Objective: Meta-analysis focusing on the role of first-trimester neutrophil-tolymphocyte ratio (N... more Objective: Meta-analysis focusing on the role of first-trimester neutrophil-tolymphocyte ratio (NLR) in the prediction of preeclampsia.
Objective: To evaluate the neutrophil-to-lymphocyte ratio (NLR) values’ possible predictive role ... more Objective: To evaluate the neutrophil-to-lymphocyte ratio (NLR) values’ possible predictive role in fatal and severe cases of COVID-19 disease in pregnant women. Design and data collection: A case-control study was conducted with the inclusion of 45 pregnant COVID-19 patients. All the data were obtained from the hospital information system of Semmelweis University by two of the authors. Results: Statistical analyses showed that NLR values were significantly higher in patients with fatal COVID-19 compared to those who survived the disease, with or without mechanical ventilation. The study also assessed whether NLR values measured on the first day of hospitalization or at their peak provided better markers of disease severity. While both the first-day and peak NLR values were evaluated in patients who did not survive the disease, only the peak NLR values had predictive value regarding patient death. Conclusion: Based on our results, the peak NLR values appear to be useful markers of C...
ObjectiveTo review of the efficacy and safety of pravastatin use for prophylaxis and treatment of... more ObjectiveTo review of the efficacy and safety of pravastatin use for prophylaxis and treatment of preeclampsia.DesignSystematic review and meta-analysis of clinical studies evaluating pravastatin for treatment and/or prophylaxis of preeclampsia.Data collectionTwo independent reviewers systematically searched data from PubMed, Scopus, Web of Science, Cochrane, Embase, and clinicaltrials.gov databases, for studies evaluating pravastatin for prevention of pre-eclampsia.ResultsFourteen studies were identified, including 1,570 pregnant women who received either pravastatin or placebo, published between 2003 and 2022. From these studies, 5 studies were identified for inclusion in the meta-analysis to evaluate the role of pravastatin use prior to 20 weeks of gestation, to prevent pre-eclampsia, Pravastatin treatment reduced the incidence of preeclampsia by 61% and premature birth by 45%. Among the newborns, there was a 45% reduction in intrauterine growth retardation (IUGR) in the treated ...
Patients facing severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infections with como... more Patients facing severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infections with comorbidities, especially patients whose immune system is weakened have higher chances to face severe outcomes. One of the main reasons behind the suppression of the immune system is iatrogenic, in patients who have autoimmune diseases and/or had an organ transplant. Although there are studies that are examining immunocompromised and/or transplanted patients with COVID-19 infection, furthermore there is a limited number of studies available which are dealing with COVID-19 in pregnant women; however, it is unique and is worth reporting when these factors are coexisting. In this study, we present the case of a 33-year-old Caucasian pregnant woman, who had a kidney transplant in 2009 and contracted the SARS-CoV-2 virus on the 26th gestational week, in 2021. After her infection, superimposed preeclampsia was diagnosed and due to the worsening flowmetric parameters, she gave birth to a premature ...
Statins are used to treat hypercholesterolemia, with several pleiotropic effects. Alongside their... more Statins are used to treat hypercholesterolemia, with several pleiotropic effects. Alongside their positive effects (for example, decreasing blood pressure), they can also bring about negative effects/symptoms (such as myopathy). Their main mechanism of action is inducing apoptosis, the key step being the release of cytochrome c from the mitochondria. This can be facilitated by oxidative stress, through which glutathione is oxidized. In this research, glutathione was used as a respiratory substrate to measure the mitochondrial oxygen consumption of rat liver with an O2 electrode. The reduction of cytochrome c was monitored photometrically. Hydrophilic (pravastatin) and lipophilic (simvastatin) statins were used for the measurements. Pravastatin reduces the reduction of cytochrome c and the oxygen consumption of the mitochondria, while simvastatin, on the other hand, increases the reduction of cytochrome c and the mitochondrial oxygen consumption. The results make it seem probable tha...
Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) ... more Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis—elevated pH, Ca2+level and Mg2+—increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In ...
Absztrakt: Bevezetés: A terhességek 3–8%-ában megjelenő praeeclampsia kezelése még jelenleg sincs... more Absztrakt: Bevezetés: A terhességek 3–8%-ában megjelenő praeeclampsia kezelése még jelenleg sincs megoldva. Praeeclampsiában elégtelen a NO szintézise, ami hozzájárulhat az emelkedett vérnyomáshoz, proteinuriához, a placenta kóros vascularisatiójához is. Praeeclampsiás placentában a csökkent NO-szintézisnek az is oka lehet, hogy a NO-szintáz affinitása csökken a tetrahidrobiopterinhez (BH4), ezzel BH4-rezisztencia alakul ki. Az utóbbi években állatmodellekben és humánvizsgálatokban is megfigyelték, hogy a pravasztatin védhet a praeeclampsia kialakulása ellen. A pravasztatin egyik ismert pleiotrop hatása az, hogy emeli a NO-szintáz aktivitását. Célkitűzés: A pravasztatin hatásának leírása a BH4-rezisztens NO-szintáz aktivitására praeeclampsiás placentában. Módszer: A NO-szintáz aktivitását placentamikroszómában mértük C14-arginin szubsztráttal egészséges (n = 9) és praeeclampsiás (n = 9) minta felhasználásával. A NO-szintáz aktivitását 0,02 µM, fiziológiás 0,2 µM és farmakológiás 50 ...
