Pattern III Non-toxigenic Bacteroides fragilis (NTBF) Strains in Brazil

Bacteroides fragilis strains isolated from faeces of diarrhoeic and healthy children were studied by polymerase chain reaction (PCR), in order to characterise them as enterotoxigenic B. fragilis -ETBF—if they have one of the three bft gene alleles (pattern I) or as non-toxigenic B. fragilis—NTBF—if there was an absence of bft gene alleles and specific sites (flanking region of B. fragilis Pathogenicity Island—BfPAI) (pattern II NTBF) or absence of alleles, but the presence of this specific sites (pattern III NTBF). All strains were previously screened for cytotoxic activity. ETBF was detected in 1.5% (1/66) of the samples, in which we could verify, concomitantly, the presence of Escherichia coli enteroaggregative (EAEC). Due to these data, ETBF could not be associated with diarrhoea. A large number of pattern III NTBF strains were observed, which could suggest future changes in the phenotype of enterovirulence of B. fragilis species in our country. These populations were also analysed by using AP-PCR and a great heterogeneity could be observed. We were not able to make a correlation between enterovirulence patterns and genetic types.     

Genetic studies with heteroduplex DNA of bacteriophage f1. Asymmetric segregation,base correction and implications for the mechanism of genetic recombination

Heteroduplex DNA of bacteriophage f1 constructed in vitro was used to transfect Escherichia coli. The progeny phage produced were analyzed by genetic means. A strongly asymmetric transfer of information was observed. This result shows that one strand—usually the minus strand—determines in large part the genotypes of progeny phage. These results are discussed in the light of the available information on DNA duplication. Evidence for an activity that corrects mismatched bases will be presented and discussed. Heteroduplex molecules which were heterozygous at the sites that govern sensitivity to B restriction and modification were constructed and analyzed in restricting and non-restricting hosts. Results of these studies give support to a model for f1 genetic recombination that envisages asymmetric heteroduplex formation as an intermediate. These results are discussed in relation to earlier data.     

Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6–8.8 mM) led to a change in the transistor’s output signal, which was determined by the appearance of H⁺ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria—Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM16—the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.     

Superoxide dismutase activity in transgenic canola

Superoxide dismutase (SOD) activity was investigated in leaves of transgenic canola plants which expressed heterologous genes of different origin, namely 1—herbicide resistance genes (bar and simultaneously bar and epsps); 2—DesC desaturase gene (desC) of cyanobacterium Synechococcus vulcanus; 3—human interferon α2b gene (huIFN-α2b); 4—esxA::fbpB ^ΔTMD fused gene, encoding ESAT-6 and Ag85b Mycobacterium tuberculosis proteins, inducing immune response against tuberculosis; 5—cyp11A1 gene of cytochrome P450_SCC from bovine adrenal cortex mitochondria. Introduction of herbicide resistance genes as well as desaturase gene of cyanobacterium and mycobacterium’s genes did not change leaf SOD activity. At the same time it was shown that cyp11A1 and huIFN-α2b canola have increased leaf SOD activity up 58 and 33%, respectively, compared with control ones in non-stress conditions. It may be a prerequisite for improved resistance of these plants to the stressors of different origin.     

Functional heterogeneity among peritoneal macrophages. II. Enzyme content of macrophage subpopulations.

Rabbit peritoneal exudate (PE) macrophages were separated into subpopulations on discontinuous density gradients of bovine serum albumin. Four such macrophage subpopulations, referred to as bands A, B, C, and D (from lightest to heaviest buoyant density), were examined for differences in enzyme content. With regard to three acid hydrolases—acid phosphatases, β-glucuronidase, and cathepsin D—cells in bands A and B had greater enzyme activity than cells in bands C and D. A similar distribution of activities was observed for acid p-nitrophenylphosphatase. Peroxidase activity was present only in band D. Lysozyme activity was greatest in band D cells and least in band A cells. Only small differences in cytochrome c oxidase activity were observed among the subpopulations. Arginase activity was found to be greater in cells from band A than cells in bands B, C, and D. Macrophage subpopulations derived from PE macrophages placed in tissue culture for 7 days and macrophage subpopulation cells cultured for 2 days showed differences in acid phosphatase content similar to those seen with freshly obtained subpopulations. These results extend previous work demonstrating heterogeneity among PE macrophages.     

