Blood glucose control and metabolic dysfunction-associated steatotic liver disease in people with type 1 diabetes.

PURPOSE: Metabolic dysfunction-associated steatotic liver disease (MASLD) may have distinctive pathophysiological features in type 1 diabetes (T1D). We evaluated the independent role of blood glucose control on MASLD in T1D. METHODS: In a cross-sectional study on 659 T1D adult patients, MASLD was assessed by the Fatty Liver Index (FLI) and the Hepatic Steatosis Index (HSI). Anthropometric, biochemical, and clinical parameters were retrieved from electronic records. Blood glucose control status was evaluated by dividing participants into subgroups according to the median value of HbA1c [7.6% (60 mmol/mol)], and this analysis was repeated excluding overweight/obese patients. RESULTS: Patients with HbA1c above 7.6% (60 mmol/mol) showed significantly higher MASLD indices (HSI 38 ± 6 vs. 36 ± 5, p < 0.001; FLI 26 ± 26 vs.19 ± 19, p < 0.001), and higher proportions of MASLD identified by HSI (57 vs. 44%, p < 0.001) and FLI (14 vs. 7%, p < 0.001) than patients with HbA1c below 7.6% (60 mmol/mol). Similar results were obtained for HSI after the exclusion of overweight/obese patients. Stepwise linear regression analysis confirmed that HbA1c was independently associated with HSI (r = 0.496, p = 0.009) and FLI (r = 0.722, p = 0.007); waist circumference with HSI (r = 0.492, p < 0.001); and waist circumference (r = 0.700, p < 0.001), HDL cholesterol (r = 0.719, p < 0.001), and LDL cholesterol (r = 0.712, p < 0.001) with FLI. CONCLUSIONS: Blood glucose control is a main factor associated with MASLD in adults with T1D, also independently of overweight and obesity. Appropriate therapeutic strategies focused on tight blood glucose control may also be needed for the prevention and treatment of MASLD in T1D.

Glucose lowering strategies and cardiovascular disease in type 2 diabetes - teachings from the TOSCA.IT study.

TOSCA.IT is an institutional, non-industry-supported, head-to-head study comparing long term cardiovascular effects, efficacy and safety of two antidiabetes drugs (pioglitazone vs sulphonylureas) used in combination with metformin in patients with type 2 diabetes mellitus. The study results show that in the absence of clinically evident cardiovascular disease both treatment strategies represent suitable alternatives; however, in consideration of the greater durability of the metabolic effects, the lower risk of hypoglycemia and the potential benefit on atherosclerotic cardiovascular disease, the combination of metformin and pioglitazone may be considered as the preferential therapeutic option. In this review the study is critically evaluated against the background of the evidence accumulated over the last decade on the impact of different glucose lowering drugs on cardiovascular events in people with type 2 diabetes.

Dietary intake and major food sources of polyphenols in people with type 2 diabetes: The TOSCA.IT Study.

PURPOSE: Proper evaluation of polyphenols intake at the population level is a necessary step in order to establish possible associations with health outcomes. Available data are limited, and so far no study has been performed in people with diabetes. The aim of this work was to document the intake of polyphenols and their major food sources in a cohort of people with type 2 diabetes and in socio-demographic subgroups. METHODS: We studied 2573 men and women aged 50-75 years. Among others, anthropometry was measured by standard protocol and dietary habits were investigated by food frequency questionnaire (EPIC). The intake of polyphenols was evaluated using US Department of Agriculture and Phenol-Explorer databases. RESULTS: The mean total polyphenol intake was 683.3 ± 5.8 mg/day. Non-alcoholic beverages represented the main food source of dietary polyphenols and provided 35.5% of total polyphenol intake, followed by fruits (23.0%), alcoholic beverages (14.0%), vegetables (12.4%), cereal products and tubers (4.6%), legumes (3.7%) and oils (2.1%); chocolate, cakes and nuts are negligible sources of polyphenols in this cohort. The two most important polyphenol classes contributing to the total intake were flavonoids (47.5%) and phenolic acids (47.4%). Polyphenol intake increased with age and education level and decreased with BMI; furthermore, in the northern regions of Italy, the polyphenol intake was slightly, but significantly higher than in the central or southern regions. CONCLUSIONS: The study documents for the first time the intake of polyphenols and their main food sources in people with diabetes using validated and complete databases of the polyphenol content of food. Compared with published data, collected in people without diabetes, these results suggest a lower intake and a different pattern of intake in people with diabetes.

