Association of Metallic and Nonmetallic Elements with Fibrin Clot Properties and Ischemic Stroke.

Objectives-Metallic elements and fibrin clot properties have been linked to stroke. We examined metallic and nonmetallic elements, fibrin clot lysis time (CLT), and maximum absorbance (Absmax) in relation to ischemic stroke. Design-A case-control study of ischemic stroke patients vs. healthy individuals. Subjects and Methods-Plasma and serum were collected from 260 ischemic stroke patients (45.0% women; age, 68 ± 12 years) and 291 healthy controls (59.7% women; age, 50 ± 17 years). Fibrin CLT and Absmax were measured using a validated turbidimetric assay. Serum elements were quantified by inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES). Data were analyzed by bivariate correlations and multiple or logistic regression. Results-In female stroke patients, copper, lithium, and aluminum were significantly lower compared with controls; in male stroke patients, potassium was lower, and beryllium was elevated. In female and male stroke patients, iron, zinc, nickel, calcium, magnesium, sodium, and silicon were significantly lower, while strontium was elevated. Positive correlations between fibrin clot properties and metals, observed in healthy controls, were lost in ischemic stroke patients. In multivariate regression analysis, fibrin CLT and/or Absmax was associated with zinc, calcium, potassium, beryllium, and silicon in stroke patients and with sodium, potassium, beryllium, and aluminum in controls. In logistic regression analysis, stroke was independently associated with lithium, nickel, beryllium, strontium, boron, and silicon and with sodium, potassium, calcium, and aluminum but not with fibrin CLT/Absmax. Conclusions-Various elements were associated with fibrin clot properties and the risk of ischemic stroke. Lithium, sodium, calcium, and aluminum abrogated the association of fibrin clot properties with ischemic stroke.

Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Homocysteine thiolactone contributes to the prognostic value of fibrin clot structure/function in coronary artery disease.

Fibrin clot structure/function contributes to cardiovascular disease. We examined sulfur-containing metabolites as determinants of fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to outcomes in coronary artery disease (CAD) patients. Effects of B-vitamin/folate therapy on CLT and Absmax were studied. Plasma samples were collected from 1,952 CAD patients randomized in a 2 x 2 factorial design to (i) folic acid, vitamins B12, B6; (ii) folic acid, vitamin B12; (iii) vitamin B6; (iv) placebo for 3.8 years in the Western Norway B-Vitamin Intervention Trial. Clot lysis time (CLT) and maximum absorbance (Absmax) were determined using a validated turbidimetric assay. Acute myocardial infarction (AMI) and mortality were assessed during a 7-year follow-up. Data were analyzed using bivariate and multiple regression. Survival free of events was studied using Kaplan Mayer plots. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional hazards models. Baseline urinary homocysteine (uHcy)-thiolactone and plasma cysteine (Cys) were significantly associated with CLT while plasma total Hcy was significantly associated with Absmax, independently of fibrinogen, triglycerides, vitamin E, glomerular filtration rate, body mass index, age, sex plasma creatinine, CRP, HDL-C, ApoA1, and previous diseases. B-vitamins/folate did not affect CLT and Absmax. Kaplan-Meier analysis showed associations of increased baseline CLT and Absmax with worse outcomes. In Cox regression analysis, baseline CLT and Absmax (>cutoff) predicted AMI (CLT: HR 1.58, 95% CI 1.10-2.28; P = 0.013. Absmax: HR 3.22, CI 1.19-8.69; P = 0.021) and mortality (CLT: HR 2.54, 95% CI 1.40-4.63; P = 0.002. Absmax: 2.39, 95% CI 1.17-4.92; P = 0.017). After adjustments for other prognostic biomarkers these associations remained significant. Cys and uHcy-thiolactone, but not tHcy, were significant predictors of AMI in Cox regression models that included CLT. Conclusions uHcy-thiolactone and plasma Cys are novel determinants of CLT, an important predictor of adverse CAD outcomes. CLT and Absmax were not affected by B-vitamin/folate therapy, which could account for the lack of efficacy of such therapy in CAD. Trial registration: URL: http://clinicaltrials.gov. Identifier: NCT00354081.

