A 27-amino-acid synthetic peptide corresponding to the NH2-terminal zinc-binding domain of endostatin is responsible for its antitumor activity

The first recombinant endostatin that elicited strong antitumor activity was expressed in Escherichia coli and administered as a suspension. Under these conditions, the protein retained its full antiangiogenic activity. Lack of requirement for a folded structure prompted us to investigate antitumor properties of synthetic peptides corresponding to different regions of endostatin. Here, we show that the entire antitumor, antimigration, and antipermeability activities of endostatin are mimicked by a 27-amino-acid peptide corresponding to the NH2-terminal domain of endostatin. This peptide contains three histidines that are responsible for zinc binding. Mutations of the zinc-binding histidines abolished its antitumor and antimigration activities, but not antipermeability properties.     

Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH

Biochemical estimation of NADH concentration is a useful method for monitoring cellular metabolism, because the NADH/NAD+ reduction-oxidation pair is crucial for electron transfer in the mitochondrial electron chain. In this article, we present a novel method for deriving functional maps of intracellular reduction-oxidation ratio in vivo via measurement of the fluorescence lifetimes and the ratio of free and protein-bound NADH using two-photon fluorescence lifetime imaging (FLIM). Through systematic analysis of FLIM data from the control cells, it was observed that there is a statistically significant decrease in the fluorescence lifetime of both free and protein-bound NADH and the contribution of protein-bound NADH as cells progress from an early to logarithmic to confluent phase. Potassium cyanide (KCN) treatment and serum starvation of cells yielded similar changes. There was a statistically significant decrease in the fluorescence lifetime of protein-bound and free NADH at the early and logarithmic phase of the growth curve and a statistically significant decrease in the contribution of protein-bound NADH relative to that observed in the control cells at all three phases of the growth curve. The imposed perturbations (confluence, serum starvation, and KCN treatment) are all expected to result in an increase in the ratio of NADH/NAD+. Our studies suggest that the fluorescence lifetime of both the free and the protein-bound components of NADH and the ratio of free to protein-bound NADH is related to changes in the NADH/NAD+ ratio.     

Evaluation of leaf extracts of Vitex negundo L. (Family: Verbenaceae) against larvae of Culex tritaeniorhynchus and repellent activity on adult vector mosquitoes

Petroleum ether (60-80 degrees C) extracts of the leaves of Vitex negundo (Verbenaceae) were evaluated for larvicidal activity against larval stages of Culex tritaeniorhynchus in the laboratory. Larvae of C. tritaeniorhynchus were found more susceptible, with LC(50) and LC(90) values of 2.4883 and 5.1883 mg/l, respectively. Human volunteers wearing special terricot (68:32) fabrics, in the form of armbands, anklets, headbands, collar, and shoulder and pocket strips impregnated with V. negundo leaf extract were used, to test their repellent efficacy at two concentrations viz., 1.5 and 2.0 mg/cm(2) under the field conditions. At 1.5-mg/cm(2) concentration, more efficacies were found and 6-h complete protection against mosquito bites was provided. Complete protections for 8 h were found at 2.0 mg/cm(2) against mosquitoes bites. These results clearly reveal that the V. negundo leaf extract served as a potential larvicidal agent against Japanese encephalitis vector C. tritaeniorhynchus and additionally acted as a promising repellent against various adult vector mosquitoes.     

A propensity score–matched analysis indicates screening for asymptomatic coronary artery disease does not predict cardiac events in kidney transplant recipients

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CTLA4Ig prevents initiation but not evolution of anti-phospholipid syndrome in NZW/BXSB mice