Bat-man disease transmission: zoonotic pathogens from wildlife reservoirs to human populations.

Bats are natural reservoir hosts and sources of infection of several microorganisms, many of which cause severe human diseases. Because of contact between bats and other animals, including humans, the possibility exists for additional interspecies transmissions and resulting disease outbreaks. The purpose of this article is to supply an overview on the main pathogens isolated from bats that have the potential to cause disease in humans.

Sex differences in food choices, adherence to dietary recommendations and plasma lipid profile in type 2 diabetes - The TOSCA.IT study.

BACKGROUND AND AIMS: Diabetic women have a more adverse plasma lipid profile than men. Sex differences in dietary habits may play a role, but are little investigated. The study evaluates the quality of diet, adherence to the nutritional recommendations of the Diabetes and Nutrition Study Group and their relation with plasma lipid in men and women with diabetes. METHODS AND RESULTS: We studied 2573 people, aged 50-75, enrolled in the TOSCA.IT study (clinicaltrials.gov; NCT00700856). Plasma lipids were measured centrally. Diet was assessed with a semi-quantitative food frequency questionnaire. Women had a more adverse plasma lipid profile than men. Women consumed significantly more legumes, vegetables, fruits, eggs, milk, vegetable oils, and added sugar, whereas men consumed more starchy foods, soft drinks and alcoholic beverages. This stands for a higher proportion (%) of energy intake from saturated fat and added sugar (12.0 ± 2.4 vs 11.5 ± 2.5 and 3.4 ± 3.2 vs 2.3 ± 3.2, P < 0.04), and a higher intake of fiber (11.2 ± 2.8 vs 10.4 ± 2.6 g/1000 Kcal/day) in women. Adherence to the recommendations for saturated fat and fiber consumption was associated with significantly lower LDL-cholesterol regardless of sex. Adherence to the recommendations for added sugars was associated with significantly lower triglycerides and higher HDL-cholesterol in men and women. CONCLUSIONS: Men and women with diabetes show significant differences in adherence to nutritional recommendations, but sex differences in plasma lipid profile are unlikely to be explained by nutritional factors. Adherence to the nutritional recommendations is associated with a better plasma lipid profile regardless of sex, thus reinforcing the importance of substituting saturated for unsaturated fat sources, increasing fiber and reducing added sugar intake.

Influence of dietary fat and carbohydrates proportions on plasma lipids, glucose control and low-grade inflammation in patients with type 2 diabetes-The TOSCA.IT Study.

PURPOSE: The optimal macronutrient composition of the diet for the management of type 2 diabetes is debated, particularly with regard to the ideal proportion of fat and carbohydrates. The aim of the study was to explore the association of different proportions of fat and carbohydrates of the diet-within the ranges recommended by different guidelines-with metabolic risk factors. METHODS: We studied 1785 people with type 2 diabetes, aged 50-75, enrolled in the TOSCA.IT Study. Dietary habits were assessed using a validated food-frequency questionnaire (EPIC). Anthropometry, fasting lipids, HbA1c and C-reactive protein (CRP) were measured. RESULTS: Increasing fat intake from <25 to ≥35 % is associated with a significant increase in LDL-cholesterol, triglycerides, HbA1c and CRP (p < 0.05). Increasing carbohydrates intake from <45 to ≥60 % is associated with significantly lower triglycerides, HbA1c and CRP (p < 0.05). A fiber intake ≥15 g/1000 kcal is associated with a better plasma lipids profile and lower HbA1c and CRP than lower fiber consumption. A consumption of added sugars of ≥10 % of the energy intake is associated with a more adverse plasma lipids profile and higher CRP than lower intake. CONCLUSIONS: In people with type 2 diabetes, variations in the proportion of fat and carbohydrates of the diet, within the relatively narrow ranges recommended by different nutritional guidelines, significantly impact on the metabolic profile and markers of low-grade inflammation. The data support the potential for reducing the intake of fat and added sugars, preferring complex, slowly absorbable, carbohydrates.

Die for the community: an overview of programmed cell death in bacteria.

