The Israel Medical Association journal : IMAJ, Mar 1, 2022
BACKGROUND The CHA2DS2-VASc score has been shown to predict systemic thromboembolism and mortalit... more BACKGROUND The CHA2DS2-VASc score has been shown to predict systemic thromboembolism and mortality in certain groups in sinus rhythm (SR), similar to its predictive value with atrial fibrillation (AF). OBJECTIVES To compare factors of inflammation, thrombosis, platelet reactivity, and turnover in patients with high versus low CHA2DS2-VASc score in SR. METHODS We enrolled consecutive patients in SR and no history of AF. Blood samples were collected for neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP), immature platelet fraction (IPF%) and count (IPC), CD40 ligand, soluble P-selectin (sP-selectin) and E-selectin. IPF was measured by autoanalyzer and the other factors by ELISA. RESULTS The study comprised 108 patients (age 58 ± 18 years, 63 women (58%), 28 (26%) with diabetes), In addition, 52 had high CHA2DS2-VASc score (³ 2 for male and ³ 3 for female) and 56 had low score. Patients with low scores were younger, with fewer co-morbidities, and smaller left atrial size. sP-selectin was higher in the high CHA2DS2-VASc group (45, interquartile ratio [IQR] 36-49) vs. 37 (IQR 28-46) ng/ml, P = 0.041]. Inflammatory markers were also elevated, CRP 3.1 mg/L (IQR 1.7-9.3) vs. 1.6 (IQR 0.78-5.4), P < 0.001; NLR 2.7 (IQR 2.1-3.8) vs. 2.1 (IQR 1.6-2.5), P = 0.001, respectively. There was no difference in E-selectin, CD40 ligand, IPC, or IPF% between the groups. CONCLUSIONS Patients in SR with high CHA2DS2-VASc score have higher inflammatory markers and sP-selectin. These findings may explain the higher rate of adverse cardiovascular events associated with elevated CHA2DS2-VASc score.
Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) act... more Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RT of human immunodeficiency virus type-1 (HIV-1) is composed of two subunits. The p51, which is the smaller subunit, shares with the larger p66 subunit the same amino-terminal part (which encompasses the DNA polymerase domain) and lacks the carboxyl-terminal segment of the p66 (which is the RNase H domain). The structure of the polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains) linked to the RNase H domain. Chemical modifications by thiol-specific reagents of cysteine 280, located in α helix I in the thumb subdomain of the polymerase domain, affect substantially only the RNase H activity. Also, the substitution of a serine for C280 did not alter any of the RT activities. Here we have systematically modified the C280 residue to either of the following residues: W, P, H, L, M, Y, Q, E or R. Only the first two mutations lead to a marked reduction in the RNase H activity, whereas none of the mutations affected the polymerase function to a significant extent. As expected, due to their impaired RNase H, the C280W and C280P mutants also had a very low DNA strand-transfer activity. It is also apparent from subunit-directed mutagenesis that each of the RT subunits contributes to the level of RNase H activity, yet the contribution of the p51 subunit to this activity is somewhat higher than that of the p66. Steady-state kinetic analyses have indicated that the RNase H activity was reduced mainly due to the sharp increase in the Km rather than changes in the kcat values. This suggests that the modifications of C280 lead to an impaired affinity of HIV-1 RT towards the RNA–DNA substrate.
Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) act... more Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RTs of HIV-1 and HIV-2 are heterodimers of p66/p51 and p68/p54 subunits, respectively. The smaller subunit lacks the C-terminal segment of the larger subunit (which is the RNase H domain). The structure of the DNA polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains), linked to the RNase H domain via the connection subdomain. The RNase H activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar. A chimeric RT in which residues 227–427 (which constitute a small part of the palm and the entire thumb and connection subdomains) of the Rod strain of HIV-2 RT were replaced by the corresponding segment from HIV-1 RT, has an RNase H activity as high as HIV-1 RT (despite the fact that the RNase H domain is derived from HIV-2 RT).We analyzed the RNase H activity of wild-type HIV-2 RT from the D-194 strain and compared it with this activity of the RT from the Rod strain of HIV-2 and HIV-1 RT. The level of this activity of both HIV-2 RT strains was low; suggesting that low RNase H activity is a general property of HIV-2 isolates. The in vitro RNase H digestion pattern of the three wild-type RTs was indistinguishable, despite the difference in the level of RNase H activity. We constructed new chimeric HIV-1/HIV-2 RTs, in which protein segments and/or subunits were exchanged. The DNA polymerase activity of the parental HIV-1 and HIV-2 RTs was similar; as expected, the specific activity of the polymerases of all the hybrid RTs were also similar. However, the RNase H specific activity of the chimeric RTs was either high (like HIV-1 RT) or low (like HIV-2 RT). The origin of the thumb subdomain in the small subunit of the chimeric RTs (residues 244–322) determines the level of the RNase H activity. The strand-transfer activity of the chimeric RTs is also affected by the thumb subdomain of the small subunit; transfer was much more efficient if this subdomain was derived from HIV-1 RT. The data can be explained from the three-dimensional structure of HIV-1 RT. The thumb of the smaller subunit contacts the RNase H domain; it is through these contacts that the thumb affects the level of the RNase H activity of RT.
