Search

Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBAD chi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The aim of the present work was to determine, in a cohort of men and women, whether ferritin and transferrin were associated with glucose metabolism and whether they were predictive of the onset of hyperglycemia (impaired fasting glycemia or type 2 diabetes) after 3 years of follow-up. RESEARCH DESIGN AND METHODS: Among 4,501 subjects from the French Data from an Epidemiological Study on the Insulin Resistance Syndrome (DESIR) cohort, 1,277 subjects (644 men and 633 women) were randomly selected for the analysis of iron biomarkers at baseline and at 3 years. In addition, to determine whether these parameters were relevant to pathological changes, all 231 subjects normoglycemic at baseline and hyperglycemic 3 years later were analyzed for iron biomarkers. RESULTS: At baseline, plasma ferritin concentrations were positively correlated with fasting insulin and fasting glucose in the 1,277 subjects. Although transferrin and ferritin were negatively correlated, transferrin was also positively correlated with fasting insulinemia. Baseline ferritin concentration was an independent predictor of an increase in insulin concentration over a 3-year period (P = 0.002). Further, baseline ferritin and transferrin were independently associated with the onset of hyperglycemia over a 3-year period in the whole population (P < 0.001 for both) and in each sex. CONCLUSIONS: Although negatively correlated, both transferrin and ferritin were positively associated with the onset of abnormalities in glucose metabolism in a prospective study. These results further support the hypothesis of a causative role of iron metabolism in the onset of insulin resistance and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Aims: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. Methods and Results: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. Conclusions: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. Significance and Impact of the Study: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase ‘structure-function’ relation. [ABSTRACT FROM AUTHOR]

Background: C-reactive protein (CRP), a marker of systemic inflammation, is a powerful predictor of adverse cardiovascular events. Respiratory impairment is also associated with cardiovascular risk. Although some studies have found an inverse relationship between lung function and markers of systemic inflammation, only one study has reported a relationship between lung function and CRP levels. In contrast, little is known about the relationship between bronchial hyperresponsiveness (BHR) and systemic inflammation. The association between lung function and CRP and between BHR and CRP has been investigated. Methods: As part of the European Community Respiratory Health Survey follow up study serum CRP levels, forced expiratory volume in 1 second (FEV1), and BHR to methacholine (&ges;20% decrease in FEY1 to <4 mg methacholine) were measured in 259 adults aged 28-56 years free of cardiovascular disease or respiratory infection. Results: Mean (SD) FEV1 (adjusted for age, sex, height, and smoking status) was lower in subjects with a high CRP level (high tertile) (3.29 .(0.44) I/s v 3.50 (0.44) l/s; p<0.01) and BHR was more frequent (41.9% v 24.9%; p=0.005) than in subjects with lower CRP levels (low+middle tertiles). Similar results were obtained when the potential confounding factors were taken into account. Similar patterns of results were found in non-smokers and in non-asthmatic subjects. Conclusions: Increased CRP levels are strongly and independently associated with respiratory impairment and more frequent BHR. These results suggest that both respiratory impairment and BHR are associated with a systemic inflammatory process. [ABSTRACT FROM AUTHOR]

The aims of the study were to evaluate the impact of a written information about treatment related risks in patient receiving blood derived or recombinant medications. Haemophiliac patients and patients with constitutional or acquired immune deficiencies are concerned by this treatment and these information. Our objectives are to evaluate the efficacy of the written information, the knowledge of the patients about these medications and the psychological, emotional impact if these information. The study is based on questionnaires which specified how the patient treat bleeding episodes, their knowledge about viral safety of blood products, the patient's perception of his or her health status and relationship with the physician. Psychological and emotional status are evaluated with the Hospital Anxiety and Depression Scale. The results show the difficulty to inform patients: if the information generate only limited anxiety in patients with haemophilia or immune deficiencies, we observe that the delivery of a written information got a mediocre effect on overall knowledge. We think that this information must be appropriate for patients and be communicated orally within the patient–physician relationship. [ABSTRACT FROM AUTHOR]

Analyzes vaccine-induced immune responses during an ongoing controlled multicenter vaccine trial. Responses elicited by vaccination; Effect of reducing serum hepatitis B virus DNA; Control of viremia during vaccine therapy.