BackgroundPravastatin, a known inducer of endothelial nitric-oxide synthase (eNOS) was demonstrat... more BackgroundPravastatin, a known inducer of endothelial nitric-oxide synthase (eNOS) was demonstrated in human placenta, however the exact mechanism of it’s action is not fully understood. Since placental NO (nitric oxide) synthesis is of primary importance in the regulation of placental blood flow, we aimed to clarify the effects of pravastatin on healthy (n = 6) and preeclamptic (n = 6) placentas (Caucasian participants).MethodsThe eNOS activity of human placental microsomes was determined by the conversion rate of C14 L-arginine into C14 L-citrulline with or without pravastatin and Geldanamycin. Phosphorylation of eNOS (Ser1177) was investigated by Western blot. Microsomal arginine uptake was measured by a rapid filtration method.ResultsPravastatin significantly increased total eNOS activity in healthy (28%,p<0.05) and preeclamptic placentas (32%,p<0.05) using 1 mM Ca2+promoting the dissociation of a eNOS from it’s inhibitor caveolin. Pravastatin and Geldanamycin (Hsp90 inhib...
The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Ty... more The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Tyr154, which was originally described as phosphorylation [Gaboriaud C, Serre L, Guy‐Crotte O, Forest E & Fontecilla‐Camps JC (1996) J Mol Biol259, 995–1010]. Here we report that Tyr154 is sulfated, not phosphorylated. Cationic and anionic trypsinogens were purified from human pancreatic juice and subjected to alkaline hydrolysis. Modified tyrosine amino acids were separated on a Dowex cation‐exchange column and analyzed by thin layer chromatography. Both human cationic and anionic trypsinogens contained tyrosine sulfate, but no tyrosine phosphate, whereas bovine trypsinogen contained neither. Furthermore, incorporation of [35S]SO4 into human cationic trypsinogen transiently expressed by human embryonic kidney 239T cells was demonstrated. Mutation of Tyr154 to Phe abolished radioactive sulfate incorporation, confirming that Tyr154 is the site of sulfation in cationic trypsinogen. Sulfated ...
Human pancreatic secretions contain two major trypsinogen isoforms, cationic and anionic trypsino... more Human pancreatic secretions contain two major trypsinogen isoforms, cationic and anionic trypsinogen, normally at a ratio of 2 : 1. Pancreatitis, pancreatic cancer and chronic alcoholism lead to a characteristic reversal of the isoform ratio, and anionic trypsinogen becomes the predominant zymogen secreted. To understand the biochemical consequences of these alterations, we recombinantly expressed and purified both human trypsinogens and documented characteristics of autoactivation, autocatalytic degradation and Ca2+-dependence. Even though the two trypsinogens are approximately 90% identical in their primary structure, we found that human anionic trypsinogen and trypsin exhibited a significantly increased (10-20-fold) propensity for autocatalytic degradation, relative to cationic trypsinogen and trypsin. Furthermore, in contrast to the characteristic stimulation of the cationic proenzyme, acidic pH inhibited autoactivation of anionic trypsinogen. In mixtures of cationic and anionic trypsinogen, an increase in the proportion of the anionic proenzyme had no significant effect on the levels of trypsin generated by autoactivation or by enterokinase at pH 8.0 in 1 mm Ca2+- conditions that were characteristic of the pancreatic juice. In contrast, rates of trypsinogen activation were markedly reduced with increasing ratios of anionic trypsinogen under conditions that were typical of potential sites of pathological intra-acinar trypsinogen activation. Thus, at low Ca2+ concentrations at pH 8.0, selective degradation of anionic trypsinogen and trypsin caused diminished trypsin production; while at pH 5.0, inhibition of anionic trypsinogen activation resulted in lower trypsin yields. Taken together, the observations indicate that up-regulation of anionic trypsinogen in pancreatic diseases does not affect physiological trypsinogen activation, but significantly limits trypsin generation under potential pathological conditions.