Leishmania donovani pteridine reductase 1: comparative protein modeling and protein–ligand interaction studies of the leishmanicidal constituents isolated from the fruits of Piper longum

Visceral leishmaniasis or kala-azar is caused by the dimorphic parasite Leishmania donovani in the Indian subcontinent. Treatment options for kala-azar are currently inadequate due to various limitations. Currently, drug discovery for leishmaniases is oriented towards rational drug design; the aim is to identify specific inhibitors that target particular metabolic activities as a possible means of controlling the parasites without affecting the host. Leishmania salvages pteridin from its host and reduces it using pteridine reductase 1 (PTR1, EC 1.5.1.33), which makes this reductase an excellent drug target. Recently, we identified six alkamides and one benzenoid compound from the n-hexane fraction of the fruit of Piper longum that possess potent leishmanicidal activity against promastigotes as well as axenic amastigotes. Based on a homology model derived for recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with these compounds to evaluate their binding affinity. A fairly good agreement between experimental data and the results of molecular modeling investigation of the bioactive and inactive compounds was observed. The amide group in the conjugated alkamides and the 3,4-methylenedioxystyrene moiety in the benzenoid compound acts as heads and the long aliphatic chain acts as a tail, thus playing important roles in the binding of the inhibitor to the appropriate position at the active site. The remarkably high activity of a component containing piperine and piperine isomers (3.36:1) as observed by our group prompted us to study the activities of all four isomers of piperine—piperine (2E,4E), isopiperine (2Z,4E), isochavicine (2E,4Z), and chavicine (2Z,4Z)—against LdPTR1. The maximum inhibitory effect was demonstrated by isochavicine. The identification of these predicted inhibitors of LdPTR1 allowed us to build up a stereoview of the structure of the binding site in relation to activity, affording significant information that should prove useful during the structure-based design of leishmanicidal drugs.     

Midgut pH in different instars of three Aedes mosquito species and the relation between pH and susceptibility of larvae to a nuclear polyhedrosis virus

Midgut pH values were determined for second, third, and fourth instar larvae of Aedes epactius, A. atropalpus, and A. scutellaris. Larvae were fed mixtures of pH indicators and kaolin clay and observed with a stereoscope to determine midgut pH. No significant difference was found in midgut pH values among the three species when the same instar was compared. However, significant differences were found in the length of the high (9.0 to 10.0) pH region when comparing the three instars for each species. Increasing age of treated A. epactius larvae caused a decrease in susceptibility to a nuclear polyhedrosis virus—from 56% infection for 12-hr-old larvae declining to 8% infection for 72-hr larvae. The increased length of the high pH region may relate to decreasing susceptibility of A. epactius to a nuclear polyhedrosis virus as the larvae age.     

Carbohydrate metabolism in several tissues of rainbow trout,Oncorhynchus mykiss,is modified during ovarian recrudescence

• 1.1. Several pathways of carbohydrate metabolism were evaluated in three different tissues—liver, gonad and kidney—of a hatchery-reared population of rainbow trout (Oncorhynchus mykiss) which characterised two different stages of their gonadal maturation, i.e. previtellogenesis and established exogenous vitellogenesis.
• 2.2. A fall in liver glycogen levels was observed during exogenous vitellogenesis. A decrease in activity of the enzymes involved in glycolysis and in the pentose phosphate shunt was also observed, suggesting that at the end of exogenous vitellogenesis the necessity of energy and reducing power has decreased compared to the situation at the onset of this period.
• 3.3. The main changes observed in gonad during vitellogenesis were the decreased activity of glycolysis and the pentose phosphate shunt as well as increased glycogen levels. The stored glycogen should be used later in association with the embryo development.
• 4.4. No major changes were observed in kidney metabolism throughout the vitellogenic process.
• 5.5. Exogenous vitellogenesis in rainbow trout is mainly associated with increased glycogen levels in the gonad and decreased metabolic activity in the liver.