The study of porcine endogenous retroviruses (PERVs) becomes increasingly important due to the po... more The study of porcine endogenous retroviruses (PERVs) becomes increasingly important due to the potential use of pig cells, tissues, and organs as a source for xenogenic cell therapy and xenotransplantation into humans. Consequently, we have constructed a plasmid that induces in bacteria the synthesis of a soluble and highly active reverse transcriptase (RT) of PERV-B. The purified PERV RT was studied biochemically in comparison with the RT of murine leukemia virus (MLV), because of the high-sequence homology between these two RTs. The data show that in several properties the two enzymes are similar, particularly regarding the monomeric subunit composition of the proteins in solution, the high resistance to deoxynucleoside analogues, and the pattern of RNA cleavage by the ribonuclease H activity (RNase H) of the RTs. However, in several cases there are apparent differences between the two RTs, most notable the divalent cation preference (Mn+2 versus Mg+2) in the DNA polymerase reactions. As already shown for viral PERV RT, the novel recombinant PERV RT exhibits a relatively high resistance to several deoxynucleoside analogue inhibitors, suggesting that they might not be very efficient in inhibiting the replication of PERV virions. Therefore, the availability of large amounts of the recombinant RT can be useful for a wide screening of novel drugs against infectious PERV.
The Israel Medical Association journal : IMAJ, Mar 1, 2022
BACKGROUND The CHA2DS2-VASc score has been shown to predict systemic thromboembolism and mortalit... more BACKGROUND The CHA2DS2-VASc score has been shown to predict systemic thromboembolism and mortality in certain groups in sinus rhythm (SR), similar to its predictive value with atrial fibrillation (AF). OBJECTIVES To compare factors of inflammation, thrombosis, platelet reactivity, and turnover in patients with high versus low CHA2DS2-VASc score in SR. METHODS We enrolled consecutive patients in SR and no history of AF. Blood samples were collected for neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP), immature platelet fraction (IPF%) and count (IPC), CD40 ligand, soluble P-selectin (sP-selectin) and E-selectin. IPF was measured by autoanalyzer and the other factors by ELISA. RESULTS The study comprised 108 patients (age 58 ± 18 years, 63 women (58%), 28 (26%) with diabetes), In addition, 52 had high CHA2DS2-VASc score (³ 2 for male and ³ 3 for female) and 56 had low score. Patients with low scores were younger, with fewer co-morbidities, and smaller left atrial size. sP-selectin was higher in the high CHA2DS2-VASc group (45, interquartile ratio [IQR] 36-49) vs. 37 (IQR 28-46) ng/ml, P = 0.041]. Inflammatory markers were also elevated, CRP 3.1 mg/L (IQR 1.7-9.3) vs. 1.6 (IQR 0.78-5.4), P < 0.001; NLR 2.7 (IQR 2.1-3.8) vs. 2.1 (IQR 1.6-2.5), P = 0.001, respectively. There was no difference in E-selectin, CD40 ligand, IPC, or IPF% between the groups. CONCLUSIONS Patients in SR with high CHA2DS2-VASc score have higher inflammatory markers and sP-selectin. These findings may explain the higher rate of adverse cardiovascular events associated with elevated CHA2DS2-VASc score.
Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) act... more Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RT of human immunodeficiency virus type-1 (HIV-1) is composed of two subunits. The p51, which is the smaller subunit, shares with the larger p66 subunit the same amino-terminal part (which encompasses the DNA polymerase domain) and lacks the carboxyl-terminal segment of the p66 (which is the RNase H domain). The structure of the polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains) linked to the RNase H domain. Chemical modifications by thiol-specific reagents of cysteine 280, located in α helix I in the thumb subdomain of the polymerase domain, affect substantially only the RNase H activity. Also, the substitution of a serine for C280 did not alter any of the RT activities. Here we have systematically modified the C280 residue to either of the following residues: W, P, H, L, M, Y, Q, E or R. Only the first two mutations lead to a marked reduction in the RNase H activity, whereas none of the mutations affected the polymerase function to a significant extent. As expected, due to their impaired RNase H, the C280W and C280P mutants also had a very low DNA strand-transfer activity. It is also apparent from subunit-directed mutagenesis that each of the RT subunits contributes to the level of RNase H activity, yet the contribution of the p51 subunit to this activity is somewhat higher than that of the p66. Steady-state kinetic analyses have indicated that the RNase H activity was reduced mainly due to the sharp increase in the Km rather than changes in the kcat values. This suggests that the modifications of C280 lead to an impaired affinity of HIV-1 RT towards the RNA–DNA substrate.
Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) act... more Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RTs of HIV-1 and HIV-2 are heterodimers of p66/p51 and p68/p54 subunits, respectively. The smaller subunit lacks the C-terminal segment of the larger subunit (which is the RNase H domain). The structure of the DNA polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains), linked to the RNase H domain via the connection subdomain. The RNase H activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar. A chimeric RT in which residues 227–427 (which constitute a small part of the palm and the entire thumb and connection subdomains) of the Rod strain of HIV-2 RT were replaced by the corresponding segment from HIV-1 RT, has an RNase H activity as high as HIV-1 RT (despite the fact that the RNase H domain is derived from HIV-2 RT).We analyzed the RNase H activity of wild-type HIV-2 RT from the D-194 strain and compared it with this activity of the RT from the Rod strain of HIV-2 and HIV-1 RT. The level of this activity of both HIV-2 RT strains was low; suggesting that low RNase H activity is a general property of HIV-2 isolates. The in vitro RNase H digestion pattern of the three wild-type RTs was indistinguishable, despite the difference in the level of RNase H activity. We constructed new chimeric HIV-1/HIV-2 RTs, in which protein segments and/or subunits were exchanged. The DNA polymerase activity of the parental HIV-1 and HIV-2 RTs was similar; as expected, the specific activity of the polymerases of all the hybrid RTs were also similar. However, the RNase H specific activity of the chimeric RTs was either high (like HIV-1 RT) or low (like HIV-2 RT). The origin of the thumb subdomain in the small subunit of the chimeric RTs (residues 244–322) determines the level of the RNase H activity. The strand-transfer activity of the chimeric RTs is also affected by the thumb subdomain of the small subunit; transfer was much more efficient if this subdomain was derived from HIV-1 RT. The data can be explained from the three-dimensional structure of HIV-1 RT. The thumb of the smaller subunit contacts the RNase H domain; it is through these contacts that the thumb affects the level of the RNase H activity of RT.
The study of porcine endogenous retroviruses (PERVs) becomes increasingly important due to the po... more The study of porcine endogenous retroviruses (PERVs) becomes increasingly important due to the potential use of pig cells, tissues, and organs as a source for xenogenic cell therapy and xenotransplantation into humans. Consequently, we have constructed a plasmid that induces in bacteria the synthesis of a soluble and highly active reverse transcriptase (RT) of PERV-B. The purified PERV RT was studied biochemically in comparison with the RT of murine leukemia virus (MLV), because of the high-sequence homology between these two RTs. The data show that in several properties the two enzymes are similar, particularly regarding the monomeric subunit composition of the proteins in solution, the high resistance to deoxynucleoside analogues, and the pattern of RNA cleavage by the ribonuclease H activity (RNase H) of the RTs. However, in several cases there are apparent differences between the two RTs, most notable the divalent cation preference (Mn+2 versus Mg+2) in the DNA polymerase reactions. As already shown for viral PERV RT, the novel recombinant PERV RT exhibits a relatively high resistance to several deoxynucleoside analogue inhibitors, suggesting that they might not be very efficient in inhibiting the replication of PERV virions. Therefore, the availability of large amounts of the recombinant RT can be useful for a wide screening of novel drugs against infectious PERV.
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