Biomedical subjects

R Tal

Publications and source records attributed to R Tal.

At least 19 recordsLinked to original sources

PubMed2004

Abdominal compartment syndrome: urological aspects.

ACS is prevalent in various surgical conditions and in a large percentage of critically ill patients. Measuring the IAP is important in the early diagnosis of ACS and can be easily done by measuring the intravesical pressure. ACS adversely affects many organ systems; the pathogenesis of renal dysfunction is probably multifactorial, from a combination of reduced cardiac output, reduced GFR mediated by secretion of renin and angiotensin, aldosterone-mediated water reabsorption, increased renal parenchymal pressure and direct compression of the renal vein. Successful treatment requires a high index of suspicion, prompt recognition and early surgical abdominal decompression.

Abdomen↗

PubMed2002

Pitfalls and complications with laparoscopic intraperitoneal expanded polytetrafluoroethylene patch repair of postoperative ventral hernia.

BACKGROUND: This study reviewed our experience with laparoscopic ventral postoperative (incisional) hernia repair. METHODS: Clinical data from the first 100 cases were analyzed retrospectively. RESULTS: Between 1997 and 2000, 64 women and 36 men (mean age, 58.4 +/- 13.6 years; range, 27-87 years) underwent laparoscopic hernioplasty. Hernias (mean diameter, 6.2 +/- 3.7 cm) were in a midline (74%), subcostal (10%), or other incision location, and were recurrent in 25%, of the patients. The mean operative time was 119 +/- 77 min. Extensive adhesiolysis was necessary in 37 cases. There was no mortality. The recorded complications included inadvertent enterotomies (n = 6), seromas (n = 11), prolonged ileus (n = 4), and prolonged fever (n = 3). Seven cases were converted; to repair accidental enterotomies (n = 4) due to difficult adhesiolysis (n = 2), or to control bleeding (n = 1). Six patients underwent reoperation because of enetric leak (n = 3) or bowel obstruction (n = 3). There were two documented recurrences (2%). The mean follow-up period was 19 months (range, 12-54 months). CONCLUSIONS: Laparoscopic intraperitoneal approach to postoperative ventral (incisional) hernia repair may be associated with significant complications and morbidity, which can be prevented in part by meticulous technique and liberal conversions. The justification of this procedure is the low recurrence rate, according to preliminary results.

Adult↗

PubMed2002

Lidocaine inhibits secretion of IL-8 and IL-1beta and stimulates secretion of IL-1 receptor antagonist by epithelial cells.

Lidocaine and related local anaesthetics have been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms underlying their therapeutic effect are poorly defined. Intestinal epithelial cells play an important role in the mucosal inflammatory response that leads to tissue damage in UC via the secretion of pro-inflammatory cytokines and chemokines. The aim of this study was to evaluate the direct immunoregulatory effect of lidocaine on pro-inflammatory cytokine and chemokine secretion from intestinal epithelial cells. HT-29 and Caco-2 cell lines were used as a model system and treated with lidocaine and related drugs. The expression of IL-8, IL-1beta and the IL-1 receptor antagonist (RA) were assessed by ELISA and quantification of mRNA. In further experiments, the effect of lidocaine on the secretion of IL-8 from freshly isolated epithelial cells stimulated with TNFalpha was tested. Lidocaine, in therapeutic concentrations, inhibited the spontaneous and TNFalpha-stimulated secretion of IL-8 and IL-1beta from HT-29 and Caco-2 cell lines in a dose-dependent manner. Similarly, suppression of IL-8 secretion was noted in the freshly isolated epithelial cells. Other local anaesthetics, bupivacaine and amethocaine, had comparable effects. Lidocaine stimulated the secretion of the anti-inflammatory molecule IL-1 RA. Both the inhibitory and the stimulatory effects of lidocaine involved regulation of transcription. The results imply that the therapeutic effect of lidocaine may be mediated, at least in part, by its direct effects on epithelial cells to inhibit the secretion of proinflammatory molecules on one hand while triggering the secretion of anti-inflammatory mediators on the other.

Adenocarcinoma↗

PubMed2001

Biochemical and immunological properties of human cardiac troponin I fragments.

