Influence of river discharge on abundance and dissemination of heterotrophic, indicator and pathogenic bacteria along the East Coast of India.

In order to examine the influence of discharge from different rivers from peninsular India and urban sewage on intensity and dissemination of heterotrophic, indicator and pathogenic bacteria, a study was carried out during peak discharge period along coastal Bay of Bengal. The coastal Bay received freshwater inputs from the river Ganges while Godavari and Krishna contributed to the south. Contrasting difference in salinity, temperature, nutrients and organic matter was observed between north and south east coast of India. The highest heterotrophic, indicator and pathogenic bacterial abundance was observed in the central coastal Bay that received urban sewage from the major city. Intensity and dissemination of heterotrophic, indicator and pathogenic bacteria displayed linear relation with magnitude of discharge. The coliform load was observed up to 100km from the coast suggesting that marine waters were polluted during the monsoon season and its impact on the ecosystem needs further studies.

γδ T Cell-Mediated Immune Responses in Disease and Therapy.

The role of γδ T cells in immunotherapy has gained specific importance in the recent years because of their prominent function involving directly or indirectly in the rehabilitation of the diseases. γδ T cells represent a minor population of T cells that express a distinct T cell receptor (TCR) composed of γδ chains instead of αß chains. Unlike αß T cells, γδ T cells display a restricted TCR repertoire and recognize mostly unknown non-peptide antigens. γδ T cells act as a link between innate and adaptive immunity, because they lack precise major histocompatibility complex (MHC) restriction and seize the ability to recognize ligands that are generated during affliction. Skin epidermal γδ T cells recognize antigen expressed by damaged or stressed keratinocytes and play an indispensable role in tissue homeostasis and repair through secretion of distinct growth factors. γδ T cell based immunotherapy strategies possess great prominence in the treatment because of the property of their MHC-independent cytotoxicity, copious amount of cytokine release, and a immediate response in infections. Understanding the role of γδ T cells in pathogenic infections, wound healing, autoimmune diseases, and cancer might provide knowledge for the successful treatment of these diseases using γδ T cell based immunotherapy. Enhancing the human Vγ9Vδ2 T cells functions by administration of aminobisphosphonates like zoledronate, pamidronate, and bromohydrin pyrophosphate along with cytokines and monoclonal antibodies shows a hopeful approach for treatment of tumors and infections. The current review summarizes the role of γδ T cells in various human diseases and immunotherapeutic approaches using γδ T cells.

Fiji School of Medicine Diploma in Pharmacy graduates, ten year analysis--where are they now?

The Fiji School of Medicine (FSM) has been delivering pharmacy education since 1944. The philosophy of education provision changed from pharmacy technician training to pharmacist education in 1991 and since that time, pharmacy graduates from FSM have influenced the practice of pharmacy in Fiji and in other countries of the Pacific Region. FSM Diploma in Pharmacy graduates now comprise forty six percent of pharmacists active in the private sector of Fiji. Ten of sixty eight Fiji pharmacy graduates (fifteen percent) since 1993 have emigrated from Fiji. This paper describes the progress to date in pharmacy education philosophy and tracks the current employment and emigration of all pharmacy graduates since the introduction of pharmacist education (replacing the then pharmacy technician training) in 1991.

Anti-HIV inhibitors based on nucleic acids: emergence of aptamers as potent antivirals.

The development of resistance and the inability of currently approved antiretroviral drugs to completely eradicate HIV-1 have led to increased focus on therapies other than small molecules. Although nucleic acid-based intervention requires complex tasks involving intracellular delivery and/or stable expression in target cells, recent advances in gene therapy methods combined with continued progress in stem cell approaches have made nucleic acid-based compounds excellent candidates for effectively inhibiting intracellular targets. Consequently, multiple nucleic acid-based therapies are being developed. These include antisense nucleic acids, peptide nucleic acids and RNA decoys, which can interfere with HIV-1 replication. More recently, RNA interference, which exploits a novel cellular pathway, has been shown to effectively reduce viral titers in cell culture and promises to be a potential candidate for suppressing HIV replication in vivo. A promising candidate in the midst of these emerging approaches is the aptamer approach, which involves the use of a class of small nucleic acid molecules isolated from combinatorial libraries by an in vitro evolution protocol termed SELEX. Aptamers exhibit exquisite specificity, high affinity and the virtual lack of immunogenicity, features that make them exceptionally well-suited to combat HIV without affecting the host. The powerful nature of these specific antagonists of protein function could lead to the development of an effective anti-HIV therapy. Several highly specific, nucleic acid aptamers targeting select HIV proteins have been described. Investigations with anti-HIV RNA aptamers have shown an effective block to viral replication. This review summarizes the existing nucleic-acid based approaches to block HIV replication and attempts to chart the current progress in the development of aptamers against HIV, their use in inhibiting the virus replication, prospects for their use in the clinic and potential drawbacks.