Programmed cell death is a process known to have a crucial role in many aspects of eukaryotes physiology and is clearly essential to their life. As a consequence, the underlying molecular mechanisms have been extensively studied in eukaryotes and we now know that different signalling pathways leading to functionally and morphologically different forms of death exist in these organisms. Similarly, mono-cellular organism can activate signalling pathways leading to death of a number of cells within a colony. The reason why a single-cell organism would activate a program leading to its death is apparently counterintuitive and probably for this reason cell death in prokaryotes has received a lot less attention in the past years. However, as summarized in this review there are many reasons leading to prokaryotic cell death, for the benefit of the colony. Indeed, single-celled organism can greatly benefit from multicellular organization. Within this forms of organization, regulation of death becomes an important issue, contributing to important processes such as: stress response, development, genetic transformation, and biofilm formation.

Lower incidence of macrovascular complications in patients on insulin glargine versus those on basal human insulins: a population-based cohort study in Italy.

BACKGROUND AND AIM: The aim of this study was to compare the use of insulin glargine and intermediate/long-acting human insulin (HI) in relation to the incidence of complications in diabetic patients. METHODS AND RESULTS: A population-based cohort study was conducted using administrative data from four local health authorities in the Abruzzo Region (900,000 inhabitants). Diabetic patients without macrovascular diseases and treated with either intermediate/long-acting HI or glargine were followed for 3-years; the incidence of diabetic (macrovascular, microvascular and metabolic) complications was ascertained by hospital discharge claims and estimated using Cox proportional hazard models. Propensity score (PS) matching was also used to adjust for significant differences in the baseline characteristics between the two groups. RESULTS: Overall, 1921 diabetic patients were included: 744 intermediate/long-acting HI and 1177 glargine users. During the 3-year follow-up, 209 (28.1%) incident events of any diabetic complication occurred in the intermediate/long-acting HI and 159 (13.5%) in the glargine group. After adjustment for covariates, glargine users had an HR (95% CI) of 0.57 (0.44-0.74) for any diabetic complication and HRs of 0.61 (0.44-0.84), 0.58 (0.33-1.04) and 0.35 (0.18-0.70) for macrovascular, microvascular and metabolic complications, respectively, compared to intermediate/long-acting HI users. PS analyses supported these findings. CONCLUSIONS: The use of glargine is associated with a lower risk of macrovascular complications compared with traditional basal insulins. However, limitations inherent to the study design including the short length of observation and the lack of data on metabolic control or diabetes duration, do not allow us to consider this association as a proof of causality.

Addition of either pioglitazone or a sulfonylurea in type 2 diabetic patients inadequately controlled with metformin alone: impact on cardiovascular events. A randomized controlled trial.

BACKGROUND AND AIMS: Metformin is the first-line therapy in type 2 diabetes. In patients inadequately controlled with metformin, the addition of a sulfonylurea or pioglitazone are equally plausible options to improve glycemic control. However, these drugs have profound differences in their mechanism of action, side effects, and impact on cardiovascular risk factors. A formal comparison of these two therapies in terms of cardiovascular morbidity and mortality is lacking. The TOSCA.IT study was designed to explore the effects of adding pioglitazone or a sulfonylurea on cardiovascular events in type 2 diabetic patients inadequately controlled with metformin. METHODS: Multicentre, randomized, open label, parallel group trial of 48 month duration. Type 2 diabetic subjects, 50-75 years, BMI 20-45 Kg/m(2), on secondary failure to metformin monotherapy will be randomized to add-on a sulfonylurea or pioglitazone. The primary efficacy outcome is a composite endpoint of all-cause mortality, nonfatal myocardial infarction, nonfatal stroke, and unplanned coronary revascularization. Principal secondary outcome is a composite ischemic endpoint of sudden death, fatal and non-fatal myocardial infarction and stroke, endovascular or surgical intervention on the coronary, leg or carotid arteries, major amputations. Side effects, quality of life and economic costs will also be evaluated. Efficacy, safety, tolerability, and study conduct will be monitored by an independent Data Safety Monitoring Board. End points will be adjudicated by an independent external committee. CONCLUSIONS: TOSCA.IT is the first on-going study investigating the head-to-head comparison of adding a sulfonylurea or pioglitazone to existing metformin treatment in terms of hard cardiovascular outcomes. REGISTRATION: Clinicaltrials.gov ID NCT00700856.

Measurement of the intrarenal arterial resistance index for the identification and prediction of diabetic nephropathy.