Cardiac troponin I (cTnI) is the inhibitory subunit of the troponin complex and is a biochemical marker for myocardial infarction (MI). It is found in human serum within 4-6 h following MI. One of us has shown [Morjana (1998) Biotechnol. Appl. Biochem. 28, 105-111] that MI patient serum TnI is cleaved at the N- and C-terminals and that the TnI fragments exist as a complex with tropinin C (TnC) and troponin T (TnT). In the present study, we have generated C-terminal truncated TnI fragments and studied their immunological and biochemical properties. Human recombinant TnI (rTnI) expressed in Escherichia coli is cleaved into a major fragment with a molecular mass of 17500 Da using CNBr. The major CNBr fragment contains the first 153 amino acids of human cTnI (TnI153). Cleavage of the rTnI with the endoproteinase Asp-N generates a smaller TnI fragment (TnI88, residues 6-96). TnI153 has higher immunological activity than that of rTnI and lower activity than that of TnI88, as judged by the Stratus II TnI Immunoassay. TnI153 exhibits biochemical and immunological properties similar to those of intact TnI. It binds TnC at a molar ratio of 1:1 and forms a ternary complex with TnC and TnT. TnC enhances the immunological activity of TnI153, but has little effect on the activity of TnI88. The TnI153-TnC complex exhibits higher immunological activity than rTnI-TnC and TnI88-TnC, and much higher activity than free rTnI, TnI153 and TnI88. The presence of TnT has no effect on the immunological activity of the TnI153-TnC complex, suggesting that the addition of TnT does not interfere with TnI153 recognition by TnI monoclonal antibodies. Free TnI153 and TnI88 degrade rapidly in human serum. TnC protects TnI153 from proteolytic degradation, but offers less protection for TnI88. The TnI88-TnC complex lost 80% of its immunological activity after incubation for 2 days in human serum at 37 degrees C. However, there was no loss in the immunological activity of the TnI153-TnC complex under the same conditions. A cTnI fragment (TnI80, residues 1-80), expressed in E. coli as a fusion protein, exhibits immunological activity and stability similar to that of TnI88.

Biomarkers↗

PubMed2001

Disaccharides derived from heparin or heparan sulfate regulate IL-8 and IL-1 beta secretion by intestinal epithelial cells.

BACKGROUND & AIMS: Intestinal epithelial cells can produce cytokines and chemokines that play an important role in the mucosal immune response. Regulation of this secretion is important to prevent inflammatory tissue damage. Disaccharides derived from heparan sulfate and heparin have been shown to down-regulate inflammation in vivo. We tested the effect of such disaccharides on cytokine secretion by intestinal epithelial cells. METHODS: Spontaneous and tumor necrosis factor (TNF)-alpha-stimulated interleukin (IL)-8 and IL-1 beta secretion and mRNA expression were assessed in HT-29 and Caco-2 intestinal epithelial cell lines in the presence of a panel of heparin and heparan sulfate disaccharides. RESULTS: Specific disaccharides suppressed spontaneous and TNF-alpha-induced mediator secretion in a dose-dependent manner. Disaccharide activity was structurally restricted. Preincubation of cells with nonsuppressing disaccharides blocked the activity of suppressing disaccharides. The number of sulfate moieties determined the ability of nonsuppressing disaccharides to block the effect of suppressive disaccharides. No suppression of mRNA expression was noted, and intracellular mediator levels were not reduced. CONCLUSIONS: Disaccharides derived from heparin and heparan sulfate regulate proinflammatory mediator secretion from intestinal epithelial cells. Dose dependence and competition by structurally diverging disaccharides suggest a receptor-mediated mechanism. Unchanged mRNA and intracellular mediator levels suggest that the disaccharides act at posttranscriptional stages.

Adjuvants, Immunologic↗

PubMed2000

Somatostatin through its specific receptor inhibits spontaneous and TNF-alpha- and bacteria-induced IL-8 and IL-1 beta secretion from intestinal epithelial cells.