BACKGROUND: A new intravenous lipid emulsion (ILE) prepared from a mixture of soybean and olive oils contains only long-chain triacylglycerols, with a low proportion (20%) of polyunsaturated fatty acids and 60% monounsaturated fatty acids. OBJECTIVE: The goal of this randomized, double-blind clinical trial was to assess in children the efficacy and safety of this new ILE compared with a control group receiving a soybean-oil emulsion. DESIGN: Eighteen children received for 2 mo 24% of nonprotein energy (1.80 g kg (-)(1) d(-)(1)) either as the new ILE or a soybean oil-based emulsion. Assessments were performed on days -30, 0, 30, and 60 and the changes (day 60 - day 0) assessed by analysis of variance. RESULTS: There were no significant differences in triacylglycerol, apolipoproteins A-I and B, or HDL cholesterol between the 2 groups, whereas total and LDL cholesterol were higher in the soybean oil group on day 60. The pattern of 20:4n-6 in erythrocyte membranes did not change significantly, nor did the ratio of 20:3n-9 to 20:4n-6. On day 60, 18:1n-9 was significantly higher in the olive oil group, the ratio of Sigma(n)-6 > C(18) + 18:3n-6 to 18:2n-6 was 2.20 +/- 0.09 in the olive oil group and 1.33 +/- 0.16 in the soybean-oil group, and Sigma(n)-3 > C(18) was 3.83 +/- 0.30 in the olive oil group and 4. 03 +/- 0.33 in the soybean-oil group. The peroxidation index was lower after the olive oil treatment. CONCLUSIONS: The olive oil-based emulsion was well tolerated, maintained a normal EFA status, and may be more suitable for prevention of lipid peroxidation than the soybean-oil-based emulsion. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS)4 such as the superoxide anion, O2•-, the hydroxyl radical, OH•, and H2O2, are constantly produced by metabolic reactions (Halliwell 1994). When they are not removed through the action of biological antioxidants, they are harmful to cells, induce membrane lipid peroxidation and damage proteins and nucleic acids. ROS are counteracted by a wide range of antioxidants synthesized in the cell, including glutathione peroxidase (GSH-PX) and reductase, superoxide dismutase (SOD) and catalase, or supplied by the diet such as vitamins E and C or flavonoids. An excess of ROS, known as oxidative stress, can occur in many circumstances including inflammation, cigarette smoking, acute physical exercise and exposure to high altitude. Effects of altitude on the generation of ROS have been extensively investigated. In rats exposed to hypobaric hypoxia “equivalent to an altitude of 5500 m”, Nakanashi et al. (1995) reported increases in oxidative stress and concentrations of the antioxidant enzymes GSH-PX and SOD. In humans, Vasankari et al. (1997) showed that levels of serum conjugated dienes (CD), a lipid peroxidation marker, were increased during living, training and racing at moderate altitude. Working and racing dogs are often subjected to high levels of metabolic stress that generate ROS. However, the antioxidant/oxidant balance at rest and after exercise for these dogs remains unknown. The search and rescue dog teams such as the canine unit of the Paris Fire Brigade have to work without advance notice or any kind of adaptation period in situations of natural catastrophes (e.g., earthquakes or land slides), very often at high altitude (e.g., Central Asia or South America). Bearing this in mind and with the objective of offering the dogs better preparation for this type of situation, we organized a dedicated expedition to Chile in 1996. Five rescue teams from Carabinieros de Chile and five from the Paris Fire Department participated in the study. They reached the volcano Licancabur, starting from sea level, in less than 24 h. They stayed 3 d at 4800 m, then they climbed the volcano, reaching an altitude of 5980 m on d 4 and returning to sea level at the end of d 5. This study was designed as an open comparative study between two groups of well-trained dogs fed different diets. Approval to use laboratory animals was given by the French Ministry of Agriculture, and the protocol complied with NIH guidelines (NRC 1985). Materials and methods. Diet. Group 1, the French dogs, received 400 g/d of a dry diet designed to cope with stress (Alpicroc, Royal Canin, France). They received also a daily supplement of 1 g of fish oil (Maxepa, Pierre Fabre, Castres, France) and 500 mg of vitamin E (α-tocopherol; Toco 500, Pharma 2000, Buc, France). Group 2, the Chilean dogs, received 600 g/d of a standard diet. Both group of dogs normally lived at sea level. Table 1 contains the composition of the two diets. Work tests. The dogs had to search for and discover two victims hidden by debris in a defined area. Lag times to find the first (T1) and second victim (T2) as well as the time interval (ΔT 5 T2 2 T1) were determined. Cardiac frequency and blood pressure were measured at rest and after exercise. On d 2 of the study, the blood draw and the search for victims took place at sea level; on d 4, they were at an altitude of 4800 m and on d 5 at an altitude of 5980 m. After the search on d 5, the dogs returned to sea level. Biological parameters. Blood samples were collected at the different altitudes 30 min after work. Plasma was separated from RBC by centrifugation at 3000 rev/min and kept frozen at -20°C until analysis. Buffy coat was removed and RBC were washed twice with saline water. Peroxidation resistance index. Washed RBC (20 µL) were submitted to oxidative stress by tert-butyl hydroperoxide (t-BOOH), a specific lipid peroxidative agent that breaks membrane lipids and causes the lysis of cells. We measured induced hemolysis time in seconds. This time is a function of (t-BOOH) concentration and RBC antioxidant capacity. RBC