Analysis of the relationship between psychological gender and risk perception style and attitudes towards safety in a group of women and men.

The aim of the research was to analyse the relationship between psychological gender and risk perception style and the manifestation of pro-safe attitudes. The study involved 507 people in the age range 20-64 years, including 257 women and 250 men. Variables were measured using the following methods: Znajmiecka-Sikora's questionnaire of attitudes towards safety (PwB), Zaleskiewicz's questionnaire of the individual risk perception style (SIRI) and Lipinska-Grobelny and Gorczycka's scale of masculinity and femininity (SMiK). The obtained results prove that psychological gender significantly differentiates risk perception style and attitudes towards safety. The feminine dimension has been shown to strengthen safe attitudes and minimize risk-taking, while the masculine dimension weakens attitudes towards safety and strengthens risk propensity.

Changes in redox plasma proteome of Pon1-/- mice are exacerbated by a hyperhomocysteinemic diet.

High-density lipoprotein (HDL), in addition to promoting reverse cholesterol transport, possesses anti-oxidative, anti-inflammatory, and antithrombotic activities, which are thought to be promoted by paraoxonase 1 (PON1), an HDL-associated enzyme. Reduced levels of PON1 are associated with increased oxidative stress and cardiovascular disease both in humans and Pon1-/- mice. However, molecular basis of these associations are not fully understood. We used label-free mass spectrometry and Ingenuity Pathway Analysis bioinformatics resources to examine plasma proteomes in four-month-old Pon1-/- mice (n = 32) and their Pon1+/+ siblings (n = 15) fed with a hyper-homocysteinemic (HHcy) diet. We found that inactivation of the Pon1 gene resulted in dysregulation of proteins involved in the maintenance of redox homeostasis in mice. Redox-responsive proteins affected by Pon1-/- genotype were more numerous in mice fed with HHcy diet (18 out of 89, 20%) than in mice fed with a control diet (4 out of 50, 8%). Most of the redox-related proteins affected by Pon1-/- genotype in mice fed with a control diet (3 out of 4, 75%) were also affected in HHcy mice, while the majority of Pon1-/- genotype-dependent redox proteins in HHcy mice (15 out of 18, 83%) were not affected by Pon1-/- genotype in control diet animals. In addition to redox-related proteins, we identified proteins involved in acute phase response, complement/blood coagulation, lipoprotein/lipid metabolism, immune response, purine metabolism, glucose metabolism, and other proteins that were dysregulated by Pon1-/- genotype in HHcy mice. Taken together, our findings suggest that Pon1 interacts with proteins involved in antioxidant defenses and other processes linked to cardiovascular disease. Dysregulation of these processes provides an explanation for the pro-oxidant and pro-atherogenic phenotypes observed in Pon1-/- mice and humans with attenuated PON1 levels.

Work safety climate. Comparison of selected occupational groups.

Implementation of effective programs to improve occupational safety should be linked to an understanding of the specific nature of the given job. The aim of the research was to compare occupational groups with different job-related specificities: industrial production line workers, retail workers and mine rescuers, in terms of their assessment of the work safety climate. The survey covered 2,995 respondents with diversified demographic characteristics. The study used an abridged version of the Safety Climate Questionnaire by Znajmiecka-Sikora (2019) to assess 10 separate safety climate dimensions. The results of the MANOVA multivariate analysis, Wilks' multivariate F-tests and univariate F tests prove that there is a statistically significant difference between the respondents representing the three occupational groups collectively in terms of global assessment of all work safety climate dimensions, and also indicate significant differences between workers belonging to the three occupational groups in terms of their assessment of the individual dimensions of the work safety climate, except the organization's occupational health and safety management policy as well as technical facilities and ergonomics, which may be due to the universality of the requirements set for organizations with regard to these two aspects of safe behavior. The differences observed in the assessment of the remaining work safety climate dimensions induces one to promote more differentiated and individualized activities, taking into account the work specificity and the nature of the threats occurring in the respective working environment of the representatives of the different occupations. The difference in assessment of the work safety climate found in the research encourages one to create practical programs for safety, not only in the procedural and technical dimension, but also in the social and psychological one.