     

Histochemical study of the effects of cadmium uptake on oxidative enzymes of intermediary metabolism in kidney of goldfish (Carassius auratus)

Uptake of cadmium and histochemical changes of oxidative enzymes—succinate dehydrogenase (E.C. 1.3.99.1), cytochrome oxidase (E.C. 1.9.3.1) and glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) —in the kidney of Carassius auratus were examined at different times after 10 ppm Cd²⁺ exposure in very alkaline tap water (346 mg/L HCO₃⁻), which determines a fast and strong precipitation of cadmium as carbonate. A low accumulation of cadmium after 7 days of treatment followed by a gradual increase until the 40th day was observed. The oxidative enzyme activity that resulted was: very weak after 7 days, higher after 14 days and lower after 40 days of treatment. The variations in the enzyme activity along with the accumulation of the metal are interpreted as an increased energy requirement followed by an impairment of mitochondria.     

Association of glutathione S-transferase omega gene polymorphisms with progression of head and neck cancer

This study investigated the influence of glutathione S-transferase omega 1 (GSTO1) and GSTO2 gene polymorphisms on susceptibility and aggressiveness of head and neck squamous cell carcinoma (HNSCC). A case–control study consisting of 300 HNSCC cases and 299 age and sex- matched normal control was performed. Genotyping of GSTO1*A140D and GSTO2*N142D polymorphisms was determined using the polymerase chain reaction—restriction fragment length polymorphism method. Our results revealed that the frequencies of GSTO1 and GSTO2 genotypes were not significantly different between HNSCC cases and controls. No significant differences were found in smoking or drinking status between cases and controls. However, HNSCC individuals with the GSTO1*D140 varient were significantly associated with nodal metastasis (OR?=?0.53, 95?%CI?=?0.31–0.91, P?=?0.020) and advanced pathological stage (OR?=?0.33,95?%CI?=?0.15–0.70, P?=?0.032), while no significant association was observed between GSTO2 genotype and clinicopathological features. Therefore, our findings suggest that the GSTO1*D140 variant genotype in individuals might play a protective role against the aggressiveness of HNSCC.     

Antimalarial activity of azadipeptide nitriles

Azadipeptide nitriles—novel cysteine protease inhibitors—display structure-dependent antimalarial activity against both chloroquine-sensitive and chloroquine-resistant lines of cultured Plasmodium falciparum malaria parasites. Inhibition of parasite’s hemoglobin-degrading cysteine proteases was also investigated, revealing the azadipeptide nitriles as potent inhibitors of falcipain-2 and -3. A correlation between the cysteine protease-inhibiting activity and the antimalarial potential of the compounds was observed. These first generation azadipeptide nitriles represent a promising new class of compounds for antimalarial drug development.     

