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J C Hastings

Publications and source records attributed to J C Hastings.

At least 19 recordsLinked to original sources
PubMed1997

Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase.

Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors.

Calcium↗
PubMed1997

Discovery and analysis of inhibitors of the human immunodeficiency integrase.

An essential step in the replication of retroviruses is the integration of a DNA copy of the viral genome into the genome of the host cell. Integration encompasses a series of ordered endonucleolytic and DNA strand transfer reactions catalyzed by the viral enzyme, integrase. The requirement for integrase activity in the propagation of HIV-1 in cell culture defines the enzyme as a potential target for chemotherapeutic intervention. We have therefore developed a non-radioisotopic microtiter plate assay which can be used to identify novel inhibitors of integrase from random chemical screens and for the bioassay driven isolation of inhibitors from natural products. This assay uncouples various steps in the reaction pathway and therefore can be exploited to characterize inhibitors. In this monograph we describe a series of modifications to the method which facilitate such mechanistic studies using as an example a series of previously described integrase inhibitors.

Antiviral Agents↗
PubMed1996

A novel antiviral agent which inhibits the endonuclease of influenza viruses.

A novel anti-influenza virus compound, flutimide, was identified in extracts of a recently identified fungal species, Delitschia confertaspora (F. Pelaez, J.D. Polishook, M. Valldosera, and J.Guarro, Mycotaxon 50:115-122, 1994). The compound, a substituted 2,6-diketopiperazine, selectively inhibited the cap-dependent transcriptase of influenza A and B viruses and had no effect on the activities of other polymerases. Similar to the 4-substituted 2,4-dioxobutanoic acids, a series of transcriptase inhibitors which we described previously (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J.Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994), this inhibitor, which is a natural product, affected neither the initiation nor the elongation of influenza virus mRNA synthesis, but it specifically targeted the cap-dependent endonuclease of the transcriptase. Additionally, the compound was inhibitory to the replication of influenza A and B viruses in cell culture. The selective antiviral properties of this compound further demonstrate the utility of influenza virus endonuclease as a target of antiviral agents.

Antiviral Agents↗
PubMed1996

Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors.

We previously identified a series of compounds which specifically inhibited the transcription of influenza A and B viruses (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J. Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994). The compounds, 4-substituted 2,4-dioxobutanoic acids, selectively targeted the cap-dependent endonuclease activity of the transcriptase complex. Additionally, several of these compounds effectively inhibited the replication of influenza virus but not other viruses in cell culture assays. Here, we report on the anti-influenza virus activities of other potent derivatives of the series evaluated in both in vitro and in vivo infectivity assays. These compounds inhibited the replication of influenza virus in yield reduction assays, with 50% inhibitory concentrations ranging from 0.18 to 0.71 microM. These 50% inhibitory concentrations were similar to those observed for inhibition of in vitro transcription (0.32 to 0.54 microM). One selected compound also elicited a dose-dependent inhibition of influenza virus replication in mice following an upper respiratory tract challenge. These studies demonstrate the antiviral efficacy of this inhibitor class and thereby establish the utility of influenza virus endonuclease as a chemotherapeutic target.

Animals↗
PubMed1996

The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates.

The integration of a DNA copy of the viral genome into the genome of the host cell is an essential step in the replication of all retroviruses. Integration requires two discrete biochemical reactions; specific processing of each viral long terminal repeat terminus or donor substrate, and a DNA strand transfer step wherein the processed donor substrate is joined to a nonspecific target DNA. Both reactions are catalyzed by a virally encoded enzyme, integrase. A microtiter assay for the strand transfer activity of human immunodeficiency virus type 1 integrase which uses an immobilized oligonucleotide as the donor substrate was previously published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini, Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA. The enzyme which binds to the immobilized donor is shown to be sufficient to catalyze strand transfer with target DNA substrates added subsequent to assembly; in the absence of the target substrate, the complex was retained on the donor in an enzymatically competent state. Assembly required high concentrations of divalent cation, with optimal activity achieved at 25 mM MnCl2. In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the divalent cation in assembly and catalysis, respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to promote the multimerization of integrase in the absence of a DNA substrate and alleviate the requirement for high concentrations of divalent cation during assembly. The superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro. Subunits were observed to exchange during the assembly reaction, suggesting that multimerization can occur either before or coincident with but not after donor binding. These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction.