High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires dele... more High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient Escherichia coli strain. As a first application of this system, the effect of the pancreatitis-associated mutation A16V on the autoactivation of human cationic trypsinogen was characterized. The use of the novel pepP knockout Escherichia coli strain should be generally applicable to the expression of recombinant proteins, which undergo unwanted N-terminal trimming by aminopeptidase P. The human pancreas produces the digestive pro-enzyme trypsinogen in three isoforms. On the basis of their relative isoelectric points and electrophoretic mobility, these are commonly referred to as cationic trypsinogen, anionic trypsinogen, and mesotrypsinogen. The isoenzymes are encoded by separate genes, the PRSS1 (protease, serine, 1), PRSS2 and PRSS3 genes (for a recent review see [1] and references therein). Cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) make up the bulk of secreted trypsinogens in the pancreatic juice, while mesotrypsinogen (PRSS3) accounts for 2-10 % [2-6]. Trypsinogens are synthesized as prepro-enzymes (pre-trypsinogens) with a signal peptide of 15 amino acids, followed by the 8 amino acid long pro-peptide, the trypsinogen activation peptide. The signal-peptide is removed upon entry into the endoplasmic reticulum lumen and the pro-enzymes are packaged into zymogen granules and eventually secreted into the pancreatic juice. Activation of trypsinogen to trypsin is achieved by proteolytic removal of the pro-peptide, which normally takes place in the duodenum catalyzed by enteropeptidase (enterokinase), a highly specialized serine protease in the brush-border membrane of enterocytes. Trypsin can also activate trypsinogen, a process termed autoactivation, which in the duodenum may have a physiological role in facilitating zymogen activation, whereas inappropriate autoactivation in the pancreas might
Homogenized first trimester human placenta exhibits both Ca(2+)-dependent (90-95 per cent) and Ca... more Homogenized first trimester human placenta exhibits both Ca(2+)-dependent (90-95 per cent) and Ca(2+)-independent (5-10 per cent) nitric oxide (NO)-synthesizing activities. Addition of tetrahydrobiopterin (BH4) to homogenates containing Ca2+ in maximally activating concentrations (&gt; 0.5 microM) results in a further 2-2.5-fold activation of NO synthesis, with half-maximal stimulation observed at 26 +/- 8.2 microM BH4 (mean +/- SEM, n = 4). Chelation of Ca2+ in the medium abolishes the stimulatory effect, indicating that only a Ca2(+)-dependent NO-synthase (NOS) isoform is activated by BH4. Based on our previous findings, we suggest that this isoform is the endothelial or Type III NOS. Importantly, BH4 has no significant effect on the Ca2(+)-dependency of NOS activity, the apparent Km values for Ca2+ are comparable in the absence (1.8 +/- 0.4 microM, mean +/- SEM, n = 6) or presence (2.5 +/- 0.6 microM, mean +/- SEM, n = 6) of 50 microM BH4. The BH4 content of these placentae is 207.4 +/- 86.7 pmol/g wet tissue (mean +/- s.d., n = 9), therefore, BH4 added to the homogenate does not simply restore the concentrations that occur endogenously. The results provide the first evidence that in the early human placenta, a constitutively expressed CA 2(+)-dependent NOS isoform is stimulated by exogenous BH4, raising the possibility that BH4 is an important regulator of NOS activity in this tissue. This novel aspect of the NO-generating pathway may have implications in the aetiology and treatment of pregnancy-induced hypertension and pre-eclampsia.
Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change ... more Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N G-nitro-L-arginine (L-NNA) was examined on NO production, NOsensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5-200 mg/kg) to 21day-old rats reduced NOS activity and NO concentration dose dependently by 70-90% and the tissue cGMP level by 60-80%. By contrast, chronic application of L-NNA between postnatal days 4-21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10-50%. Chronic treatment of 10 mg/kg L-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50 mg/kg) of L-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50 mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.
The effects of thyroid hormones (TH) on the enzyme level and activity of neuronal nitric oxide sy... more The effects of thyroid hormones (TH) on the enzyme level and activity of neuronal nitric oxide synthase (nNOS) were studied in the rat cerebral cortex during postnatal life. As revealed by arginine/citrulline conversion assay and Western blot analysis of the homogenate of the parietal cortex T4 significantly increased nNOS activity and nNOS protein level to 153 ± 25% and to 178 ± 20%, respectively. In contrast, 6-n-propyl-2-thyouracil (PTU) decreased nNOS activity and nNOS level to 45 ± 10% and to 19 ± 4%, respectively. The number of nNOS-immunoreactive neurons did not change after either T4 or PTU treatment, however, following T4 administration the percentage of intensively immunoreactive neurons increased to 85 ± 3% compared to control (65 ± 6%), whereas it decreased to 49 ± 2% after PTU treatment. Our findings indicate that abnormal TH levels differentially regulate the activity and the level of nNOS and suggest a cross-talk between the TH and NO signaling pathway in the developing cerebral cortex of rats.