Risk Factors for Hearing Impairment in Type 1 Diabetes

Objective: Studies have demonstrated that glycated hemoglobin (HbA1c) is a significant predictor of hearing impairment in type 1 diabetes. We identified additional factors associated with hearing impairment in participants with type 1 diabetes from the Diabetes Control and Complications Trial and its observational follow-up, the Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study.Methods: A total of 1,150 DCCT/EDIC participants were recruited for the Hearing Study. A medical history, physical measurements, and a self-administered hearing questionnaire were obtained. Audiometry was performed by study-certified personnel and assessed centrally. Logistic regression models assessed the association of risk factors and comorbidities with speech- and high-frequency hearing impairment.Results: Mean age was 55 ± 7 years, duration of diabetes 34 ± 5 years, and DCCT/EDIC HbA1c 7.9 ± 0.9% (63 mmol/mol). In multivariable models, higher odds of speech-frequency impairment were significantly associated with older age, higher HbA1c, history of noise exposure, male sex, and higher triglycerides. Higher odds of high-frequency impairment were associated with older age, male sex, history of noise exposure, higher skin intrinsic florescence (SIF) as a marker of tissue glycation, higher HbA1c, nonprofessional/nontechnical occupations, sedentary activity, and lower low-density-lipoprotein cholesterol. Among participants who previously completed computed tomography and carotid ultrasonography, coronary artery calcification (CAC) >0 and carotid intima-medial thickness were significantly associated with high-but not speech-frequency impairment.Conclusion: Consistent with previous reports, male sex, age, several metabolic factors, and noise exposure are independently associated with hearing impairment. The association with SIF further emphasizes the importance of glycemia—as a modifiable risk factor—over time. In addition, the macrovascular contribution of CAC is novel and important.Abbreviations: AER = albumin excretion rate; CAC = coronary artery calcification; CVD = cardiovascular disease; DCCT/EDIC = Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications; eGFR = estimated glomerular filtration rate; ETDRS = Early Treatment Diabetic Retinopathy Study; HbA1c = glycated hemoglobin; HDL = high-density lipoprotein; IMT = intima-media thickness; LDL = low-density lipoprotein; NHANES = National Health and Nutrition Examination Survey; OR = odds ratio; SIF = skin intrinsic fluorescence; T1D = type 1 diabetes     

Programmed loss of flight ability in the early adult life of the blowfly Phormia terrae novae as a possible mechanism of intraspecific niche building with respect to the duration of life

• 1.1. The age related flight performance of males and females of the blowfly Phormia terrae novae was studied and the influence of artificially fed glucose determined.
• 2.2. Maximum flight ability—measured as flight distance, duration and initial velocity—was achieved during the first week after emergence, thereafter the flight performance drops precipitously between the 12th and 17th day of adult life.
• 3.3. Males and females, fed and unfed individuals, show no difference in their time course of flight ability.
• 4.4. The possible causes of the programmed loss of flight performance were discussed with regard to histological and enzyme activity changes observed at the same and other species.
• 5.5. The biological significance of this physiological age effect was revealed.

     

Furanocoumarins: Novel topoisomerase I inhibitors from Ruta graveolens L.

Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA–topoisomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxation of plasmid in enzyme–substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme–extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA–enzyme complex was observed after enzyme–extract preincubation. The activity could be assigned to furanocoumarins—psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors.     

Regulation of Rous sarcoma virus RNA-dependent DNA polymerase isoenzymes by in vitro phosphorylation-dephosphorylation

The activity of the αβ form of Rous sarcoma virus RNA-dependent DNA polymerase was stimulated upon treatment with the protein kinase purified from the same virus. This enhancement was observed for both DNA-dependent and RNA-dependent DNA polymerase activities, whereas the RNase H activity associated with the polymerase was not affected. On the other hand, the protein kinase did not induce detectable changes in the activities of the α-polymerase isoenzyme. Treatment with Escherichia coli alkaline phosphatase resulted in a reduction of the polymerase activities of the αβ isoenzyme with no effects on RNase H as well as on the α form of the DNA polymerase. Preincubations of the αβ- and α-oncornaviral polymerase isoenzymes with two other protein kinases—from avian myeloblastosis virus and from beef heart (catalytic subunit)—had no substantial effects on DNA polymerase and RNase H activities of both polymerase isoenzymes. Both α and β subunits of the polymerase isoenzymes were phosphorylated in vitro by all three protein kinases employed, although only the β subunit was shown previously to be phosphorylated in vivo.     

Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague–Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, β-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b₅ reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.