4-fold more IFN-alpha than controls and production associated with CD11c(+) cells. Finally, rIFN-gamma and anti-CD40 combined synergistically to increase poly(IC)-inducible IFN-alpha synthetic capacity in bone marrow dendritic cells. We conclude that innate immune production of IFN-alpha is cooperatively regulated by CD40 and IFN-gamma acting on dendritic cells, suggesting a unique mechanism by which innate immune function evolves in response to specific adaptive immune signals.

Adjuvants, Immunologic↗
PubMed1997

[Rapid diagnosis of tuberculosis].

Two systems, the newly developed Mycobacteria Growth Indicator Tube (MGIT) and biphasic Septi-Chek AFB based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. The difference in the rates of isolation of bacteria between the two groups of media was more remarkable with smear-negative specimens. The isolation of the Mycobacterium tuberculosis complex by MGIT occurred 8 days previous to the isolation by the conventional Ogawa method. The mean time for detecting M. tuberculosis complex by Septi-Chek AFB was similar to those of the Ogawa method. A greater difference in isolation time was observed for mycobacteria other than M. tuberculosis (MOTT) isolates. These results indicate that the MGIT and Septi-Chek AFB systems based on liquid media are efficient for the recovery of mycobacteria. PCR and other nucleic acid amplification methods are widely used for the detection of M. tuberculosis in clinical specimens. Although the sensitivities of the Gen-Probe Amplified Mycobacteria Direct Test (MTD) and Amplicor Mycobacteria for the detection of the M. tuberculosis complex appear to be similar to the sensitivity of the culture method using the Septi-Chek AFB, the two methods should be quite useful for rapid detection of M. tuberculosis infections. On the other hand, two cooperative blind studies conducted between 6 to 9 laboratories to estimate the reliability and reproducibility of these two commercially available kits revealed the necessity of good laboratory practice and development of reference reagents to monitor the performance of the whole assay, including pretreatment of clinical specimens. Considerable progress has been made in recent years toward understanding the molecular basis of the resistance to antituberculosis drugs, isoniazid (katG, inhA, ahpC), rifampin (rpoB), pyrazinamide (pncA), streptomycin (rpsL, rrs), ethambutol (embB), and fluoroquinolones (gyrA). Most cases of resistance are related usually to simple nucleotide substitutions rather than to acquisition of new genetic elements. Multidrug-resistant isolates of M. tuberculosis arise as a consequence of sequential accumulation of mutations conferring resistance to single therapeutic agents. The basis of resistance is not able to be explained yet in a substantial percentage of strains (> 90%) for other antituberculosis drugs than rifampin. Further studies are required to fully understand the molecular mechanisms of resistance.

DNA, Bacterial↗
PubMed1975

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli. II. Localization of amino acid replacements in proteins S5 and S12 altered in double mutants resistant to neamine.

Protein S5 and S12 were isolated from 30S ribosomal subunits of two E. coli mutants highly resistant to the antibiotic neamine, and of the parental strain. Proteinchemical analyses on these proteins led to the following results: a) In protein S5 the arginine residue in peptide T2 of the parental strain is replaced by glycine in one (nea 314) or serine in the other (nea 319) of the two mutants. b) In protein S12 The proline residue in peptide T15 of the parental strain is replaced by leucine in mutant nea 314 and by glutamine in mutant nea 319. Comparison of these results with those obtained in earlier studies on other mutants with altered ribosomal proteins revealed that the amino acid replacements in neamine resistant mutants and in "revertants" from streptomycin dependence occur at the same amino acid positions of proteins S5 and S12. Therefore it is likely that both types of mutants belong to the same class.

Amino Acid Sequence↗
PubMed2026

Desert-derived Ensifer sp. SA403 enhances potato salt tolerance by reshaping rhizosphere microbiome functions and host responses.