The effect of increased processivity on overall fidelity of human immunodeficiency virus type 1 reverse transcriptase.

We previously reported that two insertions of 15 amino acids in the beta3-beta4 hairpin loop of fingers subdomain of HIV-1(NL4-3) RT confer an increased polymerase processivity. The processivity of human immunodeficiency virus (HIV) reverse transcriptase (RT) is thought to influence the fidelity of HIV-1 RT, which tends to create errors at template sites with high termination probability. Employing the two insertion variants of HIV-1 RT (FE20 and FE103), we examined the relationship between processivity, overall fidelity and error specificity. Although the overall mutation rate was unaffected by increased processivity, one of the mutants, FE103, generated significantly fewer frameshift errors. The other mutant, FE20, generated errors at hotspots not previously observed for HIV-1 RT. Our results indicate that an increase in the polymerase processivity of HIV-1 RT does not necessarily result in a decreased mutation rate and confirm that changes in processivity alter the sequence context in which the errors are made. Furthermore, our results also reveal that the mutation frequency obtained via in vitro gap-filling reactions with wild-type HIV-1(NL4-3) RT is only 2-fold higher than that obtained via a single cycle infection assay using the same, wild-type HIV-1(NL4-3) RT sequence as part of the helper pol function [Mansky and Temin: J Virol 69:5087-5094;1995].

Differential influence of nucleoside analog-resistance mutations K65R and L74V on the overall mutation rate and error specificity of human immunodeficiency virus type 1 reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) variants with the K65R or L74V substitution display resistance to several nucleoside analogs. An in vitro dNTP exclusion assay revealed an increased fidelity for K65R RT compared with wild-type RT, but little change for L74V RT. When the forward mutation rates were measured via a gap-filling assay, the K65R variant displayed an 8-fold decrease in the overall mutation rate (1.0 x 10(-3) versus 8.6 x 10(-3) for wild-type HIV-1 RT), whereas the rate for the L74V variant was closer to that for wild-type RT (5.0 x 10(-3)). The increase in overall fidelity observed for K65R RT is the largest reported for any drug-resistant HIV-1 RT variant. Nucleotide sequence analysis of lacZalpha mutants generated by variant RTs indicated that K65R RT displays uniform reduction in most types of errors, whereas L74V RT does not. Modeling the substitutions into the x-ray structure of the ternary complex revealed that the major influence of Leu(74) in stabilizing the templating base is unaffected by Val substitution, whereas the K65R substitution appears to increase the stringency of dNTP binding. It is speculated that the increased fidelity of K65R RT is due to an altered interaction with the dNTP substrate.

The ankyrin repeat-containing adaptor protein Tvl-1 is a novel substrate and regulator of Raf-1.

Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and in vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream of MEF2B with the two genes in a head to head arrangement.

The influence of 3TC-resistance mutations E89G and M184V in the human immunodeficiency virus reverse transcriptase on mispair extension efficiency.

Two nucleoside analog resistance mutations in HIV-1 reverse transcriptase (RT), E89G and M184V, were previously shown to increase the dNTP insertion fidelity of HIV-1 RT. However, forward mutation assays using a lacZ alpha reporter gene have revealed a lack of impact on the overall error rate of these variants. In an effort to investigate the basis for this discrepancy, we have examined whether the increases in misinsertion fidelity observed for E89G and M184V RTs are accompanied by an increase in mispair extension fidelity. The relative efficiencies with which the wild type, E89G, M184V and M184V/E89G HIV-1 RTs extend model template-primer duplexes containing 3'-OH terminal mismatches were measured. The calculated efficiencies of mispair extension ( f ext) were, in general, not significantly decreased from the wild type HIV-1 RT. In fact, the efficiency of extension from one of the mispaired primer-template duplexes was significantly increased for two of the mutants tested. These results suggest that amino acid substitutions that increase the fidelity of dNTP insertion do not necessarily increase misextension fidelity, and that the decreased misextension fidelity may counterbalance the increases in misinsertion fidelity observed for E89G and M184V RTs.

Virtues of being faithful: can we limit the genetic variation in human immunodeficiency virus?