Chronic pancreatitis (CP) is a common inflammatory disease of the pancreas. Mutations in the gene... more Chronic pancreatitis (CP) is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) 1 and the pancreatic secretory trypsin inhibitor (SPINK1) 2 are associated with CP. Since increased proteolytic activity due to mutated PRSS1 enhances the risk for CP, mutations in the gene encoding anionic trypsinogen (PRSS2) may also act disease predisposing. Here we analyzed PRSS2 in CP patients and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4 %) controls but only in 32/2,466 (1.3 %) patients (odds ratio 0.37; P = 1.1 × 10-8). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity due to the introduction of a novel tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby plays a protective role against chronic pancreatitis.
The aim of this study was to characterize the mechanism of the chemical interaction between L-asc... more The aim of this study was to characterize the mechanism of the chemical interaction between L-ascorbic acid (ASC) and tetrahydrobiopterin (BH 4) in vitro and to examine its effect on the activity of endothelial nitric oxide synthase (eNOS) in first trimester human placentae. At room temperature, in Tris-HCl buffer (pH 7.4), both ASC and BH 4 were readily oxidized by dissolved O 2 or H 2 O 2. BH 4 was more sensitive to auto-oxidation, while ASC was more susceptible to oxidation by H 2 O 2. Addition of 36 µmol/l BH 4 to 143 µmol/l ASC increased the initial rate of ASC oxidation 3.2-fold in a catalase-sensitive manner, indicating that enhanced ASC oxidation is partly due to the formation of H 2 O 2. In the presence of catalase, BH 4 still stimulated 1.9-fold the initial rate of ASC oxidation, suggesting that another auto-oxidation product of BH 4 , most probably quininoid-BH 2 (qBH 2), could also stimulate ASC oxidation while itself being reduced back to BH 4. ASC prevented the auto-oxidation of BH 4 in a concentrationdependent fashion, with 3 mmol/l ASC providing an almost complete stabilization of 25 µmol/l BH 4. Importantly, basal eNOS activity in placental microsomes was stimulated 2.5-fold by 0.5 µmol/l BH 4 , and 0.5 mmol/l ASC enhanced the BH 4-stimulation 1.4-fold, with a smaller effect on basal eNOS activity. Taken together, the findings support the notion that the stabilizing action of ASC on BH 4 is related to the ASC-mediated reductive reversal of the auto-oxidation process of BH 4. Moreover, we demonstrated that concentrations of ASC present in the placenta as a common vitamin C supply are sufficient to protect cellular free BH 4 and may contribute to the stimulation of placental eNOS activity.
Type III nitric oxide synthase (NOS III) is responsible for Ͼ90% of nitric oxide (NO) synthesizin... more Type III nitric oxide synthase (NOS III) is responsible for Ͼ90% of nitric oxide (NO) synthesizing activity in first trimester placentae. Enzyme activity is distributed between cytosolic (30%) and membrane-bound forms (70%), with highest specific activity observed in microsomal fractions. In the present study, the effect of tetrahydrobiopterin (BH 4) on subunit structure and activity of microsomal and cytosolic NOS III was compared. As revealed by immunoblot analysis, incubation of microsomal membranes with 50 µM final concentration BH 4 for 10 min at 37 o C resulted in a striking conversion of monomeric NOS III into a protein having the characteristics (electrophoretic mobility, resistance to sodium dodecyl sulphate) of the homodimeric form. In contrast, BH 4 induced significantly less marked changes in the NOS III dimer content of cytosolic fractions. Enzyme activity in microsomes is stimulated~6-fold upon addition of 50 µM BH 4 , while only a 2-fold activation is detectable in cytosolic fractions. Taken together, the observations suggest that BH 4 activates NOS III in the primordial human placenta by promoting its subunit assembly in the membrane, while cytosolic NOS III is relatively insensitive to BH 4. Compartment-specific action of BH 4 represents a novel mechanism which is implicated in the regulation of placental NOS activity.
The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequ... more The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic trypsinogen. A dramatic increase in autoactivation of cationic trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian trypsinogen activation peptides has evolved not only for optimal enteropeptidase recognition in the duodenum but also for efficient inhibition of trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions unrelated to digestion, as found in the group III trypsinogens of cold-adapted fishes.
1 The abbreviations used are: HP, hereditary pancreatitis; Tg, trypsinogen; Tr, trypsin; Tg*, two... more 1 The abbreviations used are: HP, hereditary pancreatitis; Tg, trypsinogen; Tr, trypsin; Tg*, two-chain trypsinogen with the Arg 117-Val 118 peptide bond cleaved; Tr*, two-chain trypsin with the Arg 117-Val 118 peptide bond cleaved; PSTI, pancreatic secretory trypsin inhibitor. 2 Z. Kukor, M. Tóth, and M. Sahin-Tóth, unpublished observations.