Soil salinization increasingly threatens global food security, and potato (Solanum tuberosum L.), a moderately salt-sensitive crop, is particularly vulnerable to saline soils. Plant growth-promoting rhizobacteria (PGPR) offer a promising strategy to improve crop performance, yet how PGPR interact with native microorganisms to enhance potato salt tolerance remains poorly understood. In this study, we identified a desert-derived PGPR strain, Ensifer sp. SA403, which substantially enhanced potato performance under high salinity across sterile, non-sterile and field conditions. Physiologically, inoculation with SA403 reduced shoot Na⁺ accumulation and increased the K⁺/Na⁺ ratio; notably, these effects were markedly stronger in non-sterile substrates than under sterile conditions, indicating that SA403-mediated ion homeostasis relies on cooperation with the resident microbiota rather than on the strain acting alone. Metagenomic profiling indicated that SA403 strain reshaped rhizosphere communities, significantly enriching beneficial taxa such as Priestia and Bradyrhizobium, and upregulated functional pathways involved in glutathione and sulfur metabolism. Furthermore, host transcriptomic analyses showed that SA403 modulated plant responses to salt stress, with differentially expressed genes enriched in jasmonic acid signaling, ethanolamine metabolism and amino-acid biosynthesis pathways. Field trials on saline soils confirmed that SA403 significantly increased seedling emergence and tuber weight. Together, our results demonstrate that SA403 functions as a biological mediator that optimizes rhizosphere microecology and coordinates ion balance and host signaling to enhance potato salt tolerance. These findings support the potential of SA403 as a robust PGPR-based tool for sustainable potato production on saline soils.

Rhizosphere↗
PubMed1996

Mechanism of differential activities of ofloxacin enantiomers.

Ofloxacin, a potent quinolone antibacterial agent, has a tricyclic ring structure with a methyl group attached to the asymmetric carbon at the C-3 position on the oxazine ring. The S isomer (DR-3355) of ofloxacin has antibacterial activity up to 2 orders of magnitude greater than that of the R isomer (DR-3354). This differential antibacterial activity was not due to different drug transport mechanisms of the two isomers but was found to be derived from the inhibitory activity against the target enzyme, DNA gyrase. Previous mechanistic studies have suggested that the bactericidal effect of the drug is mediated through the stabilization of a cleavable complex via a cooperative drug binding process to a partially denatured DNA pocket created by DNA gyrase. The drug binds to supercoiled DNA in a manner similar to that to which it binds to the enzyme-DNA complex. In the present studies, we first examined the binding of the two radiolabeled ofloxacin enantiomers to supercoiled pUC9 plasmid DNA. Surprisingly, the two enantiomers possessed similar apparent binding affinities and binding cooperatives. The major difference in binding between the two stereoisomers was the molar binding ratio: 4 for the more active S isomer versus 2 for the less active R isomer. We next examined the relative binding potencies of the stereoisomers to the DNA-DNA gyrase complex. The results of a competition assay showed that (S)-ofloxacin binds 12-fold better to the complex than (R)-ofloxacin. The binding potencies of the two enantiomers and two other quinolones correlated well with their respective concentrations causing 50% inhibition against DNA gyrase. The results are interpreted by a stacking model by using the concept of the cooperative drug-DNA binding mechanism, indicating that the potencies of quinolones cannot be determined solely by the DNA binding affinity and cooperativity but can also be determined by their capability in maximally saturating the binding site. The capability of the drug in saturating the binding pocket manifests itself in an increased efficacy at inhibiting the enzyme through a direct interaction between the drug and the enzyme. The results augment the previous suggestion that the binding pocket in the enzyme-DNA complex involves multiple receptor groups including not only DNA bases but also a gyrase subunit. The higher level of potency of (S)-ofloxacin is proposed to derive from the fact that a greater number of molecules are assembled in the pocket. This greater number of molecules optimizes the interaction between the drug and the enzyme, possibly through a contact between the C-7 substituent and the quinolone pocket on the B subunit of DNA gyrase.

