Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by <Emphasis Type="Italic">N</Emphasis>-acetyl cysteine

Induction of apoptosis in cancer cells has become the major focus of anti-cancer therapeutics development. WithaferinA, a major chemical constituent of Withania somnifera, reportedly shows cytotoxicity in a variety of tumor cell lines while its molecular mechanisms of action are not fully understood. We observed that withaferinA primarily induces oxidative stress in human leukemia HL-60 cells and in several other cancer cell lines. The withanolide induced early ROS generation and mitochondrial membrane potential (Δψ_mt) loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei. These events paralleled activation of caspases −9, −3 and PARP cleavage. WA also activated extrinsic pathway significantly as evidenced by time dependent increase in caspase-8 activity vis-à-vis TNFR-1 over expression. WA mediated decreased expression of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Furthermore, withaferinA inhibited DNA binding of NF-κB and caused nuclear cleavage of p65/Rel by activated caspase-3. N-acetyl-cysteine rescued all these events suggesting thereby a pro-oxidant effect of withaferinA. The results of our studies demonstrate that withaferinA induced early ROS generation and mitochondrial dysfunction in cancer cells trigger events responsible for mitochondrial -dependent and -independent apoptosis pathways.     

Efficient biotransformation of herbicide diuron by bacterial strain <Emphasis Type="Italic">Micrococcus</Emphasis> sp. PS-1

A Gram-positive, Micrococcus sp. strain PS-1 capable of utilizing phenylurea herbicide diuron as a sole carbon source at a high concentration (up to 250 ppm) was isolated from diuron storage site by selective enrichment study. The taxonomic characterization with 16S rRNA gene sequencing (1,477 bp) identified PS-1 as a member of Micrococcus sp. It was studied for the degradation of diuron and a range of its analogues (monuron, linuron, monolinuron, chlortoluron and fenuron). The shake flasks experiments demonstrated fast degradation of diuron (up to 96% at 250 ppm within 30 h incubation) with the addition of small quantity (0.01%) of non-ionic detergent. The relative degradation profile by the isolate was in the order of fenuron > monuron > diuron > linuron > monolinuron > chlortoluron. Further, the biochemical characterization of catabolic pathway by spectroscopic and chromatographic techniques demonstrated that the degradation proceeded via formation of dealkylated metabolites to form 3,4-dichloroaniline (3,4-DCA). It was the major metabolite formed, associated with profound increase in degradation kinetics in presence of appropriate additive.     

Anti-tumor therapy with macroencapsulated endostatin producer cells

Background  

Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors.     

3,5,4′-trihydroxy-6,7-dimethoxyflavone 3-glucoside from Sesuvium portulacastrum

A new flavonol glycoside 3,5,4′-trihydroxy-6,7-dimethoxyflavone 3-O-glucoside has been characterized from Sesuvium portulacastrum, together with its aglycone, eupalitin.     

Structural homologies among type I restriction-modification systems.

Structural homologies among different restriction systems of Escherichia coli and several Salmonella species have been investigated by immunological methods using antibodies prepared against two subunits of the E. coli K12 restriction enzyme, and by DNA hybridization experiments using different fragments of the E. coli K12 hsd genes as probes. The results with both techniques show a strong homology between the E. coli K12 and B restriction-modification systems, weaker but nevertheless marked homology between E. coli K12 and the Salmonella systems SB, SP, and SQ and, surprisingly, no homology between the E. coli K12 and A systems.     

In vitro Hormonal Regulation of Laticifer Differentiation in Calotropis procera

Cultures of Calotropis procera were maintained on MS mediumsupplemented with 4·6 µM FAP and 3 µM NAA.Laticifer initials were observed in 2-week-old cultures whichwere converted into matured, branched, non-articulated laticifersin 4 weeks. It was observed that laticifer differentiation increasedwith the age of cultures, from 2·78% (in the second passage)to 15·11% (in the 12th passage). It has been establishedthat laticifer differentiation in vitro is a cytokinin-dependentprocess and among the cytokinins, FAP was more effective thanBA and 2-iP. But the type of auxin and its concentration alsoplay an important role in modifying the effect of cytokinin.Among the different auxins used IAA was more effective for laticiferdifferentiation than IBA and NAA, while 2,4D was inhibitory.Maximum laticifer differentiation (17·01% was observedon MS medium supplemented with 4·6 µM FAP and 1µM IAA.Copyright 1995, 1999 Academic Press Calotropis procera, callus culture, laticifer, differentiation, hormonal regulation     

Both CAG repeats and inverted DNA repeats stimulate spontaneous unequal sister-chromatid exchange in Saccharomyces cerevisiae

Genomic regions containing trinucleotide repeats (TNRs) are highly unstable, as the repeated sequences exhibit a high rate of mutational change, in which they undergo either a contraction or an expansion of repeat numbers. Although expansion of TNRs is associated with several human genetic diseases, the expansion mechanism is poorly understood. Extensive studies in model organisms have indicated that instability of TNRs occurs by several mechanisms, including replication slippage, DNA repair and recombination. In all models, the formation of secondary structures by disease-associated TNRs is a critical step in the mutation process. In this report, we demonstrate that TNRs and inverted repeats (IRs) both of which have the potential to form secondary structures in vivo, increase spontaneous unequal sister-chromatid exchange (SCE) in vegetatively growing yeast cells. Our results also show that TNR-mediated SCE events are independent of RAD50, MRE11 and RAD51, whereas IR-stimulated SCEs are dependent on the RAD52 epistasis-group genes. We propose that many TNR expansion mutations occur by SCE.     

Immunosensors for pesticide analysis: antibody production and sensor development

Immunosensors, a type of affinity biosensor, are based on the binding interactions between an immobilized biomolecule (antibody/antigen) on the electronic transducer surface with the analyte of interest (antigen/antibody), resulting in a detectable signal. The sensor system takes advantage of the high selectivity provided by the molecular recognition characteristic of an antibody, which binds reversibly with a specific antigen. This review article presents the current status of immunosensors, highlighting their potential benefits and limitations for pesticide analysis. The basic criteria for generating specific antibodies against low-molecular-mass pesticides, which are usually nonimmunogenic in nature, are briefly discussed. The article also describes the fundamentals of important transducer technologies and their use in immunosensor development.     

Identification of the First ATRIP–Deficient Patient and Novel Mutations in ATR Define a Clinical Spectrum for ATR–ATRIP Seckel Syndrome

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR–Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR–ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR–ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP–deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR–deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR–ATRIP deficient sub-class of Seckel Syndrome. ATR–ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR–SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR–ATRIP Seckel Syndrome to be defined.