Genetic Attenuation of Paraoxonase 1 Activity Induces Proatherogenic Changes in Plasma Proteomes of Mice and Humans.

High-density lipoprotein (HDL), in addition to promoting reverse cholesterol transport, possesses anti-inflammatory, antioxidative, and antithrombotic activities. Paraoxonase 1 (PON1), carried on HDL in the blood, can contribute to these antiatherogenic activities. The PON1-Q192R polymorphism involves a change from glutamine (Q variant) to arginine (R variant) at position 192 of the PON1 protein and affects its enzymatic activity. The molecular basis of PON1 association with cardiovascular and neurological diseases is not fully understood. To get insight into the function of PON1 in human disease, we examined how genetic attenuation of PON1 levels/activity affect plasma proteomes of mice and humans. Healthy participants (48.9 years old, 50% women) were randomly recruited from the Poznan population. Four-month-old Pon1-/- (n = 17) and Pon1+/+ (n = 8) mice (50% female) were used in these experiments. Plasma proteomes were analyzed using label-free mass spectrometry. Bioinformatics analysis was carried out using the Ingenuity Pathway Analysis (IPA) resources. PON1-Q192R polymorphism and Pon1-/- genotype induced similar changes in plasma proteomes of humans and mice, respectively. The top molecular network, identified by IPA, affected by these changes involved proteins participating in lipoprotein metabolism. Other PON1 genotype-dependent proteomic changes affect different biological networks in humans and mice: "cardiovascular, neurological disease, organismal injury/abnormalities" in PON1-192QQ humans and "humoral immune response, inflammatory response, protein synthesis" and "cell-to-cell signaling/interaction, hematological system development/function, immune cell trafficking" in Pon1-/- mice. Our findings suggest that PON1 interacts with molecular pathways involved in lipoprotein metabolism, acute/inflammatory response, and complement/blood coagulation that are essential for blood homeostasis. Modulation of those interactions by the PON1 genotype can account for its association with cardiovascular and neurological diseases.

Cystathionine ß-synthase deficiency: different changes in proteomes of thrombosis-resistant Cbs-/- mice and thrombosis-prone CBS-/- humans.

Cystathionine ß-synthase (CBS)-deficient patients are prone to vascular thrombosis. In contrast, Cbs-/- mice show no abnormalities in blood coagulation. To identify molecular basis underlying these disparately different thrombotic phenotypes, we analyzed plasma proteomes of Cbs-/- vs. Cbs+/+ mice (8-month-old, 12/group, sex-matched) and CBS-/- vs. CBS+/+ humans (37 ± 7-year-old, 10-14/group, sex-matched) using label-free mass spectrometry. We identified 117 and 41 differentiating plasma proteins in Cbs-/- mice and CBS-/- humans, respectively. Twenty-one proteins were shared between CBS-/- humans and Cbs-/- mice, with sixteen changed in the opposite direction. Proteins involved in blood coagulation and complement/coagulation cascades represented a greater fraction of the differentiating proteins in CBS-/- patients (51%) than in Cbs-/- mice (21%). Top canonical pathways, identified by Ingenuity Pathways Analysis, such as LXR/RXR, FXR/RXR activation (- log[P-value] = 30-31) and atherosclerosis signaling (- log[P-value] = 10-11) were similarly affected in Cbs-/- mice and CBS-/- humans. The Coagulation System was affected stronger in CBS-/- humans than in Cbs-/- mice (- log[P-value] = 15 vs. 10, respectively) while acute phase response and complement system were affected stronger in Cbs-/- mice (- log[P-value] = 33 and 22, respectively) than in humans (- log[P-value] = 22 and 6, respectively). Other pathways, including IL-7 signaling and B cell development were affected only in Cbs-/- mice. Taken together, our findings suggest that differences in these processes, in particular in the Coagulation System, could account for the thrombotic phenotype in CBS-/- patients and the absence of thrombosis in Cbs-/- mice. Overall, our findings suggest that Cbs-/- mice have a better adaptive response to protect from prothrombotic effects of hyperhomocysteinemia than CBS-/- humans.