Objective: Meta-analysis focusing on the role of first-trimester neutrophil-tolymphocyte ratio (N... more Objective: Meta-analysis focusing on the role of first-trimester neutrophil-tolymphocyte ratio (NLR) in the prediction of preeclampsia.
Objective: To evaluate the neutrophil-to-lymphocyte ratio (NLR) values’ possible predictive role ... more Objective: To evaluate the neutrophil-to-lymphocyte ratio (NLR) values’ possible predictive role in fatal and severe cases of COVID-19 disease in pregnant women. Design and data collection: A case-control study was conducted with the inclusion of 45 pregnant COVID-19 patients. All the data were obtained from the hospital information system of Semmelweis University by two of the authors. Results: Statistical analyses showed that NLR values were significantly higher in patients with fatal COVID-19 compared to those who survived the disease, with or without mechanical ventilation. The study also assessed whether NLR values measured on the first day of hospitalization or at their peak provided better markers of disease severity. While both the first-day and peak NLR values were evaluated in patients who did not survive the disease, only the peak NLR values had predictive value regarding patient death. Conclusion: Based on our results, the peak NLR values appear to be useful markers of C...
ObjectiveTo review of the efficacy and safety of pravastatin use for prophylaxis and treatment of... more ObjectiveTo review of the efficacy and safety of pravastatin use for prophylaxis and treatment of preeclampsia.DesignSystematic review and meta-analysis of clinical studies evaluating pravastatin for treatment and/or prophylaxis of preeclampsia.Data collectionTwo independent reviewers systematically searched data from PubMed, Scopus, Web of Science, Cochrane, Embase, and clinicaltrials.gov databases, for studies evaluating pravastatin for prevention of pre-eclampsia.ResultsFourteen studies were identified, including 1,570 pregnant women who received either pravastatin or placebo, published between 2003 and 2022. From these studies, 5 studies were identified for inclusion in the meta-analysis to evaluate the role of pravastatin use prior to 20 weeks of gestation, to prevent pre-eclampsia, Pravastatin treatment reduced the incidence of preeclampsia by 61% and premature birth by 45%. Among the newborns, there was a 45% reduction in intrauterine growth retardation (IUGR) in the treated ...
Patients facing severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infections with como... more Patients facing severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infections with comorbidities, especially patients whose immune system is weakened have higher chances to face severe outcomes. One of the main reasons behind the suppression of the immune system is iatrogenic, in patients who have autoimmune diseases and/or had an organ transplant. Although there are studies that are examining immunocompromised and/or transplanted patients with COVID-19 infection, furthermore there is a limited number of studies available which are dealing with COVID-19 in pregnant women; however, it is unique and is worth reporting when these factors are coexisting. In this study, we present the case of a 33-year-old Caucasian pregnant woman, who had a kidney transplant in 2009 and contracted the SARS-CoV-2 virus on the 26th gestational week, in 2021. After her infection, superimposed preeclampsia was diagnosed and due to the worsening flowmetric parameters, she gave birth to a premature ...
Statins are used to treat hypercholesterolemia, with several pleiotropic effects. Alongside their... more Statins are used to treat hypercholesterolemia, with several pleiotropic effects. Alongside their positive effects (for example, decreasing blood pressure), they can also bring about negative effects/symptoms (such as myopathy). Their main mechanism of action is inducing apoptosis, the key step being the release of cytochrome c from the mitochondria. This can be facilitated by oxidative stress, through which glutathione is oxidized. In this research, glutathione was used as a respiratory substrate to measure the mitochondrial oxygen consumption of rat liver with an O2 electrode. The reduction of cytochrome c was monitored photometrically. Hydrophilic (pravastatin) and lipophilic (simvastatin) statins were used for the measurements. Pravastatin reduces the reduction of cytochrome c and the oxygen consumption of the mitochondria, while simvastatin, on the other hand, increases the reduction of cytochrome c and the mitochondrial oxygen consumption. The results make it seem probable tha...
Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) ... more Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis—elevated pH, Ca2+level and Mg2+—increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In ...
Absztrakt: Bevezetés: A terhességek 3–8%-ában megjelenő praeeclampsia kezelése még jelenleg sincs... more Absztrakt: Bevezetés: A terhességek 3–8%-ában megjelenő praeeclampsia kezelése még jelenleg sincs megoldva. Praeeclampsiában elégtelen a NO szintézise, ami hozzájárulhat az emelkedett vérnyomáshoz, proteinuriához, a placenta kóros vascularisatiójához is. Praeeclampsiás placentában a csökkent NO-szintézisnek az is oka lehet, hogy a NO-szintáz affinitása csökken a tetrahidrobiopterinhez (BH4), ezzel BH4-rezisztencia alakul ki. Az utóbbi években állatmodellekben és humánvizsgálatokban is megfigyelték, hogy a pravasztatin védhet a praeeclampsia kialakulása ellen. A pravasztatin egyik ismert pleiotrop hatása az, hogy emeli a NO-szintáz aktivitását. Célkitűzés: A pravasztatin hatásának leírása a BH4-rezisztens NO-szintáz aktivitására praeeclampsiás placentában. Módszer: A NO-szintáz aktivitását placentamikroszómában mértük C14-arginin szubsztráttal egészséges (n = 9) és praeeclampsiás (n = 9) minta felhasználásával. A NO-szintáz aktivitását 0,02 µM, fiziológiás 0,2 µM és farmakológiás 50 ...