Anti-Infective Agents↗
PubMed2006

Triad3A regulates ubiquitination and proteasomal degradation of RIP1 following disruption of Hsp90 binding.

Toll-like receptors (TLRs) play a crucial role in innate immunity by recognizing microbial pathogens. Triad3A is an E3 ubiquitin-protein ligase that interacts with the Toll/interleukin-1 receptor domain of TLRs and promotes their proteolytic degradation. In the present study, we further investigated its activity on signaling molecules downstream of TLRs and tumor necrosis factor (TNF) receptor 1. Triad3A promoted down-regulation of two TIR domain-containing adapter proteins, TIRAP and TRIF, as well as a RIP1 but had no effect on other adapter molecules in either the TLRs or TNF-alpha signaling pathways. Multiple sequence alignment analysis suggested that RIP1 contains a TIR homologous domain, and mutation of amino acid residues in this domain identified three residues critical for its interaction with Triad3A. Moreover, Triad3A acted as a negative regulator in TNF-alpha signaling. Reduction of Triad3A expression by small interference RNAs rendered cells hyperresponsive to TNF-alpha stimulation. Conversely, overexpression of Triad3A in cells blocked TNF-alpha-induced cell activation. This negative regulation was effected independently of changes in the cellular protein level of RIP1. Further studies indicated that RIP1 formed a complex with Triad3A and heat shock protein 90 (Hsp90), which is a chaperone protein capable of maintaining the stability of its client proteins. Treatment of cells with geldanamycin to disrupt the Hsp90 complex led to proteasomal degradation of RIP1. Depletion of Triad3A by small interference RNA treatment inhibited geldanamycin-activated ubiquitination and proteolytic degradation of RIP1. These results suggest that Triad3A is an E3 ubiquitin-protein ligase to RIP1 and that Hsp90 and Triad3A cooperatively maintain the homeostasis of RIP1.

Adaptor Proteins, Vesicular Transport↗
PubMed1991

A possible novel pathway of regulation by murine T helper type-2 (Th2) cells of a Th1 cell activity via the modulation of the induction of nitric oxide synthase on macrophages.

Murine peritoneal macrophages activated with interferon (IFN)-gamma and lipopolysaccharide (LPS) produce high levels of nitric oxide (NO) and are efficient in killing the intracellular protozoan parasites Leishmania major in vitro. Earlier studies have shown that NO, whose synthesis in murine macrophages is catalyzed by an inducible enzyme NO synthase, plays a major effector role in the host resistance against microbial infection. We now shown that both the NO synthesis and the leishmanicidal activity can be inhibited by prior treatment of the cells with recombinant interleukin 4 (IL4). IL4 treatment had no effect on the binding of IFN-gamma to macrophages but prevented the induction of NO synthase in these cells activated with IFN-gamma and LPS. Since IFN-gamma is produced by murine T helper type-1 (Th1) cells, whereas IL4 is secreted by Th2 cells, these results suggest a novel pathway by which Th2 cells regulate an activity of Th1 cells, namely by inhibiting the induction of NO synthase. These results may also account for the mechanism by which the disease-promoting Th2 cells counteract the host-protective effect of Th1 cells in leishmaniasis and other intracellular parasitic diseases.

Amino Acid Oxidoreductases↗
PubMed1995

Antimicrobial resistance and type distribution of Streptococcus pneumoniae isolates causing systemic infections in Germany, 1992-1994.