Serum Proteome Alterations in Human Cystathionine ß-Synthase Deficiency and Ischemic Stroke Subtypes.

Ischemic stroke induces brain injury via thrombotic or embolic mechanisms involving large or small vessels. Cystathionine ß-synthase deficiency (CBS), an inborn error of metabolism, is associated with vascular thromboembolism, the major cause of morbidity and mortality in affected patients. Because thromboembolism involves the brain vasculature in these patients, we hypothesize that CBS deficiency and ischemic stroke have similar molecular phenotypes. We used label-free mass spectrometry for quantification of changes in serum proteomes in CBS-deficient patients (n = 10) and gender/age-matched unaffected controls (n = 14), as well as in patients with cardioembolic (n = 17), large-vessel (n = 26), or lacunar (n = 25) ischemic stroke subtype. In CBS-deficient patients, 40 differentially expressed serum proteins were identified, of which 18 were associated with elevated homocysteine (Hcy) and 22 were Hcy-independent. We also identified Hcy-independent differentially expressed serum proteins in ischemic stroke patients, some of which were unique to a specific subtype: 10 of 32 for cardioembolic vs. large-vessel, six of 33 for cardioembolic vs. lacunar, and six of 23 for large-vessel vs. lacunar. There were significant overlaps between proteins affected by CBS deficiency and ischemic stroke, particularly the cardioembolic subtype, similar to protein overlaps between ischemic stroke subtypes. Top molecular pathways affected by CBS deficiency and ischemic stroke subtypes included acute phase response signaling and coagulation system. Similar molecular networks centering on NFκB were affected by CBS deficiency and stroke subtypes. These findings suggest common mechanisms involved in the pathologies of CBS deficiency and ischemic stroke subtypes.

Sex affects N-homocysteinylation at lysine residue 212 of albumin in mice.

The modification of protein lysine residues by the thioester homocysteine (Hcy)-thiolactone has been implicated in cardiovascular and neurodegenerative diseases. However, only a handful of proteins carrying Hcy on specific lysine residues have been identified and quantified in humans or animals. In the present work, we developed a liquid chromatography/mass spectrometry targeted assay, based on multiple reaction monitoring, for quantification of N-Hcy-Lys212 (K212Hcy) and N-Hcy-Lys525 (K525Hcy) sites in serum albumin in mice. Using this assay, we found that female (n = 20) and male (n = 13) Cbs-/- mice had significantly elevated levels of K212Hcy and K525Hcy modifications in serum albumin relative to their female (n = 19) and male (n = 17) Cbs+/- littermates. There was significantly more K212Hcy modification in Cbs-/- males than in Cbs-/- females (5.78 ± 4.21 vs. 3.15 ± 1.38 units, P = 0.023). Higher K212Hcy levels in males than in females were observed also in Cbs+/- mice (2.72 ± 0.81 vs. 1.89 ± 1.07 units, P = 0.008). In contrast, levels of the K525Hcy albumin modification were similar between males and females, both in Cbs-/- and Cbs+/- mice. These findings suggest that the sex-specific K212Hcy modification in albumin might have an important biological function in mice that is not affected by the Cbs genotype.

L-Arginine and vitamin C attenuate pro-atherogenic effects of high-fat diet on biomarkers of endothelial dysfunction in rats.