BackgroundPravastatin, a known inducer of endothelial nitric-oxide synthase (eNOS) was demonstrat... more BackgroundPravastatin, a known inducer of endothelial nitric-oxide synthase (eNOS) was demonstrated in human placenta, however the exact mechanism of it’s action is not fully understood. Since placental NO (nitric oxide) synthesis is of primary importance in the regulation of placental blood flow, we aimed to clarify the effects of pravastatin on healthy (n = 6) and preeclamptic (n = 6) placentas (Caucasian participants).MethodsThe eNOS activity of human placental microsomes was determined by the conversion rate of C14 L-arginine into C14 L-citrulline with or without pravastatin and Geldanamycin. Phosphorylation of eNOS (Ser1177) was investigated by Western blot. Microsomal arginine uptake was measured by a rapid filtration method.ResultsPravastatin significantly increased total eNOS activity in healthy (28%,p<0.05) and preeclamptic placentas (32%,p<0.05) using 1 mM Ca2+promoting the dissociation of a eNOS from it’s inhibitor caveolin. Pravastatin and Geldanamycin (Hsp90 inhib...
The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Ty... more The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Tyr154, which was originally described as phosphorylation [Gaboriaud C, Serre L, Guy‐Crotte O, Forest E & Fontecilla‐Camps JC (1996) J Mol Biol259, 995–1010]. Here we report that Tyr154 is sulfated, not phosphorylated. Cationic and anionic trypsinogens were purified from human pancreatic juice and subjected to alkaline hydrolysis. Modified tyrosine amino acids were separated on a Dowex cation‐exchange column and analyzed by thin layer chromatography. Both human cationic and anionic trypsinogens contained tyrosine sulfate, but no tyrosine phosphate, whereas bovine trypsinogen contained neither. Furthermore, incorporation of [35S]SO4 into human cationic trypsinogen transiently expressed by human embryonic kidney 239T cells was demonstrated. Mutation of Tyr154 to Phe abolished radioactive sulfate incorporation, confirming that Tyr154 is the site of sulfation in cationic trypsinogen. Sulfated ...
Human pancreatic secretions contain two major trypsinogen isoforms, cationic and anionic trypsino... more Human pancreatic secretions contain two major trypsinogen isoforms, cationic and anionic trypsinogen, normally at a ratio of 2 : 1. Pancreatitis, pancreatic cancer and chronic alcoholism lead to a characteristic reversal of the isoform ratio, and anionic trypsinogen becomes the predominant zymogen secreted. To understand the biochemical consequences of these alterations, we recombinantly expressed and purified both human trypsinogens and documented characteristics of autoactivation, autocatalytic degradation and Ca2+-dependence. Even though the two trypsinogens are approximately 90% identical in their primary structure, we found that human anionic trypsinogen and trypsin exhibited a significantly increased (10-20-fold) propensity for autocatalytic degradation, relative to cationic trypsinogen and trypsin. Furthermore, in contrast to the characteristic stimulation of the cationic proenzyme, acidic pH inhibited autoactivation of anionic trypsinogen. In mixtures of cationic and anionic trypsinogen, an increase in the proportion of the anionic proenzyme had no significant effect on the levels of trypsin generated by autoactivation or by enterokinase at pH 8.0 in 1 mm Ca2+- conditions that were characteristic of the pancreatic juice. In contrast, rates of trypsinogen activation were markedly reduced with increasing ratios of anionic trypsinogen under conditions that were typical of potential sites of pathological intra-acinar trypsinogen activation. Thus, at low Ca2+ concentrations at pH 8.0, selective degradation of anionic trypsinogen and trypsin caused diminished trypsin production; while at pH 5.0, inhibition of anionic trypsinogen activation resulted in lower trypsin yields. Taken together, the observations indicate that up-regulation of anionic trypsinogen in pancreatic diseases does not affect physiological trypsinogen activation, but significantly limits trypsin generation under potential pathological conditions.