A prospective study of pneumococcal infections was performed in cooperation with 40 clinical microbiology laboratories in Germany. Minimal inhibitory concentration (MIC) values for 844 strains of Streptococcus pneumoniae, isolated from patients with systemic infections, were determined in tests with penicillin, tetracycline, erythromycin, chloramphenicol, cefotaxime, and clindamycin by a standard broth microdilution method; 1.8% of pneumococcal isolates exhibited reduced susceptibility to penicillin (MIC, > or = 0.1 micrograms/mL). The Etest, which was used to confirm the level of resistance to penicillin, proved to be a reliable and easily performed method for determination of MICs. The rates of resistance to clindamycin, erythromycin, tetracycline, and chloramphenicol were 1.4%, 3.2%, 11.0%, and 1.9%, respectively. Resistance to cefotaxime was not observed. Typing of a randomly selected subgroup of all strains (n = 115) showed types 1 (9.6%), 14 (8.7%), 3 (7.8%), and 23F (7.8%) to be the most prevalent types in Germany. At least 86.1% of these pneumococcal strains belonged to capsular types included in the 23-valent vaccine.

Adult↗
PubMed2000

Association between measures of milk quality and risk of violative antimicrobial residues in grade-A raw milk.

OBJECTIVE: To determine whether bulk-tank standard plate counts or plate loop counts and bulk-tank somatic cell counts (SCC) were associated with detection of violative antimicrobial residues in milk from dairy cattle. DESIGN: Longitudinal study. PROCEDURE: Information for 1994 through 1997 was obtained from a large milk marketing cooperative that operated in multiple states throughout the northeastern and midwestern United States (16,831 herd-years of information from 6,546 farms) and from the Ohio Department of Agriculture Grade-A Milk Certification Program (12,042 herd-years of information from 4,022 farms). Data were analyzed by use of multivariate logistic regression. RESULTS: For both data sets, odds that a violative antibiotic residue would be detected increased as mean SCC for the herd-year increased. Standard plate counts and plate loop counts were not associated with odds that a violative antibiotic residue would be detected. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study suggested that the odds that a violative antibiotic residue would be found in bulk-tank milk increased as mean SCC for the herd-year increased. This suggests that management practices that would be expected to influence SCC may also influence the risk of antibiotic residue violations.

Animals↗
PubMed1991

[Therapeutic effect of ofloxacin on intractable pulmonary tuberculosis and ofloxacin resistance of tubercle bacilli isolated from the patients. Chest DIsease Cooperative Study Unit of National Sanatoriums in Kinki District].

Ofloxacin, a synthetic antibacterial pyridone-carboxylic acid derivative, was used in the treatment of intractable pulmonary tuberculosis. In this study, the therapeutic effect of Ofloxacin on pulmonary tuberculosis and Ofloxacin resistance were analyzed. All patients had been hospitalized in eight national sanatoria in Kinki district, and were excreting tubercle bacilli resistant to various anti-tuberculosis drugs agents. Ofloxacin was given to 118 patients orally at a daily dose of 300 mg to 600 mg for more than 3 months. A few anti-tuberculosis drugs, which had failed in the negative conversion of bacilli previously, were used in combination. By Ofloxacin, 23 patients (19.5%) showed negative conversion of tubercle bacilli in sputum culture within 5 months, and they remained culture-negative for at least 6 months after conversion. Side-effects were observed in 2 patients. One complained of arthralgia and the other felt abdominal fullness. But both were not serious. From these results, it was concluded that Ofloxacin was effective for intractable pulmonary tuberculosis. The resistance of tubercle bacilli to Ofloxacin increased significantly after it was used.

Adult↗
PubMed1997

Economic costs and trade impacts of microbial foodborne illness.

This article presents the economic costs of foodborne diseases for selected countries, the approaches used to calculate these costs, and a discussion on the interaction between microbial food safety issues and international trade in food. The human illness costs due to foodborne pathogens are estimated most completely in the United States of America, where, each year, 7 foodborne pathogens (Campylobacter jejuni, Clostridium perfringens, Escherichia coli O157:H7. Listeria monocytogenes, Salmonella, Staphylococcus aureus, and Toxoplasma gondii) cause an estimated 3.3-12.3 million cases of foodborne illness and up to 3900 deaths. These 7 pathogens are found in animal products and cost the United States an estimated $6.5-$34.9 billion (1995 US$) annually. The presence of foodborne pathogens in a country's food supply not only affects the health of the local population, but also represents a potential for spread to pathogens to visitors to the country and to consumers in countries which import food products. With more complete data on foodborne illnesses, deaths, costs and international trade rejections in each country, indicators could be developed by which changes in food safety can be monitored.