High-fat diet (HFD) is known to cause endothelial dysfunction and contribute to atherosclerosis progression. The objective of this study was to evaluate the efficacy of L-arginine (L-Arg) and vitamin C supplementation as a potentially useful strategy for modulation of serum homocysteine (Hcy) levels, tumor necrosis factor alpha (TNF-α), oxidative stress, and insulin resistance induced by HFD in rats. Six weeks-old female and male Wistar rats were divided into five groups of twelve rats each and treated for six weeks with: group 1, standard diet; group 2, HFD; group 3, HFD supplemented with L-Arg (20g/kg diet); group 4, HFD supplemented with L-Arg (20g/kg diet) plus vitamin C (100mg/kg diet); group 5, HFD supplemented with vitamin C (100mg/kg diet). HFD significantly elevated TNF-α, reduced total antioxidant status (TAS), and increased insulin resistance (HOMA-IR). Significant increases of total cholesterol (TCH), LDL cholesterol (LDL), triglyceride (TG) and a decrease of HDL cholesterol (HDL) were observed in HFD rats. Supplementation with l-Arg prevented the decrease of TAS and the increases in HOMA-IR, LDL, and TG levels. Moreover, Hcy and TNF-α levels were reduced in L-Arg supplemented group. Supplementation with vitamin C significantly atenuated TAS decrease and lowered LDL levels. L-Arg plus vitamin C enhanced L-Arg effect on TAS and protected against TNF-α increase. Western blot analysis showed that l-Arg supplementation of HFD rats reduced the level of protein carbonyls. Taken together, these findings indicate that supplemental l-arginine and/or vitamin C, by their abilities to modulate biomarkers of HFD-induced endothelial dysfunction, are anti-atherogenic.

Paraoxonase 1 deficiency and hyperhomocysteinemia alter the expression of mouse kidney proteins involved in renal disease.

SCOPE: Hyperhomocysteinemia (HHcy) is associated with kidney disease and leads to atherosclerosis and thrombosis. Paraoxonase 1 (Pon1), a hydrolase that participates in homocysteine (Hcy) metabolism and is carried in the circulation on high-density lipoprotein, has also been linked to kidney disease and atherothrombosis. Pon1-knockout mice are susceptible to atherosclerosis and exhibit a kidney-associated phenotype, polyuria or urine dilution. We hypothesize that HHcy and Pon1 deficiency are toxic to kidney function because they impair metabolic pathways important for normal kidney homeostasis. METHODS AND RESULTS: We examined changes in the mouse kidney proteome induced by Pon1 gene deletion and dietary HHcy, using 2D IEF/SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. We found that the expression of ten mouse kidney proteins was altered by the Pon1(-/-) genotype or HHcy. Proteins involved in metabolism of lipid (ApoA-I), protein (Hspd1), carbohydrate (Pdhb, Fbp1-isoform2, Eno1), and energy (Ndufs8, Ldhd) were down-regulated. Proteins involved in lipid transport (Pebp1), oxidative stress response (Prdx2), and cellular detoxification (Glo1) were up-regulated. The kidney proteins altered by HHcy or Pon1 are also altered in renal disease. CONCLUSION: Our findings suggest that excess Hcy is toxic because it deregulates the expression of proteins involved in diverse cellular processes-from lipid, protein, carbohydrate, and energy metabolisms to detoxification and antioxidant defenses-that are essential for normal kidney homeostasis. Dysregulation of these processes can account for the involvement of HHcy and reduced Pon1 in kidney disease. Our findings also show that Pon1 plays an important role in maintaining normal kidney homeostasis.

Identification of N-homocysteinylation sites in plasma proteins.

A protocol for the identification of N-homocysteinylation sites in plasma proteins is described. Human plasma or purified fibrinogen is subjected to trypsin digestion and analysis of N-Hcy-peptides by liquid chromatography/mass spectroscopy (LC/MS). Human fibrinogen is isolated from the plasma by the glycine precipitation method. Identification of N-Hcy-Lys-peptides in tryptic digests of in vivo-derived samples is facilitated by the use of N-Hcy-albumin and N-Hcy-fibrinogen synthesized in vitro from commercially available human proteins. This protocol allows identification of N-homocysteinylation sites at Lys4, Lys12, Lys137, and Lys525 in albumin directly in trypsin-digested human serum samples. N-Hcy-Lys562, N-Hcy-Lys344, and N-Hcy-Lys385 were identified in human fibrinogen from patients with cystathionine ß-synthase deficiency. The protocol can be completed in 4 days.

Analysis of site-specific N-homocysteinylation of human serum albumin in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry.