Human immunodeficiency virus (HIV) infections are characterized by a high degree of viral variation. The genetic variation is thought to be a combined effect of a high error rate of reverse transcriptase (RT), viral genomic recombination, the selection forces of the human immune system, the requirement for growth in multiple cell types during pathogenesis, and persistent immune activation associated with HIV disease. This hypermutability gives the virus an ability to escape mechanisms of innate immune surveillance and therapeutic interventions. Indeed, HIV variants that are resistant to drugs that antagonize both the HIV protease and RT enzymes are well described. Furthermore, there are seemingly no procedures to restrict this disarming property of HIV to mutate rapidly. Recently we have shown that some of the drug-resistant RTs display an increased in vitro polymerase fidelity. The question is whether this finding will stimulate new approaches that will not only help the immune system to deal with the virus more efficiently but also to reduce or delay resistance to various classes of anti-HIV drugs. The pros and cons of this concept and the influence of viral replication rates and viral fitness on HIV variability are discussed.

The influence of 3TC resistance mutation M184I on the fidelity and error specificity of human immunodeficiency virus type 1 reverse transcriptase.

A common target for therapies against human immuno-deficiency virus type 1 (HIV-1) is the viral reverse transcriptase (RT). Treatment with the widely used nucleoside analog (-)-2', 3'-deoxy-3'-thiacytidine (3TC) leads to the development of resistance-conferring mutations at residue M184 within the YMDD motif of RT. First, variants of HIV with the M184I substitution appear transiently, followed by viruses containing the M184V substitution, which persist and become the dominant variant for the duration of therapy. In the three-dimensional crystal structure of HIV-1 RT complexed with double-stranded DNA, the M184 residue lies in the vicinity of the primer terminus, near the incoming dNTP substrate. Recent studies have shown that 3TC resistance mutations, including M184I, increase the nucleotide insertion and mispair extension fidelity. Therefore, we have examined the effects of the M184I mutation on the overall polymerase fidelity of HIV-1 RT via an M13-based forward mutation assay. We found the overall error rate of the M184I variant of HIV-1 RT to be 1.7 x 10(-5) per nucleotide. This represents a 4-fold increase in fidelity over wild-type HIV-1Hxb2RT (7.0 x 10(-5) per nucleotide) and a 2.5-fold increase in fidelity over the M184V variant (4.3 x 10(-5) per nucleotide). Of the nucleoside analog resistance mutations studied using the forward assay, the M184I variant has shown the greatest increase in fidelity observed to date. Interestingly, the M184I variant RT displays significantly altered error specificity, both in terms of error rate at specific sites and in the overall ratio of substitution to frameshift mutations in the entire target.

The impact of multidideoxynucleoside resistance-conferring mutations in human immunodeficiency virus type 1 reverse transcriptase on polymerase fidelity and error specificity.

Variants of human immunodeficiency virus type 1 (HIV-1) that are highly resistant to a number of nucleoside analog drugs have been shown to develop in some patients receiving 2',3'-dideoxy-3'-azidothymidine therapy in combination with 2',3'-dideoxycytidine or 2',3'-dideoxyinosine. The appearance, in the reverse transcriptase (RT), of the Q151M mutation in such variants precedes the sequential appearance of three or four additional mutations, resulting in a highly resistant virus. Three of the affected residues are proposed to lie in the vicinity of the template-primer in the three-dimensional structure of the HIV-1 RT-double-stranded DNA complex. The amino acid residue Q151 is thought to be very near the templating base. The nucleoside analog resistance mutations in the beta9-beta10 (M184V) and the beta5a (E89G) strands of HIV-1 RT were previously shown to increase the fidelity of deoxynucleoside triphosphate insertion. Therefore, we have examined wild-type HIV-1BH10 RT and two nucleoside analog-resistant variants, the Q151M and A62V/V75I/F77L/F116Y/Q151M (VILYM) RTs, for their overall forward mutation rates in an M13 gapped-duplex assay that utilizes lacZ alpha as a reporter. The overall error rates for the wild-type, the Q151M, and the VILYM RTs were 4.5 x 10(-5), 4.0 x 10(-5), and 2.3 x 10(-5) per nucleotide, respectively. Although the mutant RTs displayed minimal decreases in the overall error rates compared to wild-type RT, the error specificities of both mutant RTs were altered. The Q151M RT mutant generated new hot spots, which were not observed for wild-type HIV-1 RT previously. The VILYM RT showed a marked reduction in error rate at two of the predominant mutational hot spots that have been observed for wild-type HIV-1 RT.

Increased misincorporation fidelity observed for nucleoside analog resistance mutations M184V and E89G in human immunodeficiency virus type 1 reverse transcriptase does not correlate with the overall error rate measured in vitro.