fatty acids. Lipids were extracted from RBC by a mixture of isopropanol chloroform; BHT at 0.05% was added to prevent in vitro peroxidation. RBC phospholipids were separated using a Sep-Pak column. Transesterification of fatty acids was performed by BF3/methanol at 70°C. Fatty acid methyl esters were determined by gas liquid chromatography (GLC) on a polar capillary column. Plasma vitamin E. Plasma vitamin E was measured by reverse- phase HPLC. Statistical analysis. Data are expressed as means ± SD. Data were analyzed by two-factor ANOVA (group and altitude), and significant differences between groups were determined by Student-Neuman-Keuls test using SAS (SAS Institute, Cary, NC). Significance was set at P < 0.05. Results. Effect of altitude on work capacity. At sea level (baseline), cardiac frequency (beats/min) was not different in the two groups at rest or after work (Table 2). It increased slightly with altitude and returned close to baseline values on d 5. Times required to find victims were much longer in Group 2 than Group 1, at baseline and at high altitude. Peroxidation resistance index. RBC hemolysis time was not different in the two groups at baseline, and was significantly decreased at 4800 m, indicating that RBC became less resistant to induced peroxidation (Table 3). RBC from Group 2 were more sensitive than those from Group 1 at 5980 m. Plasma vitamin E. Plasma vitamin E baseline values tended to be higher in Group 1 than in Group 2 due to daily supplementation of Group 1 dogs (Table 4). High altitude induced a marked (significant) increase in plasma tocopherol concentrations in Group 1, whereas there was only a moderate increase in Group 2. RBC fatty acid composition. RBC fatty acid composition was not modified by high altitude and remained constant throughout the study (Table 5). Group 1 dogs had higher levels of (n-3) fatty acids and lower levels of (n-6) fatty acids than Group 2 dogs. These differences reflected dietary intake. Discussion. The acute exposure to high altitude, without adaptation, induces a physiologic stress that reduces oxygen supply to cells. In our study, hypobaric-hypoxic conditions led to enhanced cardiac frequency, at rest and after exercise, and decreased work efficiency. However, these parameters were less affected in dogs supplemented with fish oil and vitamin E than in control dogs. Fish oils have been reported to have a beneficial effect on cardiovascular diseases, presumably via increased membrane fluidity (Driss et al. 1988) and an increased prostacyclin/thromboxane A2 ratio (Schmidt and Dyerberg 1994). Therefore, the better performance of supplemented dogs could be related to (n-3) fatty acid–induced endothelial vasorelaxation and enhanced red cell deformability, both conditions that improve oxygen uptake by muscle cells. Moreover, vitamin E could also have played a protective role against membrane peroxidation in response to high altitude–induced stress. Lipid peroxidation is usually assessed by techniques based on the determination of malondialdehyde (MDA) or CD (Esterbauer and Cheeseman, 1990), which are known to give controversial results because of their poor sensitivity and lower specificity. In our study, red cell membrane polyunsaturated fatty acid (PUFA) levels were used as a biomarker of lipid peroxidation. PUFA levels were not lowered under our experimental conditions, indicating that lipid peroxidation was not initiated or that ROS production had not yet overwhelmed the antioxidant capacities of red cell membranes. Our results are consistent with those of Radak et al. (1997) who did not detect lipid peroxidation in rats exposed to high altitude during a period of 20 d. They reported that tissue amino acids were more sensitive to oxidative modification than PUFA. The peroxidation resistance index test used in our study gives a better understanding of the oxidant/antioxidant balance of red cells. The hemolysis time was not different in the two groups at sea level and was considerably decreased at 4800 m. The altitude lowering effect was more pronounced after heavy exercise such as climbing the volcano. However, in this particular case, resistance to induced peroxidation was greater in dogs fed vitamin E than in those not fed vitamin E. Enzymatic and nonenzymatic antioxidants can be produced as an adaptive response to cellular oxidative stress. Ji (1993) reported increased levels of SOD and GSH, and Pincemail et al. (1998) reported increased plasma vitamin E in response to oxidative stress. The exact mechanism of such an increase has not been elucidated in detail, but redistribution from cellular compartments is highly probable. This notion is supported by the higher peroxidation susceptibility of red cells observed in our study. The clinical manifestations induced by altitude hypoxia were similar to those reported in racing sled dogs (Grandjean and Sept 1991). Only few cases of diarrhea, myoglobinuria and rhabdomyolysis occurred. These disappeared immediately after returning to sea level. Whether they were related to oxidative stress or not requires more investigation. ROS, generated by work during a short-term exposure to high altitude, induced biological and physiologic modifications, which were partly prevented in dogs fed dry diet and supplemented with fish oil and vitamin E. The French dogs performed equally well at sea level and at 4800 m, whereas the Chilean dogs did not perform as well at the higher altitude as at sea level. The performance of both groups of dogs decreased at the higher altitude. Despite minor or severe pathologic changes, these supplemented dogs were able to perform search and rescue tasks without further adaptation. [ABSTRACT FROM AUTHOR]