Cost of Illness↗
PubMed1997

The effect of monitoring of antibiotic use on decreasing antibiotic resistance in the hospital.

In Greece, antibiotic over-consumption and high resistance rates run in parallel. In the spring of 1989 surveillance of 12500 Gram-negative strains, derived from 55 hospitals from all over Greece, revealed that resistance rates of Pseudomonas aeruginosa, Enterobacter spp., Klebsiella spp. and Acinetobacter spp. to antimicrobial agents introduced after 1985 exceeded 50%. As a consequence, the application of (1) rules of hospital hygiene, (2) educational small group programs, and (3) an antibiotic policy aiming to restrict antibiotic use, was decided in Laiko General Hospital. Since 1989, imipenem, the newer quinolones, vancomycin, aztreonam and third-generation cephalosporins were only ordered to the hospital pharmacy after completion of a specific request form, which since 1991 has been more detailed and which can be signed only by physicians with interest in infectious diseases. In 1991, in cooperation with the pharmacy, an audit program was added requiring a final inspection of the already approved request forms by an infectious diseases specialist. Any disagreement was discussed with the physicians in charge. Consumption data were analysed monthly and discussed with each department. Newer antibiotic consumption in a selected month (November) of three consecutive years, before (1991) and after the application of the audit program (1992-1995) has been analysed. Results reveal a decrease in consumption of restricted antibiotics, especially in surgical departments and in kidney transplantation units, without simultaneous increase in consumption of the non-restricted compounds. Since 1994, resistance has decreased remarkably. However, the resistance of quinolones is increasing steeply. Consequently, for the last 12 months quinolones have been removed from the hospital formulary. An audit program requires close co-operation of physicians, pharmacists and, particularly, of surgeons, in the application of a correct prophylaxis regimen. It seems to be efficacious in reducing both resistance rates and total antibiotic consumption.

Anti-Bacterial Agents↗
PubMed2001

[Anti-tuberculosis drug resistance in Japan and in the world].

In 1994, the World Health Organization (WHO) and the International Union against Tuberculosis and Lung Disease (IUATLD) launched a global project on anti-tuberculosis drug resistance surveillance. The results from the first 4 years (1994-1997) and the second 4 years (1996-1999) of the projects were reported in 1998 and 2000, respectively. These surveillance results showed that resistance to anti-tuberculosis drugs is a global problem. The reports also showed that there were several hot spots around the world where prevalence of multidrug resistant tuberculosis (MDR-TB, defined as resistance to at least isoniazid and rifampin) was particularly high and could possibly threaten control programs. The Tuberculosis Research Committee of Japan (Ryoken) has conducted nationwide surveys for drug resistant tuberculosis at 2- or 5-yearly intervals since 1957. The 1997 survey showed that among patients with no prior treatment, resistance to any of the four drugs was found in 10.3%, and the prevalence of primary MDR was 0.8%. The prevalence of drug resistance in the previously treated cases was 42.4% for any of the four drugs and 19.7% for MDR, indicating a high prevalence rate compared with those reported in the global project. Compared with the previous survey in 1992, the current survey shows increased prevalence of drug resistance in both new and re-treatment cases. No significant differences in resistance rates by sex, age group, nationality, district, and/or accompanying diseases were observed in any of the new or re-treatment cases. Other factors associated with the high prevalence in re-treatment cases remain to be determined. A total of 78 hospitals in various districts of Japan participated the cooperative study. Each collaborating laboratory sent all the isolated mycobacterial cultures to the Research Institute of Tuberculosis (RIT). In the local laboratories, the absolute concentration method using 1% Ogawa egg slant, its modified methods using a 48-well plate and a 16-well plate, combination of above 2 or 3 methods, and other method were used for drugsusceptibility testing, and the proportion method using 1% Ogawa egg slant was used in the RIT. The results in the local laboratories were compared with those in the RIT. There was no significant difference in the concordance rates according to the test drugs among methods for drug susceptibility testing used in the local laboratories. Relatively lower concordance rates were seen in the laboratories using the Microtiter method related to high overestimation rates, compared with those in the laboratories using the standard method and Well-pack method. However, relatively lower concordance rates (less than 90%) were seen in the laboratories using any of the three methods, indicating that there are variations among facilities.