Intestinal epithelial cells secrete proinflammatory cytokines and chemokines that are crucial in mucosal defense. However, this secretion must be tightly regulated, because uncontrolled secretion of proinflammatory mediators may lead to chronic inflammation and mucosal damage. The aim of this study was to determine whether somatostatin, secreted within the intestinal mucosa, regulates secretion of cytokines from intestinal epithelial cells. The spontaneous as well as TNF-alpha- and Salmonella-induced secretion of IL-8 and IL-1beta derived from intestinal cell lines Caco-2 and HT-29 was measured after treatment with somatostatin or its synthetic analogue, octreotide. Somatostatin, at physiological nanomolar concentrations, markedly inhibited the spontaneous and TNF-alpha-induced secretion of IL-8 and IL-1beta. This inhibition was dose dependent, reaching >90% blockage at 3 nM. Furthermore, somatostatin completely abrogated the increased secretion of IL-8 and IL-1beta after invasion by Salmonella. Octreotide, which mainly stimulates somatostatin receptor subtypes 2 and 5, affected the secretion of IL-8 and IL-1beta similarly, and the somatostatin antagonist cyclo-somatostatin completely blocked the somatostatin- and octreotide-induced inhibitory effects. This inhibition was correlated to a reduction of the mRNA concentrations of IL-8 and IL-1beta. No effect was noted regarding cell viability. These results indicate that somatostatin, by directly interacting with its specific receptors that are expressed on intestinal epithelial cells, down-regulates proinflammatory mediator secretion by a mechanism involving the regulation of transcription. These findings suggest that somatostatin plays an active role in regulating the mucosal inflammatory response of intestinal epithelial cells after physiological and pathophysiological stimulations such as bacterial invasion.

Caco-2 Cells↗

PubMed2000

Follow-up of sperm concentration and motility in patients with lymphoma.

Lymphomas are a group of diseases, prevalent at reproductive age. Fertility is notoriously reduced among lymphoma patients. This study evaluates pre- and post-treatment semen concentration and motility, and factors associated with semen quality deterioration. We followed-up 33 patients with non-Hodgkin's lymphoma or with Hodgkin's disease during the years 1987-1997 who were referred for semen cryopreservation. Pretreatment semen analysis, and hormonal profile were recorded at diagnosis and at least 1 year after completion of the treatment, and compared. Medical records for disease type, disease stage and treatment protocols were related to long-term sperm outcome. Hormonal concentrations were not predictive of post-treatment sperm concentration. In patients with localized disease, initial sperm concentration and motility tended to be preserved, compared with patients with widespread disease (P: = 0. 016). In Hodgkin's disease patients, treatment with the adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) protocol was superior to the mechloretamine, vincristine, procarbazine and prednisone with ABV protocol regarding germinal toxicity (P: = 0.0008). The post-treatment sperm outcome was better in patients treated with local irradiation than in those who did not undergo irradiation (P: = 0.0027). No predictive tools for post-treatment fertility were found and, therefore, every patient with a lymphoma should have his semen cryopreserved at diagnosis.

Adolescent↗

PubMed1999

Human gastrin: a Helicobacter pylori--specific growth factor.

BACKGROUND & AIMS: Helicobacter pylori resides within the gastric mucosa, a niche hostile to other microorganisms. Human gastrin levels are elevated after infection and return to normal after eradication. The aim of this study was to test the direct effect of gastrin on the growth of H. pylori. METHODS: H. pylori and control bacteria were grown with gastrin or control peptides and growth rate was determined. (125)I-labeled gastrin was used to determine uptake. RESULTS: Human gastrin stimulated H. pylori growth in a specific, dose-dependent manner. Gastrin shortened the lag time, increased growth rate in the logarithmic phase, and increased final bacterial concentration at the stationary phase. These effects were shown over a wide concentration range, including physiological luminal and serum levels. Labeled gastrin uptake was inhibited by unlabeled gastrin. Controls consisting of cholecystokinin and pentagastrin inhibited gastrin uptake but did not stimulate growth. In contrast, somatostatin and epidermal growth factor had no effect on either gastrin uptake or bacterial growth. These results suggest a structurally restricted, receptor-mediated, gastrin-specific effect. CONCLUSIONS: Human gastrin is a specific growth factor for H. pylori and may have a role in the adaptation of H. pylori to its unique habitat.

Campylobacter jejuni↗

PubMed1999

Wrist and forearm postures and motions during typing.