NZW "x" BXSB F1 mice develop SLE that is associated with an anti-phospholipid syndrome characterized by anti-cardiolipin antibodies, thrombocytopenia and small coronary artery thrombosis. This syndrome is immune mediated and, dependent, on CD4+T cells. To determine whether disease in these mice can be treated with blockade of T cell costimulation we treated them with the CD28 antagonist CTLA4Ig at 9 or 12 weeks of age. CTLA4Ig completely prevented both SLE nephritis and myocardial infarcts if it was given at 9 weeks of age, before anti-cardiolipin antibodies could be detected in the serum and prevented both B cell expansion and activation and the development of peripheral monocytosis. If treatment was delayed until 12 weeks of age after cardiolipin antibodies had arisen but before the onset of clinical disease, CTLA4Ig had very little effect on disease progression. These findings indicate that CD4+T cell activation through CD28 is critical for disease initiation in this model but plays little role in disease progression or tissue damage. These findings have relevance to the treatment of anti-phospholipid syndrome in humans.     

Uptake of 2-NBDG as a method to monitor therapy response in breast cancer cell lines

This study quantifies uptake of a fluorescent glucose analog, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG), in a large panel of breast cancer cells and demonstrates potential to monitor changes in glycolysis caused by anticancer and endocrine therapies. Expressions of glucose transporter (GLUT 1) and hexokinase (HK I), which phosphorylates 2-NBDG, were measured via western blot in two normal mammary epithelial and eight breast cancer cell lines of varying biological subtype. Fluorescence intensity of each cell line labeled with 100 μM 2-NBDG for 20 min or unlabeled control was quantified. A subset of cancer cells was treated with anticancer and endocrine therapies, and 2-NBDG fluorescence changes were measured. Expression of GLUT 1 was necessary for uptake of 2-NBDG, as demonstrated by lack of 2-NBDG uptake in normal human mammary epithelial cells (HMECs). GLUT 1 expression and 2-NBDG uptake was ubiquitous among all breast cancer lines. Reduction and stimulation of 2-NBDG uptake was demonstrated by perturbation with anticancer agents, lonidamine (LND), and α-cyano-hydroxycinnamate (α-Cinn), respectively. LND directly inhibits HK and significantly reduced 2-NBDG fluorescence in a subset of two breast cancer cell lines. Conversely, when cells were treated with α-Cinn, a drug used to increase glycolysis, 2-NBDG uptake was increased. Furthermore, tamoxifen (tam), a common endocrine therapy, was administered to estrogen receptor positive and negative (ER+/−) breast cells and demonstrated a decreased 2-NBDG uptake in ER+ cells, reflecting a decrease in glycolysis. Results indicate that 2-NBDG uptake can be used to measure changes in glycolysis and has potential for use in early drug development.     

Concomitant autoimmunity in myasthenia gravis--lack of association with IgA deficiency

A marked increase in concomitant autoimmune diseases has previously been noted in patients with myasthenia gravis (MG). We show that these diseases occur both before and after the onset of MG and that the process is not influenced by thymectomy. IgA deficiency (IgAD), which is strongly associated with the same HLA haplotype as early onset MG, has recently been suggested to be an autoimmune disease. However, there was no increase in the prevalence of IgAD in a large cohort of Swedish MG patients.     

Effect of site-directed asparagine to isoleucine substitutions at the N-linked E1 glycosylation sites on rubella virus viability.

The role of three N-linked glycosylation sites in rubella virus (RV) E1 protein on virion release was analyzed by transfecting Vero 76 cells with infectious RV RNA (Robo302WT) containing isoleucine substitutions at N76, N177, and N209 (individually and in combinations). RV RNAs were detected and found to retain substitutions in the transfected cells, but RV capsid indicative of infection was undetectable, except for in Robo302WT and Robo302-N177I transfected cells. Only culture supernatants of Robo302WT and Robo302-N177I RNA transfected cells were positive for RV, suggestive of the virion release into the culture medium. Further, detection of intracellular RV E1 and newly released virion-associated E1 was possible only from cells previously incubated with Robo302-N177I and Robo302WT culture supernatants, suggesting that N177I substituted virus retained infectivity. These results suggest that while glycosylation at N177 is not critical, N76I and N209I mutations are lethal to RV viability.