Nucleoside analog-resistant variants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that displayed higher in vitro polymerase fidelity were previously identified via nucleotide insertion and mispair extension assays. To evaluate the contribution of increased nucleotide insertion and primer extension fidelities on the overall error rate of HIV-1 RT, we have measured the impact of two such mutations, E89G and M184V, on DNA copying fidelity in an M13 phage-based forward mutation assay. Using this assay, we observed mutation frequencies of 8.60 x 10(-3), 6.26 x 10(-3), 5.53 x 10(-3), and 12.30 x 10(-3) for wild-type, E89G, M184V, and double-mutant E89G/M184V HIV-1 RTs, respectively. Therefore, the overall polymerase fidelities of wild-type, E89G, M184V, and E89G/M184V HIV-1 RTs are similar (less than twofold differences) for DNA-dependent DNA synthesis. Thus, rather large increases in fidelity of deoxynucleoside triphosphate insertion and mispair extension observed previously appear not to influence the overall error rate of these mutants. However, a qualitative analysis of the mutations induced revealed significant differences in the mutational spectra between the wild-type and mutant enzymes.

Insertions into the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase reveal a role for fingers subdomain in processive polymerization.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) displays a characteristic poor processivity during DNA polymerization. Structural elements of RT that determine processivity are poorly understood. The three-dimensional structure of HIV-1 RT, which assumes a hand-like structure, shows that the fingers, palm, and thumb subdomains form the template-binding cleft and may be involved in determining the degree of processivity. To assess the influence of fingers subdomain of HIV-1 RT in polymerase processivity, two insertions were engineered in the beta3-beta4 hairpin of HIV-1NL4-3 RT. The recombinant mutant RTs, named FE20 and FE103, displayed wild type or near wild type levels of RNA-dependent DNA polymerase activity on all templates tested and wild type or near wild type-like sensitivities to dideoxy-NTPs. When polymerase activities were measured under conditions that allow a single cycle of DNA polymerization, both of the mutants displayed 25-30% greater processivity than wild type enzyme. Homology modeling the three-dimensional structures of wild type HIV-1NL4-3 RT and its finger insertion mutants revealed that the extended loop between the beta3 and beta4 strands protrudes into the cleft, reducing the distance between the fingers and thumb subdomains to approximately 12 A. Analysis of the models for the mutants suggests an extensive interaction between the protein and template-primer, which may reduce the degree of superstructure in the template-primer. Our data suggest that the beta3-beta4 hairpin of fingers subdomain is an important determinant of processive polymerization by HIV-1 RT.

Long-term reduction of serum bilirubin levels in Gunn rats by retroviral gene transfer in vivo.

Conjugation with glucuronic acid, mediated by bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (bilirubin-UGT), is essential for efficient biliary excretion of bilirubin. Inherited absence of this enzyme activity results in the potentially lethal Crigler-Najjar syndrome type I in humans and lifelong hyperbilirubinemia in Gunn rats. To develop a gene therapy for bilirubin-UGT deficiency, we constructed a high-titer replication-deficient amphotropic recombinant retrovirus (MFG-S hB-UGT1) capable of transferring the gene encoding bilirubin-UGT1, the principal bilirubin-UGT isoform in human liver. To stimulate hepatocyte proliferation, Gunn rats were subjected to 66% hepatectomy. After 24 hours, the portal vein, the hepatic artery, and the inferior vena cava above and below the hepatic vein were clamped, and the portal vein and the isolated segment of the vena cava were cannulated. The liver was perfused with the MFG-S hB-UGT1 preparation through the portal vein at 5 ml/min for 10 minutes, then circulation was restored. Control rat livers were perfused with a recombinant retrovirus expressing Escherichia coli beta-galactosidase. In MFG-S hB-UGT1-perfused rats, but not in controls, expression of human bilirubin-UGT1 was shown by immunotransblotting, bilirubin-UGT assay of liver homogenates, and biliary excretion of bilirubin diglucuronide and monoglucuronide. Mean serum bilirubin levels decreased by 20% to 25% in 3 weeks and remained at that level throughout the study period (18 months). This is the first report of long-term amelioration of inherited jaundice by retrovirus-directed gene therapy in an animal model for Crigler-Najjar syndrome.

Effects of mutations in Pr160gag-pol upon tRNA(Lys3) and Pr160gag-plo incorporation into HIV-1.