Base Sequence↗
PubMed1995

Inhibition of human immunodeficiency virus integrase by bis-catechols.

The human immunodeficiency virus type 1 (HIV-1) integrase protein is required for the productive infection of T-lymphoid cells in culture (R. L. LaFemina, C. L. Schneider, H. L. Robbins, P. L. Callahan, K. LeGrow, E. Roth, W. A. Schleif, and E. A. Emini, J. Virol. 66:7414-7419, 1992). This observation suggests that chemical inhibitors of integrase may prevent the spread of HIV in infected individuals. In our search for such potential chemotherapeutic agents, we observed that beta-conidendrol inhibits both the sequence-dependent and sequence-independent endonucleolytic activities of integrase with comparable potencies in vitro (50% inhibitory concentration, 500 nM). Structurally related compounds tested for their abilities to inhibit integrase generated a limited structure-activity analysis which demonstrated that potency is associated with the bis-catechol structure: two pairs of adjacent hydroxyls on separate benzene rings. beta-Conidendrol did not inhibit several other endonucleases and/or phosphoryltransferases. Although beta-conidendrol was not effective in preventing HIV-1 infection in cell culture, the in vitro data demonstrate that it is possible to identify selective agents targeted against this essential HIV-1 function.

Base Sequence↗
PubMed1994

Viral long terminal repeat substrate binding characteristics of the human immunodeficiency virus type 1 integrase.

A DNA binding assay was developed for the human immunodeficiency virus type 1 (HIV-1) integrase. The assay was capable of defining discrete complexes between the enzyme and the viral long terminal repeat (LTR) substrate. DNA binding reflected the sequence requirements previously demonstrated for the enzyme's 3'-end processing activity. Binding exhibited a nonlinear dependence on integrase concentration, suggesting that the enzyme functions as a multimer. The oligomeric state was investigated by UV-photo-cross-linking of integrase-LTR oligonucleotide complexes using DNA substrates substituted with 5-bromo-2'-deoxycytidine within the integrase recognition sequence. In the absence of divalent cation, integrase cross-linked to the LTR oligonucleotide as a single species whose mobility by SDS-polyacrylamide gel electrophoresis was consistent with the formation of tetramers. Using these techniques, analysis of the binding properties of integrase mutants demonstrated that the catalytic and sequence-specific DNA binding activities of the enzyme are distinct, involving residues within the conserved "DD(35)E" and zinc finger motifs, respectively.

Base Sequence↗
PubMed1993

Clinical significance of epicardial pacing wire cultures.

Routine cultures of epicardial pacing wires removed 5 to 10 days postoperatively were obtained in 205 adults who underwent cardiac operations through median sternotomy. The study was conducted in a double-blind prospective fashion in which clinicians were unaware of culture results. With the exception of 10 out-of-town patients who were followed up only until the day of hospital discharge, the patients were followed for at least 6 weeks (195 patients) for evidence of poststernotomy wound infections. Deep wound infection rate was slightly less than 1% in this patient population, with less than 0.5% having had superficial wound problems. Of the 205 patients, 27 had positive epicardial pacing wires cultures, with a total of 30 microbial isolates. Of 30 isolates, 26 were consistent with local skin flora (Staphylococcus, Streptococcus, Enterococcus, and diphtheroids). Wound infection developed in none of these patients. The remaining four cultures were of either Enterobacter or Serratia. In two of these four patients deep sternal infections developed. In the remaining 178 patients whose wire cultures were negative, no deep sternal infections developed. The fact that all clinically manifested deep sternal infections were associated with positive epicardial pacing wires cultures suggests that epicardial pacing wires cultures may be useful in the treatment of high-risk patients or of those in whom deep sternal infections are suspected.

Academic Medical Centers↗
PubMed1990

Hepatic encephalopathy induced by small bowel obstruction in a noncirrhotic child with portal vein thrombosis.

