Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus:characterization of catalytic activity

Objective:To study characteristics of phospholipases C(PLCs),their importance for producing microorganisms us well us the potential of their use for industrial purposes.Methods:PLC from Bacillus cereus(B.cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomanas aeruginosa(P.aeruginosa) D183 of Gram-negative ones.Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis.Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method.Results:Maximum activity was at pH 7 and 8 and 40 ℃ for P.aeruginosa PLC,and pH 8-10 and 37 ℃ for B.cereus PLC.Both PLCs were inhibited by Pi at 5 mM or higher,whereas,PLC from B.cereus only was inhibited by EDTA.Activity of P.aeruginosa PLC was not affected by removing Zn~(2+) ions from reaction mixture or their replacement with Ca~(2+),Ba~(2+),Mg~(2+) or Mn~(2+)ions.Vis-a-vis,activity of B.cereus PLC was found to be metal ion dependent PLCs from both isolates were relatively thermostable and showed maximum affinity toward phosphatidylcholine.Sphingomyelin and phosphatidylethanolamine were not good substrates and phosphatidylinositol,phosphatidylserine,phosphatidylglycerol and cardiolipin could be considered nonsubstrates.Conclusions:Human body physiological conditions could favor activity of P.aeruginosa and B.cereus PLCs.These enzymes may participate in phosphate scavenging and virulence of producing isolates but not in autolysis.PLCs from both isolates are potential candidates for industrial use.     

Selective Cytotoxicity and Combined Effects of Camptothecin or Paclitaxel with Sodium-R-Alpha Lipoate on A549 Human Non-Small Cell Lung Cancer Cells

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer and remains the deadliest form of cancer in the United States and worldwide. New therapies are highly sought after to improve outcome. The effect of sodium-R-alpha lipoate on camptothecin- and paclitaxel-induced cytotoxicity was evaluated on A549 NSCLC and BEAS-2B “normal” lung epithelial cells. Combination indices (CI) and dose reduction indices (DRI) were investigated by studying the cytotoxicity of sodium-R-alpha lipoate (0–16 mM), camptothecin (0–25 nM) and paclitaxel (0–0.06 nM) alone and in combination. 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium-bromide (MTT) was used to assess cytotoxicity. The combinational cytotoxic effects of sodium-R-alpha lipoate with camptothecin or paclitaxel were analyzed using a simulation of dose effects (CompuSyn® 3.01). The effects of sodium-R-alpha lipoate on camptothecin- and paclitaxel-induced cytotoxicity varied based on concentrations and treatment times. It was found that sodium-R-alpha lipoate wasn't cytotoxic toward BEAS-2B cells at any of the concentrations tested. For A549 cells, CIs [(additive (CI = 1); synergistic (CI < 1); antagonistic (CI < 1)] were lower and DRIs were higher for the camptothecin/sodium-R-alpha-lipoate combination (CI = ～0.17–1.5; DRI = ～2.2–22.6) than the paclitaxel/sodium-R-alpha-lipoate combination (CI = ～0.8–9.9; DRI = ～0.10–5.8) suggesting that the camptothecin regimen was synergistic and that the addition of sodium-R-alpha lipoate was important for reducing the camptothecin dose and potential for adverse effects.     

Beliefs about hydroxyurea in youth with sickle cell disease

Background

Hydroxyurea reduces complications and improves health-related quality of life (HRQOL) in sickle cell disease (SCD) patients, however adherence remains suboptimal. Understanding patients’ views of hydroxyurea is critical to optimize adherence, particularly in adolescents and young adults (AYA). Study objectives were to assess beliefs about hydroxyurea using the Beliefs about Medicines Questionnaire (BMQ), and to examine the relationship of patients’ beliefs to their hydroxyurea adherence and HRQOL.

Evaluating the size criterion for PI-RADSv2 category 5 upgrade: is 15 mm the best threshold?

Purpose

The purpose of the study was to determine if the ≥ 15 mm threshold currently used to define PIRADS 5 lesions is the optimal size threshold for predicting high likelihood of clinically significant (CS) cancers.

Materials

Three hundred and fifty-eight lesions that may be changed from category 4 to 5 or vice versa on the basis of the size criterion (category 4: n = 288, category 5: n = 70) from 255 patients were evaluated. Kendall’s tau-b statistic accounting for inter-lesion correlation, generalized estimation equation logistic regression, and receiver operating curve analysis evaluated two lesion size-metrics (lesion diameter and relative lesion diameter—defined as lesion diameter/prostate volume) for ability to identify CS (Gleason grade ≥ 3 + 4) cancer at targeted biopsy. Optimal cut-points were identified using the Youden index. Analyses were performed for the whole prostate (WP) and zone-specific sub-cohorts of lesions in the peripheral and transition zones (PZ and TZ).

