Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1

Fensulfothion (O,O-diethyl O-[4-(methylsulfinyl)phenyl]phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide. Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp. strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon. P. alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid. The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules. However, utilization of the phosphoric acid ester and ethanol by P. alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester. The pathway of degradation of fensulfothion by P. alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.     

Book review of evolutionary genetics: Concepts,analysis, and practice

Recent technological advances have expanded and increased the resolution of studies in evolutionary biology, creating a need for a modern textbook that highlights the latest developments in the field. Evolutionary Genetics: Concepts, Analysis, and Practice, by Glenn‐Peter Sætre and Mark Ravinet (2019), as well as the book's accompanying online tutorials, provide a clear, up‐to‐date, and enjoyable introduction to evolutionary biology and genetics that explains fundamental evolutionary concepts, illustrates recent exciting findings, and offers hands‐on experience in analysing and interpreting genomic data. The book's accessible nature and emphasis on developing practical skills make it a valuable resource for undergraduate courses on evolutionary biology.     

Identification of a novel 3,5-disubstituted pyridine as a potent, selective, and orally active inhibitor of Akt1 kinase

Based on lead compounds 2 and 3 a series of 3,5-disubstituted pyridines have been designed and evaluated for inhibition of AKT/PKB. Modifications at the 3 position of the pyridine ring led to a number of potent compounds with improved physical properties, resulting in the identification of 11g as a promising, orally active Akt inhibitor. The synthesis, structure-activity relationship studies, and pharmacokinetic data are presented in this paper.     

Synthesis and structure-activity relationship of 3,4'-bispyridinylethylenes: discovery of a potent 3-isoquinolinylpyridine inhibitor of protein kinase B (PKB/Akt) for the treatment of cancer

Structure-based design and synthesis of the 3,4'-bispyridinylethylene series led to the discovery of 3-isoquinolinylpyridine 13a as a potent PKB/Akt inhibitor with an IC(50) of 1.3nM against Akt1. Compound 13a shows excellent selectivity against distinct families of kinases such as tyrosine kinases and CAMK, and displays poor to marginal selectivity against closely related kinases in the AGC and CMGC families. Moreover, 13a demonstrates potent cellular activity comparable to staurosporine, with IC(50) values of 0.42 and 0.59microM against MiaPaCa-2 and the Akt1 overexpressing FL5.12-Akt1, respectively. Inhibition of phosphorylation of the Akt downstream target GSK3 was also observed in FL5.12-Akt1 cells with an EC(50) of 1.5microM. The X-ray structures of 12 and 13a in complex with PKA in the ATP-binding site were determined.     

Electrochemical assay of the nitrate and nitrite reductase activities of Rhizobium japonicum

This work describes an electrochemical method for the determination of the nitrate and nitrite reductase activities of Rhizobium japonicum. The advantage of the method lies in the use of whole cells for the analysis and we earlier developed this protocol for the assay of NO. The results obtained are comparable to the spectrophotometric Griess assay. As the method is based on electrochemical reduction, the commonly interfering biological components like ascorbic acid, uric acid, dopamine, etc., will not interfere with the analysis. This method can be extended to the fabrication of biosensors for nitrate and nitrite using the same principle.     

Weight Loss Reduces Liver Fat and Improves Hepatic and Skeletal Muscle Insulin Sensitivity in Obese Adolescents

Obesity in adolescents is associated with metabolic risk factors for type 2 diabetes, particularly insulin resistance and excessive accumulation of intrahepatic triglyceride (IHTG). The purpose of this study was to evaluate the effect of moderate weight loss on IHTG content and insulin sensitivity in obese adolescents who had normal oral glucose tolerance. Insulin sensitivity, assessed by using the hyperinsulinemic–euglycemic clamp technique in conjunction with stable isotopically labeled tracer infusion, and IHTG content, assessed by using magnetic resonance spectroscopy, were evaluated in eight obese adolescents (BMI ≥95th percentile for age and sex; age 15.3 ± 0.6 years) before and after moderate diet‐induced weight loss (8.2 ± 2.0% of initial body weight). Weight loss caused a 61.6 ± 8.5% decrease in IHTG content (P = 0.01), and improved both hepatic (56 ± 18% increase in hepatic insulin sensitivity index, P = 0.01) and skeletal muscle (97 ± 45% increase in insulin‐mediated glucose disposal, P = 0.01) insulin sensitivity. Moderate diet‐induced weight loss decreases IHTG content and improves insulin sensitivity in the liver and skeletal muscle in obese adolescents who have normal glucose tolerance. These results support the benefits of weight loss therapy in obese adolescents who do not have evidence of obesity‐related metabolic complications during a standard medical evaluation.     

Acclimatization of tissue cultured plantlets: from laboratory to land

The ultimate success of micropropagation on a commercial scale depends on the ability to transfer plants out of culture on a large scale, at low cost and with high survival rates. During field transfer the in vitro grown plantlets are unable to compete with soil microbes and to cope with the environmental conditions. The in vitro culture conditions result in the plantlets with altered morphology, anatomy and physiology. In order to increase growth and reduce mortality in plantlets at the acclimatisation stage, efforts are focused on the control of both physical and chemical environment and biohardening of micropropagated plantlets. This review describes the abiotic and biotic stresses and current developing methods for the acclimatization of microshoots.     