Elevated levels of homocysteine (Hcy) are associated with cardiovascular and neurodegenerative diseases in humans. Hcy becomes a component of human proteins as a result of N-homocysteinylation of protein lysine residues by Hcy-thiolactone, which affects the protein's structure and function, and contributes to Hcy-related pathology. Albumin is the major target for N-homocysteinylation in human blood in vivo. Previous work has identified Lys-525 as a predominant site of N-homocysteinylation in vitro and in vivo. Here we show that Lys-4, Lys-12, Lys-137, Lys-159, Lys-205, and Lys-212 of human albumin are susceptible to N-homocysteinylation in vitro and provide evidence that two of those residues, Lys-137 and Lys-212, in addition to Lys-525, are N-homocysteinylated in vivo in human plasma.

Direct monitoring of albumin lysine-525 N-homocysteinylation in human serum by liquid chromatography/mass spectrometry.

A posttranslational protein modification by homocysteine-thiolactone (N-homocysteinylation) is linked to human vascular and neurodegenerative diseases. Although chemical and immunological methods are available to detect and quantify the extent of protein N-homocysteinylation, the determination of site-specific N-homocysteinylation in vivo remains challenging. Here we describe a liquid chromatography/mass spectrometry method that monitors the extent of N-homocysteinylation at albumin lysine-525 in vivo directly in human serum. Using this method, we found that the extent of lysine-525 N-homocysteinylation was significantly increased in patients with cystathionine beta-synthase deficiency.

Homocysteine editing and growth inhibition in Escherichia coli.

In Escherichia coli homocysteine (Hcy) is metabolically converted to the thioester Hcy-thiolactone in ATP-consuming reactions catalysed by methionyl-, isoleucyl- and leucyl-tRNA synthetases. Here we show that growth inhibition caused by supplementation of E. coli cultures with Hcy is accompanied by greatly increased accumulation of Hcy-thiolactone. Energy dissipation for Hcy editing increases 100-fold in the presence of exogenous Hcy and reaches one mole of ATP unproductively dissipated for Hcy-thiolactone synthesis per each mole of ATP that is consumed for methionine activation. Inhibiting Hcy-thiolactone synthesis with isoleucine, leucine or methionine accelerates bacterial growth in Hcy-supplemented cultures. Growth rates in Hcy-inhibited cultures are inversely related to the accumulation of Hcy-thiolactone. We also show that the levels of protein N-linked Hcy modestly increase in E. coli cells in Hcy-supplemented cultures. The results suggest that Hcy editing restrains bacterial growth.

[Decoding errors--function of the ribosomal E site]. / Bledy w odszyfrowywaniu informacji genetycznej--rola rybosomalnego miejsca E.

In the sixties of the last (20th) century James Watson presented a two-sites model for the ribosome comprising the P site for the peptidyl transfer RNA (tRNA) before peptide-bond formation and the A site, where decoding takes place according to the codon exposed there. Twenty years later the E site, which was specific for deacylated tRNA was detected. However, despite having three tRNA binding sites, only two tRNAs occupy the ribosome simultanously during protein synthesis: at the A and P sites before translocation (PRE state) and at the P and E sites after translocation (POST state). The importance of having two tRNAs in the POST state has been revealed during the last 25 years. It has been shown, that the E-tRNA prevents misincorporation of aminoacids and loss of reading frame. In other words, the ribosomal E site is a guard for accuracy of the polypeptide synthesized in the ribosomes.

Protective mechanisms against homocysteine toxicity: the role of bleomycin hydrolase.

Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.

[The role of homocysteine thiolactone in some of human diseases]. / Rola tiolaktonu homocysteiny w niektórych chorobach czlowieka.

In the present article we discuss the most recent data regarding the role of homocysteine, its cyclic thioester--homocysteine thiolactone and the process of protein N-homocysteinylation in human disease. The protective role of thiolactonase/paraoxonase enzyme, carried on high density lipoproteins (HDL) in human blood, as well as the influence of structural modifications on HDL function are discussed. We also describe the effect of vitamin therapy (folic acid, vitamins: B6, B12) used for lowering the homocysteine level in humans as well.