Results

Lesion diameter showed a modest correlation with Gleason grade (WP: τB = 0.21, p < 0.0001; PZ: τB = 0.13, p = 0.02; TZ: τB = 0.32, p = 0.001), and association with CS cancer detection (WP: AUC = 0.63, PZ: AUC = 0.59, TZ: AUC = 0.74). Empirically derived thresholds (WP: 14 mm, PZ: 13 mm, TZ: 16 mm) performed similarly to the current ≥ 15 mm standard. Lesion relative lesion diameter improved identification of CS cancers compared to lesion diameter alone (WP: τB = 0.30, PZ: τB = 0.24, TZ: τB = 0.42, all p < 0.0001). AUC also improved for WP and PZ lesions (WP: AUC = 0.70, PZ: AUC = 0.68, and TZ: AUC = 0.74).

Conclusions

The current ≥ 15 mm diameter threshold is a reasonable delineator of PI-RADS category 4 and category 5 lesions in the absence of extraprostatic extension to predict CS cancers. Additionally, relative lesion diameter can improve identification of CS cancers and may serve as another option for distinguishing category 4 and 5 lesions.

     

The association of RBC sodium-lithium countertransport (Vmax) with left ventricular mass in African American women

In Caucasian hypertensives and diabetics, increased RBC sodium-lithium countertransporter activity (SLC) is a marker for end-organ complications of vascular disease. A subgroup of African Americans with high Vmax for SLC show strong correlations with dyslipidaemia, insulin resistance, microalbuminuria and higher blood pressure. The purpose of our study was to determine if Vmax in premenopausal African American women correlates with left ventricular mass (LVM) before the onset of clinically diagnosed hypertension. Non-diabetic African American women (n = 35, mean age 31 years) were evaluated for cardiovascular disease risk factors, including anthropometric and blood pressure measurements, oral glucose tolerance test (OGTT), and euglycaemic hyperinsulinaemic clamp for insulin sensitivity. Fasting blood specimens were assayed for SLC activity (Vmax) and lipids. Cardiac structure was determined by 2-D echocardiography. LVM was calculated by the cube root formula and adjusted for height (LVM index). Vmax correlated significantly with average systolic blood pressure (r = 0.45, P = 0.007), diastolic blood pressure (r = 0.48, P = 0.004), mean blood pressure (r = 0.48, P = 0.003) and LVM index (r = 0.40, P = 0.02). Vmax was also associated with fasting insulin (r = 0.39, P = 0.01), the sum of insulin (r = 0.52, P = 0.002), and insulin sensitivity adjusted for fat-free mass (r = -0.55, P = 0.001). There was no statistically significant relationship between Vmax and body mass or lipids. Vmax for SLC correlates with cardiac structure in premenopausal African American women. Vmax is also associated with insulin sensitivity and insulin resistance in this non-diabetic sample. SLC activity may be useful in identifying a subgroup of young African American women with left ventricular hypertrophy and insulin resistance before the onset of clinically diagnosed hypertension and diabetes. Journal of Human Hypertension (2000) 14, 213-219.     

Prevention and treatment of fungal infections in bone marrow transplantation

There has not been as much success in the prevention and treatment of invasive fungal infections, particularly aspergillosis, compared to the prevention and treatment of cytomegalovirus infection and graft-versus-host disease in bone marrow transplant (BMT) recipients. Allogeneic BMT recipients who develop graft-versus-host disease and remain immunosuppressed for long periods are at major risk for development of these infections. Prevention of environmental exposure, antifungal chemoprophylaxis, and attempts at early diagnosis are essential for the reduction of mortality from invasive fungal infections. Chest computerized axial tomography is extremely useful in diagnosing pulmonary aspergillosis. However, microbiologic or histologic identification of infection remains essential. Unfortunately, the response to therapy in BMT recipients remains suboptimal. With the development of the lipid formulations of amphotericin B, the newer azoles, and the echinocandins, safer and more efficacious options have become available. The optimal use of antifungal agents or their combinations remains to be determined.     

Promoter Methylation of LEP and LEPR before and after Bariatric Surgery: A Cross-Sectional Study

IntroductionDNA methylation constitutes one important epigenetic mechanism that regulates gene expression in human cells. With regard to obesity, bariatric surgery-induced weight loss has been associated with promoter methylation changes in several genes. Hyperleptinemia is a characteristic feature of obesity. The underlying regulating mechanisms have not yet been completely elucidated.MethodsWe investigated the methylation of the promoters of the leptin gene (LEP) and the leptin receptor gene (LEPR) as well as leptin expression in pre- and postbariatric surgery patients using a comparative cross-sectional design.ResultsOur results revealed significantly higher LEP promoter methylation patterns in prebariatric surgery patients compared to postoperatively. DNA methylation of the LEPR promoter was significantly higher in the postoperative group. Moreover, we found significantly higher leptin serum levels in patients before the bariatric surgery than afterwards.DiscussionThese findings strengthen the suggestion that there is an association between LEP expression and LEP methylation in obesity. We suggest that the epigenetic profile of LEP might be influenced by leptin serum levels in the form of a regulating feedback mechanism.