Awkward upper extremity postures and repetitive wrist motions have been identified by some studies as risk factors for upper extremity musculoskeletal disorders during keyboard work. However, accurate body postures and joint motions of typists typing on standardized workstations are not known. A laboratory study was conducted to continuously measure wrist and forearm postures and motions of 25 subjects while they typed for 10-15 min at a standard computer workstation adjusted to the subjects' anthropometry. Electrogoniometers continuously recorded wrist and forearm angles. Joint angular velocities and accelerations were calculated from the postural data. The results indicate that wrist and forearm postures during typing were sustained at non-neutral angles; mean wrist extension angle was 23.4 +/- 10.9 degrees on the left and 19.9 +/- 8.6 degrees on the right. Mean ulnar deviation was 14.7 +/- 10.1 degrees on the left and 18.6 +/- 5.8 degrees on the right. More than 73% of subjects typed with the left or right wrist in greater than 15 degrees extension and more than 20% typed with the left or right wrist in greater than 20 degrees ulnar deviation. Joint angles and motions while typing on an adjusted computer workstation were not predictable based on anthropometry or typing speed and varied widely between subjects. Wrist motions are rapid and are similar in magnitude to wrist motions of industrial workers performing jobs having a high risk for developing cumulative trauma disorders. The magnitude of the dynamic components suggests that wrist joint motions may need to be evaluated as a risk factor for musculoskeletal disorders during typing.

Adult↗

PubMed1998

Improving the binding affinity of an antibody using molecular modeling and site-directed mutagenesis.

Activated Factor X releases F1.2, a 271-amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine-amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (K(D)) 1.22 x 10(-6) M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.

Alanine↗

PubMed1998

Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes.

Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of beta-1,4-glucan (cellulose) synthase in Acetobacter xylinum. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1, cdg2, and cdg3. Within each cdg operon, a pdeA gene lies upstream of a dgc gene. cdg1 contains two additional flanking genes, cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1 contributes 80% of cellular DGC and PDEA activities and cdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.

Amino Acid Sequence↗

PubMed1998

Expression and equilibrium denaturation of cardiac troponin I: stabilization of a folding intermediate during denaturation by urea.

Human cardiac troponin I has been expressed at high level in Escherichia coli as a fusion protein by using the expression vector Ptac114. The expressed protein forms primarily intracellular inclusion bodies that are solubilized in the presence of 8 M urea. The purified troponin I is recognized by anti-(human cardiac troponin I) monoclonal antibodies. Equilibrium denaturation of recombinant human troponin I and bovine troponin I is compared by monitoring changes in the protein's fluorescence and CD characteristics. At pH 7.5 the equilibrium denaturation of both proteins by urea occurs in two distinct steps involving at least three major conformational states: native, intermediate and fully denatured. The biphasic profile in the presence of urea is observed by both fluorescence and CD spectroscopy. In the intermediate state the native tertiary structure is largely disrupted and 40% of the secondary structure is conserved, as suggested by near-UV and far-UVCD respectively. Thermal denaturation of troponin I, as followed by fluorescence, shows a loss in the signal that is not reversible after heating to 90 degrees C. In the presence of a constant amount of urea (not greater than 0.5 M) the thermal denaturation becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by urea. The fluorescence of 1-anilino-8-naphthalenesulphonate produced on binding troponin I decreases in the presence of increasing concentrations of urea up to 3 M; at higher urea concentrations no further change in the remaining signal is observed. Kinetic studies show at least two phases of renaturation for troponin I previously denatured with 8 M urea, whereas only a single phase is detected for the renaturation process in the presence of 3 M urea. The results suggest the occurrence of a stable folding intermediate, the formation of which might be related to the two-domain architecture of troponin I.

Anilino Naphthalenesulfonates↗

PubMed1994

3,5,8-Trihydroxy-4-quinolone, a novel natural inhibitor of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2.