High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires dele... more High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient Escherichia coli strain. As a first application of this system, the effect of the pancreatitis-associated mutation A16V on the autoactivation of human cationic trypsinogen was characterized. The use of the novel pepP knockout Escherichia coli strain should be generally applicable to the expression of recombinant proteins, which undergo unwanted N-terminal trimming by aminopeptidase P. The human pancreas produces the digestive pro-enzyme trypsinogen in three isoforms. On the basis of their relative isoelectric points and electrophoretic mobility, these are commonly referred to as cationic trypsinogen, anionic trypsinogen, and mesotrypsinogen. The isoenzymes are encoded by separate genes, the PRSS1 (protease, serine, 1), PRSS2 and PRSS3 genes (for a recent review see [1] and references therein). Cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) make up the bulk of secreted trypsinogens in the pancreatic juice, while mesotrypsinogen (PRSS3) accounts for 2-10 % [2-6]. Trypsinogens are synthesized as prepro-enzymes (pre-trypsinogens) with a signal peptide of 15 amino acids, followed by the 8 amino acid long pro-peptide, the trypsinogen activation peptide. The signal-peptide is removed upon entry into the endoplasmic reticulum lumen and the pro-enzymes are packaged into zymogen granules and eventually secreted into the pancreatic juice. Activation of trypsinogen to trypsin is achieved by proteolytic removal of the pro-peptide, which normally takes place in the duodenum catalyzed by enteropeptidase (enterokinase), a highly specialized serine protease in the brush-border membrane of enterocytes. Trypsin can also activate trypsinogen, a process termed autoactivation, which in the duodenum may have a physiological role in facilitating zymogen activation, whereas inappropriate autoactivation in the pancreas might
Homogenized first trimester human placenta exhibits both Ca(2+)-dependent (90-95 per cent) and Ca... more Homogenized first trimester human placenta exhibits both Ca(2+)-dependent (90-95 per cent) and Ca(2+)-independent (5-10 per cent) nitric oxide (NO)-synthesizing activities. Addition of tetrahydrobiopterin (BH4) to homogenates containing Ca2+ in maximally activating concentrations (&gt; 0.5 microM) results in a further 2-2.5-fold activation of NO synthesis, with half-maximal stimulation observed at 26 +/- 8.2 microM BH4 (mean +/- SEM, n = 4). Chelation of Ca2+ in the medium abolishes the stimulatory effect, indicating that only a Ca2(+)-dependent NO-synthase (NOS) isoform is activated by BH4. Based on our previous findings, we suggest that this isoform is the endothelial or Type III NOS. Importantly, BH4 has no significant effect on the Ca2(+)-dependency of NOS activity, the apparent Km values for Ca2+ are comparable in the absence (1.8 +/- 0.4 microM, mean +/- SEM, n = 6) or presence (2.5 +/- 0.6 microM, mean +/- SEM, n = 6) of 50 microM BH4. The BH4 content of these placentae is 207.4 +/- 86.7 pmol/g wet tissue (mean +/- s.d., n = 9), therefore, BH4 added to the homogenate does not simply restore the concentrations that occur endogenously. The results provide the first evidence that in the early human placenta, a constitutively expressed CA 2(+)-dependent NOS isoform is stimulated by exogenous BH4, raising the possibility that BH4 is an important regulator of NOS activity in this tissue. This novel aspect of the NO-generating pathway may have implications in the aetiology and treatment of pregnancy-induced hypertension and pre-eclampsia.
Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change ... more Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N G-nitro-L-arginine (L-NNA) was examined on NO production, NOsensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5-200 mg/kg) to 21day-old rats reduced NOS activity and NO concentration dose dependently by 70-90% and the tissue cGMP level by 60-80%. By contrast, chronic application of L-NNA between postnatal days 4-21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10-50%. Chronic treatment of 10 mg/kg L-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50 mg/kg) of L-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50 mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.
The effects of thyroid hormones (TH) on the enzyme level and activity of neuronal nitric oxide sy... more The effects of thyroid hormones (TH) on the enzyme level and activity of neuronal nitric oxide synthase (nNOS) were studied in the rat cerebral cortex during postnatal life. As revealed by arginine/citrulline conversion assay and Western blot analysis of the homogenate of the parietal cortex T4 significantly increased nNOS activity and nNOS protein level to 153 ± 25% and to 178 ± 20%, respectively. In contrast, 6-n-propyl-2-thyouracil (PTU) decreased nNOS activity and nNOS level to 45 ± 10% and to 19 ± 4%, respectively. The number of nNOS-immunoreactive neurons did not change after either T4 or PTU treatment, however, following T4 administration the percentage of intensively immunoreactive neurons increased to 85 ± 3% compared to control (65 ± 6%), whereas it decreased to 49 ± 2% after PTU treatment. Our findings indicate that abnormal TH levels differentially regulate the activity and the level of nNOS and suggest a cross-talk between the TH and NO signaling pathway in the developing cerebral cortex of rats.