Immunoproteomic Identification of Human T Cell Antigens of Mycobacterium tuberculosis That Differentiate Healthy Contacts from Tuberculosis Patients

Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis. To identify the antigens of M. tuberculosis that differentiated between tuberculosis (TB) patients and healthy contacts based on T cell reactivity, the culture filtrate of in vitro grown M. tuberculosis was fractionated by two-dimensional liquid phase electrophoresis and tested for the ability to stimulate T cells in a whole blood assay. This approach separated the culture filtrate into 350 fractions with sufficient protein quantity (at least 200 μg of protein) for mass spectrometry and immunological analyses. High levels of interferon-γ (IFN-γ) secretion were induced by 105 fractions in healthy contacts compared with TB patients (p < 0.05). Most interesting was the identification of 10 fractions that specifically induced strong IFN-γ production in the healthy contact population but not in TB patients. Other immunological measurements showed 42 fractions that induced significant lymphocyte proliferative responses in the healthy contact group compared with the TB patients. The tumor necrosis factor-α response for most of the fractions did not significantly differ in the tested groups, and the interleukin-4 response was below the detectable range for all fractions and both study groups. Proteomic characterization of the 105 fractions that induced a significant IFN-γ response in the healthy contacts compared with the TB patients led to the identification of 59 proteins of which 24 represented potentially novel T cell antigens. Likewise, the protein identification in the 10 healthy “contact-specific fractions” revealed 16 proteins that are key candidates as vaccine or diagnostic targets.Tuberculosis (TB)1 is a major health problem throughout the world. A recent World Health Organization report shows that TB has been increasing at a rate of 1% per year, and an estimated 9.2 million new cases arise each year (1). Although TB is preventable, there has been an increase in its incidence in recent years. Re-emergence of TB is mainly due to its association with human immunodeficiency virus infection (2) and also due to the occurrence of multidrug-resistant strains of the causative agent, Mycobacterium tuberculosis (3).Vaccination of general population is cost effective and represents one of the best biological measures for disease control. The current vaccine against tuberculosis, Bacille Calmette-Guérin (BCG), has been administered to more people than any other vaccine. The side effects of BCG are tolerable, and it prevents miliary and meningeal tuberculosis in young children. In striking contrast, it affords limited and highly variable protection (0–80%) against pulmonary TB (4). Thus, BCG does not seem to be a satisfactory vaccine (5, 6) and necessitates exploration of newer strategies to improve BCG or to develop a more effective vaccine.One of the potential strategies for the development of an improved TB vaccine involves the use of the proteins secreted by M. tuberculosis during growth. There is evidence that proteins actively secreted by M. tuberculosis during growth induce cell-mediated immune responses by causing expansion of specific interferon-γ (IFN-γ)-producing T lymphocytes that are capable of recognizing and exerting antimicrobial effects against infected macrophages (7). The importance of IFN-γ pathways in host defense against M. tuberculosis was clarified by experimental studies on IFN-γ knock-out mice as well as the identification and characterization of humans with mutations in IFN-γ receptor (8, 9).Several studies have been carried out to define the secreted proteome of M. tuberculosis. The earliest study aimed at the identification of mycobacterial culture filtrate proteins, using chromatography and N-terminal sequencing to identify eight culture filtrate proteins (10). Later, many studies used two-dimensional (2D) PAGE combined with sensitive mass spectrometric methods for identification of proteins. The above mentioned approaches have identified nearly 300 culture filtrate proteins (11–13).Identification of T cell antigens in a complex mixture was first done by a T cell Western blot method (14). Later, two-dimensional separation methods were used that involved protein separation by either IEF (15) or chromatography (16) in the first dimension and preparative SDS-PAGE followed by whole gel elution (17) in the second dimension. Mouse T cell antigens of M. tuberculosis were identified using this method (15). Mycobacterial antigens that induce an immune response in healthy household contacts and treated TB patients were also mapped using this approach (16).In the present study, 2D liquid phase electrophoresis (LPE) along with an in vitro IFN-γ assay and LC-MS/MS were used to identify potential human T cell antigens. Systematic screening of the M. tuberculosis culture filtrate (CF) proteome and comparative evaluation of cellular immune responses between TB patients and healthy contacts led to the identification of 59 proteins in the most immunogenic 2D LPE fractions. Twenty-four potentially novel T cell antigens were identified, and 16 proteins were identified in 10 2D LPE fractions that differentiated healthy contacts from TB patients based on IFN-γ responses.     

Accumulation of the common mitochondrial DNA deletion induced by ionizing radiation

Point mutations and deletions in mitochondrial DNA (mtDNA) accumulate as a result of oxidative stress, including ionizing radiation. As a result, dysfunctional mitochondria suffer from a decline in oxidative phosphorylation and increased release of superoxides and other reactive oxygen species (ROS). Through this mechanism, mitochondria have been implicated in a host of degenerative diseases. Associated with this type of damage, and serving as a marker of total mtDNA mutations and deletions, the accumulation of a specific 4977-bp deletion, known as the common deletion (Delta-mtDNA(4977)), takes place. The Delta-mtDNA(4977) has been reported to increase with age and during the progression of mitochondrial degeneration. The purpose of this study was to investigate whether ionizing radiation induces the formation of the common deletion in a variety of human cell lines and to determine if it is associated with cellular radiosensitivity. Cell lines used included eight normal human skin fibroblast lines, a radiosensitive non-transformed and an SV40 transformed ataxia telangiectasia (AT) homozygous fibroblast line, a Kearns Sayre Syndrome (KSS) line known to contain mitochondrial deletions, and five human tumor lines. The Delta-mtDNA(4977) was assessed by polymerase chain reaction (PCR). Significant levels of Delta-mtDNA(4977) accumulated 72 h after irradiation doses of 2, 5, 10 or 20 Gy in all of the normal lines with lower response in tumor cell lines, but the absolute amounts of the induced deletion were variable. There was no consistent dose-response relationship. SV40 transformed and non-transformed AT cell lines both showed significant induction of the deletion. However, the five tumor cell lines showed only a modest induction of the deletion, including the one line that was deficient in DNA damage repair. No relationship was found between sensitivity to radiation-induced deletions and sensitivity to cell killing by radiation.