BACKGROUND AND AIMS: High intrarenal resistance index (RI) predicts renal function in several conditions; its use in the prediction of diabetic nephropathy (DN) is little explored. We aimed (1) to compare RI in diabetic and non diabetic hypertensive patients, and (2) to evaluate whether high RI is associated with clinical signs of DN and its progression over time. DESIGN: observational, prospective. PARTICIPANTS: 92 type 2 diabetic patients and 37 non-diabetic controls aged 40-70, with hypertension and normal renal function. We measured ultrasound RI and, among others, creatinine, estimated glomerular filtration rate and urinary albumin excretion rate (AER) at baseline and after 4.5 years follow-up. Progression of albuminuric state (i.e., transition from baseline normo-microalbuminuria to follow-up micro-macroalbuminuria) was evaluated. RI was significantly higher in diabetic than non-diabetic participants (0.69+/-0.05 vs 0.59+/-0.05, p<0.001). Diabetic patients with RI>or=0.73, i.e., above the 80th percentile of the RI distribution, had significantly higher baseline AER and a more frequent progression of the albuminuric state compared to patients with RI<0.73 (27.7microg/mg [12.1-235.4] vs 15.1microg/mg [8.6-33.4]; 52.9% vs 9.5%, respectively). AER increased significantly from baseline to follow-up in patients with RI>or=0.73 (from 27.7microg/mg [12.1-235.4] to 265.0microg/mg [23.8-1018.1], p<0.01), but not in those with RI<0.73 (from 15.1microg/mg [8.6-33.4] to 16.1microg/mg [10.7-67.2], ns). OR for progression of albuminuric state, adjusted for established predictors of DN, including baseline AER, was 5.01 (1.4-17.7, 95% CI) for patients with RI>or=0.73 vs <0.73. Findings were confirmed in patients with normoalbuminuria at baseline. CONCLUSIONS: In diabetic patients, high RI (>or=0.73) is associated with features of DN and its progression over time, independent of albuminuria.

Fasting plasma free fatty acid concentrations and Pro12Ala polymorphism of the peroxisome proliferator-activated receptor (PPAR) gamma2 gene in healthy individuals.

BACKGROUND: The Pro12Ala polymorphism of the peroxisome proliferator-activated receptor (PPAR) gamma gene has been associated in some, but not all, studies with lower body mass index (BMI) and improved insulin sensitivity; how an altered transcriptional activity of PPARgamma2 could influence insulin sensitivity is currently unclear. The free fatty acids (FFAs) released from adipose tissue triglycerides via lipolysis are key mediators of impaired insulin sensitivity; however, no study has described the relationship of the Pro12Ala mutation with circulating levels of FFAs under physiological conditions. OBJECTIVE: To investigate in a population-based sample of Caucasians the relation of the Pro12Ala polymorphism with plasma concentrations of FFAs and other markers of lipid and glucose metabolism described as components of the insulin resistance syndrome. SUBJECTS: Four hundred and thirty-eight nondiabetic employees of the Italian Telephone Company, aged 35-65 years, randomly selected from a total population of 3900 participants in a company-sponsored health screening. MEASUREMENTS: The Pro12Ala polymorphism of the PPARgamma was studied together with plasma FFAs, insulin, glucose, triglycerides, high density lipoprotein (HDL) cholesterol, blood pressure and anthropometry. The Homeostatic Model Assessment (HOMA) index was calculated as a measure of insulin resistance. RESULTS: Carriers and noncarriers of the Pro12Ala polymorphism showed very similar circulating levels of FFA (0.46 +/- 0.2 vs. 0.47 +/- 0.2, NS); plasma glucose, triglycerides, HDL cholesterol and blood pressure were also similar in the two groups with or without the polymorphism. To allow for the possible confounding effect of obesity, a separate analysis was conducted in overweight (BMI > or = 25 kg/m(2)) and normal-weight people (BMI < 25 kg/m(2)). Circulating plasma FFA concentrations, as well as triglycerides, blood pressure and HOMA, were significantly higher in overweight than normal-weight, as expected, but no significant differences were detected between carriers and noncarriers of the Pro12Ala polymorphism within each BMI group (0.49 +/- 0.2 vs. 0.48 +/- 0.2, NS, and 0.44 +/- 0.2 vs. 0.47 +/- 0.2, NS, in overweight and normal-weight, respectively). The Pro12Ala polymorphism was also analysed across increasing quartiles of FFA concentrations and no relationship was observed between the frequency of the polymorphism and FFA values (overall chi2 = 0.48, NS). CONCLUSION: This study does not show any relationship between the Pro12Ala polymorphism of the PPARgamma gene and fasting FFAs in the general population. The possibility of a different handling of FFAs under different conditions (i.e. postprandial) cannot be excluded and remains to be explored.