Hepatitis B virus infection is responsible for both morbidity and mortality in kidney transplant recipients. Adenine arabinoside 5'-monophosphate (ARA-AMP), a synthetic punne nucleotide with anti-viral activity, leads to a sustained interruption of HBV replication in approximately 40% of immunocompetent patients. We report the results of a pilot study using ARA-AMP to treat HBV-related chronic active hepat itis in kidney transplant recipients. Ten patients (2 females and 8 males, mean age 44 years, mean time post-transplantation 163 months) received a 28-day course of ARA-AMP intramuscu larly: 5 mg/kg twice daily for the first 5 days during hospitalization and subsequently 5 mg/kg once daily at home for the remaining 23 days. Mean follow-up was 18 months, ranging from 7 to 28 months. All patients but one had biopsy-proven chronic hepatitis, including five cases of cirrhosis. All patients had been chronic HBs Ag carriers for more than 1 year and had active replication as assessed by the presence of serum HBV DNA (mean titre, 270 pg/ml, ranging from 12 to 997 pg/mi, Genostics method). HBe Ag was present in 7 of the 10 patients. Pretreatment creatinine was normal. In four of the 10 patients, HBV DNA became undetectable respectively 1, 1, 5, and 11 months after beginning ARA-AMP. In five patients, HBV DNA decreased during ARA-AMP therapy but subsequently increased although no change was noted during the follow-up period. In two partial responders, a second 4-week course of ARA-AMP was given 8 and 9 months after the first course, and resulted in a sustained HBV DNA loss 1 and 4 months later: HBV DNA reappeared 3 months later in the former. Two of the responders relapsed with reappearance of HBV replication 2 and 22 months after treatment: HBV DNA disappeared 1 year later in the former. ARA-AMP was well tolerated in nine patients, without side-effects in five and mild muscular pain in four, although one patient com plained of severe but reversible peripheral neuropathy Renal function remained stable in all patients during follow-up. Our results suggest that a 4-week course of ARA AMP interrupts HBV replication in 40% of kidney recipients with chronic active hepatitis. Repetition of the treatment may enhance the efficacy of ARA-AMP in partial responders. Efficacy and tolerance seem therefore to be at least comparable to what has been previously reported in immunocompetent patients. [ABSTRACT FROM PUBLISHER]

Objective: To determine whether hepatitis C virus is involved in hepatitis-associated aplastic anemia.Design: Retrospective analysis.Setting: Bone marrow transplantation unit.Patients: One hundred and eighteen patients with severe aplastic anemia, including 19 with hepatitis-associated aplasia, 61 with aplastic anemia of undetermined cause, and 38 with aplastic anemia related to an inherited syndrome or an acquired etiology.Measurements and Main Results: There was no statistically significant difference in antihepatitis C virus antibodies between hepatitis-related aplastic anemia (15.8%; 95% CI, 4% to 36%) and aplasia of unknown (9.8%; CI, 5% to 22%) or known (7.9%; CI, 2% to 22%) cause. The antihepatitis C virus levels did not differ according to the cause of aplastic anemia. There was no relation between hepatitis C and hepatitis B virus serologies, regardless of cause.Conclusions: Hepatitis C virus is not a frequent cause of non-A, non-B hepatitis-associated aplastic anemia. Either a non-A, non-B, non-C hepatitis virus is involved in non-A, non-B hepatitis-related aplasia or hepatitis C virus prevalence is underestimated in patients with hepatitis-related aplasia, possibly as a result of immunologic defects. [ABSTRACT FROM AUTHOR]