During HIV-1 viral assembly, both Pr160gag-pol and primer tRNA(Lys3) are packaged into the virus. tRNA(Lys) packaging (both tRNA(Lys3) and tRNA(Lys1,2) is dependent upon the presence of RT sequences within Pr160gag-pol. In this work, we have monitored the effect of Pr160gag-pol mutations upon incorporation of tRNA(Lys3) and Pr160gag-pol into HIV-1 produced from COS-7 cells transfected with mutant HIV-1 proviral DNAs. Mutations include carboxy deletions of Pr160gag-pol and small amino acid insertions and replacements within the various functional domains of the reverse transcriptase (RT). tRNA(Lys3) incorporation was monitored both by 2D PAGE of viral RNA, and by hybridization with tRNA(Lys3)-specific DNA probes. Our data indicates: (1) deletion of integrase sequence has a moderate effect upon select tRNA(Lys3) packaging, while carboxy terminal deletions extending further into the RNase H and connection domains more strongly reduce viral tRNA(Lys3) content; (2) tRNA(Lys3) incorporation is strongly reduced by small inframe amino acid insertions or replacements in the carboxy region of the thumb domain and the amino half of the connection domain of RT, but tRNA(Lys3) incorporation is altered little, or not at all, by similar amino acid insertional mutations within other RT domains, such as the fingers, palm, RNase H, the amino portion of the thumb, and the carboxy region of the connection domain. The inability of connection domain mutant virus to incorporate tRNA(Lys3) and to properly process precursor proteins in the virus is due to the inability of mutant Pr160gag-pol to be incorporated into the virus. These mutant precursor proteins are maintained at levels in the cytoplasm similar to wild-type.

Higher fidelity of RNA-dependent DNA mispair extension by M184V drug-resistant than wild-type reverse transcriptase of human immunodeficiency virus type 1.

Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) has low fidelity compared with RTs of other retroviruses and cellular DNA polymerases. We and others have previously found that the fidelity of DNA-dependent DNA polymerization (DDDP) of M184V-mutated HIV-1 RT is significantly higher than that of wild-type RT. Viruses containing the M184V substitution are highly resistant to (-)-2'-dideoxy-3'-thiacytidine (3TC) in vitro and in patients treated with 3TC monotherapy. It was of interest to determine the fidelity of RNA-dependent DNA polymerization (RDDP) of M184V RT compared with wild-type because this step occurs first in reverse transcription; errors made during this step may be copied in subsequent polymerization steps. Using an in vitro mispaired primer extension assay, M184V-mutated RT exhibited 3-49-fold decreased frequency of mispair extension compared with wild-type RT. Fidelity differences between M184V and wild-type RT were most marked in extension of A:G (49-fold) and A:C (16-fold) mispairs, with only a marginal (3-fold) decrease in the extension of A:A mispairs. RT containing a methionine to isoleucine (M184I) mutation showed only slight increases in RDDP fidelity compared with wild-type, ranging from 1.5- to 6-fold increases. Of the three RTs tested, wild-type RT was the most error-prone, with mispair extension frequencies ranging from 6.674 x 10(-1) to 7.454 x10(-2).

The nucleoside analog-resistant E89G mutant of human immunodeficiency virus type 1 reverse transcriptase displays a broader cross-resistance that extends to nonnucleoside inhibitors.

The alteration of a glutamic acid (E) to a glycine (G) amino acid residue at position 89 (E89G alteration) in the human immunodeficiency virus type 1 reverse transcriptase confers decreased susceptibility to several nucleoside analog inhibitors. Because the nonnucleoside inhibitor-binding pocket is adjacent to the deoxynucleoside triphosphate substrate-binding site, the impact of the E89G reverse transcriptase has decreased susceptibility to TIBO R82150, nevirapine, and to a lesser extent, delavirdine. Human immunodeficiency viruses bearing the same mutation displayed decreased susceptibility to inhibition by these compounds in a cell culture virus replication assay.

Increased polymerase fidelity of E89G, a nucleoside analog-resistant variant of human immunodeficiency virus type 1 reverse transcriptase.

Nucleoside analog resistance in human immunodeficiency virus type 1 results from mutations in reverse transcriptase (RT) that allow the enzyme to discriminate against such analogs. To evaluate the possible impact of such mutations on the ability of human immunodeficiency virus RT to selectively incorporate Watson-Crick base-paired deoxynucleotide triphosphates (dNTPs) over incorrectly paired dNTPs, we have measured the fidelity of dNTP insertion by the E89G variant of RT in in vitro reaction mixtures containing synthetic template primers. The E89G RT was previously shown to be resistant to several ddNTPs and to phosphonoformic acid. Our results show that the mutant enzyme displays a lower level of efficiency of misinsertion than did the wild-type RT for every mispair tested (ranging from 2- to 17-fold.