Chronic pancreatitis (CP) is a common inflammatory disease of the pancreas. Mutations in the gene... more Chronic pancreatitis (CP) is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) 1 and the pancreatic secretory trypsin inhibitor (SPINK1) 2 are associated with CP. Since increased proteolytic activity due to mutated PRSS1 enhances the risk for CP, mutations in the gene encoding anionic trypsinogen (PRSS2) may also act disease predisposing. Here we analyzed PRSS2 in CP patients and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4 %) controls but only in 32/2,466 (1.3 %) patients (odds ratio 0.37; P = 1.1 × 10-8). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity due to the introduction of a novel tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby plays a protective role against chronic pancreatitis.
The aim of this study was to characterize the mechanism of the chemical interaction between L-asc... more The aim of this study was to characterize the mechanism of the chemical interaction between L-ascorbic acid (ASC) and tetrahydrobiopterin (BH 4) in vitro and to examine its effect on the activity of endothelial nitric oxide synthase (eNOS) in first trimester human placentae. At room temperature, in Tris-HCl buffer (pH 7.4), both ASC and BH 4 were readily oxidized by dissolved O 2 or H 2 O 2. BH 4 was more sensitive to auto-oxidation, while ASC was more susceptible to oxidation by H 2 O 2. Addition of 36 µmol/l BH 4 to 143 µmol/l ASC increased the initial rate of ASC oxidation 3.2-fold in a catalase-sensitive manner, indicating that enhanced ASC oxidation is partly due to the formation of H 2 O 2. In the presence of catalase, BH 4 still stimulated 1.9-fold the initial rate of ASC oxidation, suggesting that another auto-oxidation product of BH 4 , most probably quininoid-BH 2 (qBH 2), could also stimulate ASC oxidation while itself being reduced back to BH 4. ASC prevented the auto-oxidation of BH 4 in a concentrationdependent fashion, with 3 mmol/l ASC providing an almost complete stabilization of 25 µmol/l BH 4. Importantly, basal eNOS activity in placental microsomes was stimulated 2.5-fold by 0.5 µmol/l BH 4 , and 0.5 mmol/l ASC enhanced the BH 4-stimulation 1.4-fold, with a smaller effect on basal eNOS activity. Taken together, the findings support the notion that the stabilizing action of ASC on BH 4 is related to the ASC-mediated reductive reversal of the auto-oxidation process of BH 4. Moreover, we demonstrated that concentrations of ASC present in the placenta as a common vitamin C supply are sufficient to protect cellular free BH 4 and may contribute to the stimulation of placental eNOS activity.
Type III nitric oxide synthase (NOS III) is responsible for Ͼ90% of nitric oxide (NO) synthesizin... more Type III nitric oxide synthase (NOS III) is responsible for Ͼ90% of nitric oxide (NO) synthesizing activity in first trimester placentae. Enzyme activity is distributed between cytosolic (30%) and membrane-bound forms (70%), with highest specific activity observed in microsomal fractions. In the present study, the effect of tetrahydrobiopterin (BH 4) on subunit structure and activity of microsomal and cytosolic NOS III was compared. As revealed by immunoblot analysis, incubation of microsomal membranes with 50 µM final concentration BH 4 for 10 min at 37 o C resulted in a striking conversion of monomeric NOS III into a protein having the characteristics (electrophoretic mobility, resistance to sodium dodecyl sulphate) of the homodimeric form. In contrast, BH 4 induced significantly less marked changes in the NOS III dimer content of cytosolic fractions. Enzyme activity in microsomes is stimulated~6-fold upon addition of 50 µM BH 4 , while only a 2-fold activation is detectable in cytosolic fractions. Taken together, the observations suggest that BH 4 activates NOS III in the primordial human placenta by promoting its subunit assembly in the membrane, while cytosolic NOS III is relatively insensitive to BH 4. Compartment-specific action of BH 4 represents a novel mechanism which is implicated in the regulation of placental NOS activity.
The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequ... more The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic trypsinogen. A dramatic increase in autoactivation of cationic trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian trypsinogen activation peptides has evolved not only for optimal enteropeptidase recognition in the duodenum but also for efficient inhibition of trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions unrelated to digestion, as found in the group III trypsinogens of cold-adapted fishes.
1 The abbreviations used are: HP, hereditary pancreatitis; Tg, trypsinogen; Tr, trypsin; Tg*, two... more 1 The abbreviations used are: HP, hereditary pancreatitis; Tg, trypsinogen; Tr, trypsin; Tg*, two-chain trypsinogen with the Arg 117-Val 118 peptide bond cleaved; Tr*, two-chain trypsin with the Arg 117-Val 118 peptide bond cleaved; PSTI, pancreatic secretory trypsin inhibitor. 2 Z. Kukor, M. Tóth, and M. Sahin-Tóth, unpublished observations.
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Papers by Zoltan Kukor