Adult↗
PubMed2001

Augmentation of human immunodeficiency virus type 1 subtype E (CRF01_AE) multiple-drug resistance by insertion of a foreign 11-amino-acid fragment into the reverse transcriptase.

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of the pol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3'-azido-3'-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the beta3-beta4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.

Adult↗
PubMed1999

Synergistic fungistatic effects of lactoferrin in combination with antifungal drugs against clinical Candida isolates.

Because of the rising incidence of failures in the treatment of oropharyngeal candidosis in the case of severely immunosuppressed patients (mostly human immunodeficiency virus [HIV]-infected patients), there is need for the development of new, more effective agents and/or compounds that support the activity of the common antifungal agents. Since lactoferrin is one of the nonspecific host defense factors present in saliva that exhibit antifungal activity, we studied the antifungal effects of human, bovine, and iron-depleted lactoferrin in combination with fluconazole, amphotericin B, and 5-fluorocytosine in vitro against clinical isolates of Candida species. Distinct antifungal activities of lactoferrin were observed against clinical isolates of Candida. The MICs generally were determined to be in the range of 0.5 to 100 mg. ml(-1). Interestingly, in the combination experiments we observed pronounced cooperative activity against the growth of Candida by using lactoferrin and the three antifungals tested. Only in a limited concentration range was minor antagonism detected. The use of lactoferrin and fluconazole appeared to be the most successful combination. Significant reductions in the minimal effective concentrations of fluconazole were found when it was combined with a relatively low lactoferrin concentration (1 mg/ml). Such combinations still resulted in complete growth inhibition, while synergy of up to 50% against several Candida species was observed. It is concluded that the combined use of lactoferrin and antifungals against severe infections with Candida is an attractive therapeutic option. Since fluconazole-resistant Candida species have frequently been reported, especially in HIV-infected patients, the addition of lactoferrin to the existing fluconazole therapy could postpone the occurrence of species resistance against fluconazole. Clinical studies to further elucidate the potential utility of this combination therapy have been initiated.

Amphotericin B↗
PubMed2006

The biological and toxin weapons convention.

The Convention on the Prohibition of the Development, Production, and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on their Destruction prohibits the development, production, acquisition, transfer, stockpiling and use of microbial or other biological agents, or toxins in a manner which has no justification for prophylactic, protective or other peaceful purposes. It also bans weapons, equipment or means of delivery designed to use such agents or toxins for hostile purposes or in armed conflict. It covers biological and toxin weapons against humans, animals and plants. This article provides a brief history of the Convention and presents an overview of its five Review Conferences; the 'Ad Hoc Group of Governmental Experts, open to all States Parties, to Identify and Examine Potential Verification Measures from a Scientific and Technical Standpoint' (usually referred to as VEREX); and efforts to develop a legally binding instrument to strengthen the Convention, as well as the annual meetings of experts and States Parties which have taken place over the last three years. Issues of particular relevance to the World Organisation for Animal Health (OIE) are highlighted throughout, demonstrating their longstanding and fruitful contributions to ensuring that veterinary science is used only for the benefit of mankind.

Animal Diseases↗