A case of hepatic encephalopathy in a noncirrhotic child, who has undergone a previous mesocaval shunt for extrahepatic portal vein thrombosis, is presented. Hepatic encephalopathy developed 5 years after the operation and is believed to have been precipitated by the presence of small bowel obstruction. Exploratory laparotomy and lysis of an adhesion relieved the obstruction and led to the resolution of the encephalopathy.

Adolescent↗
PubMed1989

Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types.

To characterize the host range of different strains of HIV-1, we have used four types of cells, primary monocyte-derived macrophages (MDM), primary PBL, a promonocyte cell line (U937), and a CD4+ T cell line (SUP-T1). These cells were infected with three prototype strains of HIV-1, a putative lymphocyte-tropic strain (IIIB), and two putative monocyte-tropic strains (SF162 and DV). Infections were monitored by assays for infectious virus, for cell-free and cell-associated viral antigen (p24), and for the proportion of cells infected by immunohistochemical staining. It was concluded that: (a) the use of four different cell types provides a useful biological matrix for distinguishing the tropism of different strains of HIV-1; this matrix yields more information than the infection of any single cell type. (b) A monocyte-tropic strain of HIV-1, such as strain SF162, shows a reciprocal host range when compared with a lymphocyte-tropic strain such as IIIB; strain SF162 replicates well in primary MDM but not in U937 or SUP-T1 cells, while strain IIIB replicates well in both U937 and SUP-T1 cells but not in MDM. (c) Both lymphocyte-tropic and monocyte-tropic strains of HIV-1 replicate well in PBL. (d) The promonocyte cell line, U937, and the T cell line, SUP-T1, differ markedly from primary cells, such as MDM and PBL, in their ability to support the replication of different strains of HIV-1; these cell lines cannot be used as surrogates for primary cells in host range studies of HIV-1 strains.

Antibodies, Monoclonal↗
PubMed1989

Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.

Animals↗
PubMed1987

Complement component C3b binds directly to purified glycoprotein C of herpes simplex virus types 1 and 2.

Cells infected with herpes simplex virus type 1 (HSV-1), but not HSV-2, express on their surfaces a receptor for the complement component C3b. Receptor activity is markedly enhanced by treatment of the infected cells with neuraminidase. Employing a direct binding assay, consisting of purified HSV glycoproteins immobilized on nitrocellulose and iodinated C3b as a probe, we found that C3b binds directly to gC-1, as well as to gC-2, but not to gB or gD from either serotype. C3b binding was enhanced by treatment of gC-1 or gC-2 with neuraminidase. Endo F or endo H treatment of gC-1 had no effect on C3b binding. However, treatment of gC-2 with these endoglycosidases had a marked negative effect on C3b binding. These results suggest that N-linked oligosaccharides are involved in binding of C3b to gC-2, but not gC-1. Alternatively, removal of N-linked oligosaccharides from gC-2 might adversely affect polypeptide conformation. Glycoprotein C-2 also differs from gC-1 in its effects on the complement cascade. Whereas gC-1 accelerated the decay of the alternative pathway C3 convertase and impaired the efficiency of lysis by the components C5 through C9, gC-2 stabilized the active C3 convertase and had little effect on the late-acting components. The dissimilarity of gC-1 and gC-2 with regard to their effects on the complement cascade may have implications regarding the role of these glycoproteins in confronting the host immune response.

Animals↗
PubMed1986

Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates.

The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV-1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo.

Antibodies, Monoclonal↗
PubMed1975

Effect of suture materials on healing skin wounds.