The natural product of the Red Sea sponge Verongia sp., identified as 3,5,8-trihydroxy-4-quinolone, was found to be a potent inhibitor of the RNA-directed DNA synthesis of the reverse transcriptases (RTs) of human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively). This inhibition was unaffected by the nature of the primer template used for DNA synthesis. The DNA-dependent DNA polymerase activity was inhibited to a lesser extent, whereas the ribonuclease H (RNase H) function associated with both HIV RTs was only slightly inhibited. The inhibition by the trihydroxyquinolone is reversible and noncompetitive with respect to both substrates--dTTP and the template primer poly(rA)n.oligo(dT)12-18. The inhibitor binds HIV-1 RT with a high affinity (Ki = 0.46 microM). This compound was shown also to inhibit the catalytic activities of the RT of murine leukemia virus, establishing the general inhibitory effect on retroviral RTs. Introductions of acetyl or methoxy moieties at positions with potential activity have generated three synthetic analogs of the natural compound. Only one analog, 5,8-dimethoxy-4-quinolone, exhibited an inhibition potency similar to that of the unmodified compound. Analysis of the three analogs has led us to the conclusion that the hydroxyl group at the ortho position to the carbonyl group in the pyridinone ring is a key structural element for the inhibitory activity. Thus, it could well be that the inhibitor interacts with the enzyme through a hydrogen bond of this hydroxyl group. We hope that the identification of the inhibitory site of the compound might be an important step toward the rational design of new potent anti-HIV RT drugs.

Animals↗

PubMed1994

Enzymatic properties of two mutants of reverse transcriptase of human immunodeficiency virus type 1 (tyrosine 181-->isoleucine and tyrosine 188-->leucine), resistant to nonnucleoside inhibitors.

A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes: nucleoside analogs and nonnucleoside inhibitors. The nonnucleoside inhibitors are quite specific for the polymerase activity of HIV-1 RT; they do not affect the polymerase activity of HIV-2 RT or the ribonuclease H (RNase H) activity of either HIV-1 RT or HIV-2 RT. Structural, biochemical, and genetic analyses showed that this group of inhibitors binds in a hydrophobic pocket near the polymerase active site. Mutations in amino acids that line this hydrophobic pocket, for example at tyrosine 181, tyrosine 188, or lysine 103, lead to enzymes that are resistant to the nonnucleoside inhibitors. We have investigated the enzymatic properties of two mutants of HIV-1 RT in which residues 181 and 188 were replaced by the corresponding amino acids in HIV-2 RT (tyrosine 181-->isoleucine and tyrosine 188-->leucine). The two tyrosine mutants closely resemble the wild-type HIV-1 RT in almost all the catalytic functions tested, including the heat stability, sensitivity of the DNA polymerase activity to inhibition by deoxynucleoside analogs, inhibition by the zinc chelator o-phenanthroline, and the Km values calculated for the DNA polymerase activity. There is, however, a slight difference in the effect of orthophenanthroline on the RNase H activity. In addition, there is a subtle disparity in the fidelity of DNA synthesis (analyzed by a mispair extension assay), thus indicating that these mutant RTs are not likely to confer any selective advantages or disadvantages to the variant virions over wild-type virus.

Amino Acid Sequence↗

PubMed1993

Hexaprenoid hydroquinones, novel inhibitors of the reverse transcriptase of human immunodeficiency virus type 1.

Activity against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in the organic extract of the Red Sea sponge Toxiclona toxius was traced by us to five novel natural compounds, namely toxiusol [1], shaagrockol B [3], shaagrockol C [4], toxicol A [6], all of which are sulfated hexaprenoid hydroquinones, and toxicol B [7], the p-hydroquinone derivative of compound 6. The hydrolysis of the two sulfated compounds 1 and 4 yielded the corresponding hydroquinones designated as compounds 2 and 5, and further oxidation of compound 7 afforded the corresponding p-quinone derivative, compound 8. All compounds exhibited inhibitory activity of both DNA polymerizing functions of HIV-1 RT but failed to inhibit the RT-associated ribonuclease H activity. Toxiusol [1] was found to be the most potent inhibitor of the RNA-dependent DNA polymerase function (with 50% inhibition obtained at 1.5 microM and 95% inhibition at 4.6 microM), whereas the DNA-dependent DNA polymerase was significantly less sensitive to the inhibitor (with 50% inhibition achieved at 6.6 microM and 95% inhibition only at 41.6 microM). The fact that compound 1 discriminates between the two DNA polymerase activities of the RT offers new prospects for developing potent and highly specific anti-RT compounds, since the RNA-dependent DNA polymerase activity of RT is the only unique function that is not expressed at significant levels in uninfected mammalian cells.