Bacterial peptide methionine sulphoxide reductase: co-induction with glutathione S-transferase during chemical stress conditions.

Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes. A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) [Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem. J. 335, 573-579]. This raises the question of whether the products of these two genes may be involved in a common cellular protection function. To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51 kDa GST fusion protein. Following cleavage with thrombin and purification, the soluble 24 kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds. Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated. The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity. MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol. Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds [Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem. J. 346, 553-559]; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.

Evaluation of the role of two conserved active-site residues in beta class glutathione S-transferases.

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His(106) and Lys(107) of PmGST B1-1 to investigate their possible role in the enzyme's catalytic activity. His(106) was mutated to Ala, Asn and Phe, and Lys(107) to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His(106) and Lys(107) in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.

Functional analysis of the evolutionarily conserved proline 53 residue in Proteus mirabilis glutathione transferase B1-1.

The role of the evolutionarily conserved residue Pro-53 in Proteus mirabilis glutathione transferase B1-1 has been examined by replacing it with a serine residue using site-directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro-53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications.

BACKGROUND: Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS: Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS: The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Site-directed mutagenesis of the Proteus mirabilis glutathione transferase B1-1 G-site.

In order to investigate the roles of near N-terminus Tyr, Cys, and Ser residues in the activity of bacterial glutathione transferase (GSTB1-1) site-directed mutagenesis was used to replace the following residues: Tyr-4, Tyr-5, Ser-9, Cys-10, Ser-11, and Ser-13. The results presented here show that, unlike all other alpha, mu, pi, theta and sigma classes of glutathione transferases so far investigated, GSTB1-1 does not utilise any Tyr, Ser or Cys residue to activate glutathione. These results also suggest that the bacterial glutathione transferases may require classification into their own class.

Identification of an N-capping box that affects the alpha 6-helix propensity in glutathione S-transferase superfamily proteins: a role for an invariant aspartic residue.

We have identified an N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) that is strictly conserved, at the beginning of alpha 6 helix, in all glutathione S-transferases (GSTs) and most of the related superfamily proteins. By using CD and peptide modelling we have demonstrated that the capping box residues have an important role in determining the helical conformation adopted by this fragment in the hydrophobic environment of the protein. This is an example in which a local motif, contributing to nucleation of a structural element essential to the global folding of the protein, is strictly conserved in a superfamily of homologous proteins.

Effect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis.

When Proteus mirabilis was cultured anaerobically in the presence of nitrate as terminal electron acceptor, a dramatic reduction of glutathione transferase production occurred. The analysis of the glutathione affinity purified materials in terms of substrate specificity, SDS-PAGE pattern, IEF pattern and immunoblotting revealed that a significantly different glutathione transferase pattern also occurred: two new glutathione transferase forms with an isoelectric point at pH 4.8 and 5.0 appeared. Their N-terminal amino acid sequence analysis as well as the ability to bind to a glutathione affinity column indicate that major differences between anaerobic and aerobic glutathione transferase forms are mainly located in the C-terminal region of the primary structure. In contrast, no significant changes occurred in the production of glutathione transferase isoenzymes when P. mirabilis was grown anaerobically in the absence of a terminal electron acceptor. These results support the idea that bacterial glutathione transferase expression is not strictly related to the absence of oxygen stress.

Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis.

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421-425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli sigma 70 consensus promoter and to promoters of P. mirabilis cat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-beta-D-thiogalactopyranoside and growth at 37 degrees C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.

Helicobacter pylori isolated from stomach corpus and antrum: comparison of DNA patterns.

The genomic DNA of Helicobacter pylori was studied in strains isolated from two different sites of the stomach: the corpus and the antrum. 70 strains of H. pylori were found in 36 patients; 34 out of the 36 patients harboured the strain in both districts analysed. Restriction endonuclease analysis with Hae III and Hind III was used to compare the DNA patterns of strains isolated from the anatomical sites studied. Two pairs of DNA samples were not digested by these enzymes. 27 of the 32 pairs of the digested DNA appeared similar to each other. The analysis of chromosomal DNA in the remaining five pairs showed different electrophoretic patterns. These results indicate that the gastric mucosa can be colonized, at the same time, by strains of H. pylori with different genomic patterns, and this aspect can be important for epidemiological studies.