A systematic comparison has been made of six suture materials used as subcuticular closure of abdominal incisions in dogs. The effect on wound healing was measured by mechanical, biochemical, and histologic methods. No difference was demonstrated in wound breading strength among wounds closed with different suture materials up to 28 days postoperatively. At 70 days, wounds sutured with nonabsorbable sutures were weaker than those closed with absorbable sutures, but this difference probably was due to a much higher incidence of infection in wounds closed with nonabsorbable sutures. It was our observation that monofilament sutures were superior to multifilament sutures with regard to the incidence of wound infection. By five days, the rate of collagen synthesis in wounds was increased over that of normal skin and remained elevated throughout the 120 day observation period. Suture material had no effect on collagen synthesis. The rate of noncollagenous protein synthesis in the wound was not altered throughout the entire observation period and did not differ from that measured in normal skin. Catgut, both plain and chromic, produced only a mild cellular reaction in dogs after 21 days as contrasted with the intense inflammatory reaction reported by others in rats and rabbits. We observed neither plain nor chromic catgut was absorbed rapidly in dogs; intact sutures were frequently observed at 120 days. Polyglactin, a synthetic absorbable suture, produced a moderate tissue reaction and uniformly disappeared between the twenty-eighth and seventieth days. Silk and Mersilene showed the highest rate of wound infection and the most intense and prolonged tissue reaction. Prolene, a monofilament suture, produced only a mild to moderate tissue reaction. These results taken in conjunction with those of other investigators suggest a marked species difference in the reaction to suture materials, particularly catgut, and suggest caution in transferring these observations to human beings.

Animals↗
PubMed1975

The effect of suture materials on healing wounds of the bladder.

The healing pattern of bladder tissue and its response to various suture materials were studied and compared with data reported previously on skin, stomach and colon wounds. Tensile strength, biochemical data and histologic reactions were used as parameters of wound healing measurement. The healing pattern of urinary bladder wounds resembles that observed for stomach and colon. However, wounds of the urinary bladder wall regain strength equal to unwounded bladder wall within 21 days, whereas wounds of stomach and colon do not achieve more than 70 per cent of unwounded tissue strength after 120 days. The rate of collagen synthesis in bladder wounds reaches a peak at five days and returns to that of normal unwounded tissue by 70 days. Colon and stomach wounds, on the other hand, showed elevated rates of collagen synthesis even at 120 days. As with colon and stomach, absorbable sutures appear to lower the strength of both the wound and unwounded tissue. However, in stomach and colon this effect is only observed during the first 21 days of healing; in the bladder it persists throughout the 120 day observation period. No histologic evidence of smooth muscle regeneration was seen in bladder wounds. Noncollagenous protein synthesis was not increased in the wound, also suggesting smooth muscle regeneration does not occur. Silk and Mersilene sutures produced the greatest tissue reaction; if the sutures were exposed in the lumen of the bladder, stone formation was observed. This was not observed with monofilament polypropylene sutures. Plain catgut produced the least tissue reaction, but both plain aions comparable with chromic catgut but were regularly absorbed between 28 and 70 days. Since the bladder is well healed by 21 days, polyglactin-910 may be an ideal suture for use in the bladder.

Absorption↗
PubMed1975

Effect of suture materials on healing wounds of the stomach and colon.

Wound healing in the stomach and colon of mongrel dogs was studied using physical, histologic and chemical techniques to evaluate effects of various suture materials on the healing pattern and to compare visceral wounds with skin wounds. The general pattern of healing for stomach and colon wounds is the same in rats, rabbits and dogs. However, the extent of healing, the percentage of strength of normal tissue attained by the wound, is less in the dog than in the rat or rabbit. Prolene and plain and chromic catgut produce the least and silk produces the greatest cellular reaction in the stomach and colon. We believe the effect of absorbable sutures on strength of stomach and colon wounds, while statistically significant, is not of any great clinical significance except perhaps in severely debilitated patients. A trial of Prolene sutures for anastomotic closure would seem indicated. The healing of stomach and colon wounds differs from the pattern observed for skin wounds. Breaking strength increases rapidly for 14 to 21 days postwounding and then does not change significantly. The wounds are metabolically more active than skin wounds since the rate of collagen synthesis is markedly elevated even after 120 days and the rate of noncollagenous protein synthesis is twice as great in the wound as it is in normal tissue. Colon and stomach wounds closed with absorbable sutures are weaker at 14 through 28 days than similar wounds closed with nonabsorbable sutures. Yet, when strength of wound is expressed as a percentage of strength of unwounded tissue, there is no difference between wounds blosed with absorbable or nonabsorbable sutures. Thus, it would appear that there is a general diminution of the strength of normal stomach and colon wall up to 6 centimeters from wounds which have been closed with absorbable sutures.

Animals↗