Animals↗

PubMed1993

Specific inhibition of the reverse transcriptase of human immunodeficiency virus type 1 and the chimeric enzymes of human immunodeficiency virus type 1 and type 2 by nonnucleoside inhibitors.

We have studied the effects of four nonnucleoside inhibitors, including the novel natural product inhibitor calanolide A, on molecular chimeras containing complementary segments of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs). All four compounds specifically inhibited the DNA polymerase activity of HIV-1 RT but had no apparent effect on the RNase H activity of this enzyme or on the DNA polymerase or RNase H activity of HIV-2 RT. Three of these compounds showed the generally expected patterns of resistance and susceptibility with the various chimeric RTs. However, the inhibition patterns of the chimeric RTs by calanolide A provided evidence that there is a segment between residues 94 and 157 in HIV-1 RT that is critical for inhibition. However, the data also suggest that there may be a second segment located between amino acids 225 and 427 in HIV-1 RT that is also important for specifying susceptibility to the drug.

Antiviral Agents↗

PubMed1992

The effects of cysteine mutations on the catalytic activities of the reverse transcriptase of human immunodeficiency virus type-1.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) has only 2 cysteine residues at positions 38 and 280. In order to investigate the role of these cysteines in the structure and function of the enzyme, we have previously modified each of the cysteines to serines employing site-directed mutagenesis. Two of the mutant forms of HIV-1 RT, the single mutant of cysteine 280 and a double mutant with both cysteines modified, were purified. In the present study we have compared the catalytic properties of the DNA-polymerizing and the ribonuclease H (RNase H) functions of the two mutant RTs to those of the native enzyme. The results indicate that the single mutant RT closely resembles the wild type enzyme in almost all the catalytic functions tested. The double cysteine mutant RT, on the other hand, exhibits several unique features. First, the specific activities of the RNA- and DNA-directed DNA synthesis are significantly lower than the corresponding activities of the other two enzymes. This probably results from the lower Vmax values exhibited by the double mutant RT, since the Km values calculated for all enzymes were similar. Second, the most outstanding differences are associated with the RNase H activity of the double mutant RT. The specific activity of RNase H is about 4-fold higher than the wild type and the single mutant RTs. Furthermore, the heat stability of the RNase H function of the double mutated RT is at least 15-fold higher than that of the other two RTs. The substantial resistance to heat denaturation is apparent only for the RNase H activity, since the DNA polymerizing function of the double mutant RT is as sensitive to heat denaturation as the other two proteins.

Amino Acid Sequence↗

PubMed1992

The effects of cysteine mutations on the reverse transcriptases of human immunodeficiency virus types 1 and 2.

Chemical modification of HIV-1 and HIV-2 (human immunodeficiency virus, types 1 and 2) reverse transcriptases (RT) with three thiol reactive compounds selectively inhibits the RNase H function of the enzyme. HIV-1 RT has 2 cysteines (at positions 38 and 280); HIV-2 RT has 3 (38, 280, 445). Both of the cysteines in HIV-1 RT are in the polymerase domain. To investigate the role of the cysteines in the structure and function of the HIV RTs, we have converted each cysteine to serine and made combinations of the mutations. Since HIV-1 RT has alanine at position 445, we have also substituted alanine for serine at this position in HIV-2 RT. Neither of the single mutations in HIV-1 RT nor the double mutation mimics the effects of the chemical modification. The serine 280 mutation has little effect on either polymerase or RNase H; the serine 38 mutation affects both activities, as does the 38/280 double mutant. The 38 and 280 serine mutations in HIV-2 RT resemble the equivalent mutations in HIV-1 RT. Substitution of serine or alanine at position 445 (which lies in the RNase H domain) diminishes, but does not abolish, the RNase H activity of HIV-2 without affecting polymerase activity. The RNase H activity of a mutant HIV-1 RT with serine at position 280 is completely resistant to inactivation by the three thiol reactive compounds we tested, which demonstrates that cysteine 280 is the critical residue. We suggest that the reason the mutation (cysteine 280 to serine) does not mimic the chemical modification is because the chemical modification produces a greater change in the structure of the protein. We also suggest that position 280 lies at or near the important points of contact between the RNase H and polymerase domains, so that chemical modification of this position, which lies within the polymerase domain, distorts the RNase H domain.

Cysteine↗