Mutational analysis of the hMSH6 gene in familial and early-onset colorectal and endometrial cancer in Israeli patients

Familial colorectal cancer (CRC) is noted in about 15% of incident CRC cases, and at times is hallmarked by an age at diagnosis less than 50 years. Familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC) account for about 40% of familial cases. Thus, the majority of familial and early-onset CRC remain genetically elusive. Similarly, the majority of familial and early onset endometrial cancer (EC), the most prevalent extracolonic tumor in HNPCC, are genetically undefined. An attractive candidate is the hMSH6 gene. Israeli patients with early onset (age under 50 years) (n = 44) and familial nonsyndromic (n = 23) CRC, and women with familial clustering of EC or CRC (n = 12), and those diagnosed with EC at, or under, the age of 50 years (n = 5) were genotyped for germ-line mutations within the hMSH6 gene. Exon-specific PCR was followed by denaturing gradient gel electrophoresis (DGGE) analysis, complemented by DNA sequencing of abnormally migrating fragments. No patients displayed a truncating mutation, and 1 CRC patient harbored a novel missense mutation (V878A). In addition, 6 previously described polymorphisms were detected. In conclusion, mutations in the hMSH6 gene occur uncommonly in Israeli patients with familial and early-onset CRC and EC.     

Light-driven movements of the trifoliate leaves of bean (Phaseolus vulgaris L.). Activity of blue light and red light

The puhrinule of the terminal leaflet in the trifoliate leafof bean (Phaseolus vulgaris L.) responds to its continuous exposureto directional overhead light by increasing the elevation ofits attached lamina. Blue light drives this response, but theeffectiveness of unfiltered white light equalled, or exceededthe effectiveness of blue light at equivalent irradiances (200–800µmol m^–2 s^–1). Adding red light to blue lightenhanced the initial rate of response, and increased its steady-state.These effects of red light increased with irradiance. Adding200–800 µmol m^–2 s^–1 red light to 50µmol m^–2 s^–1 blue light was more effectivein enhancing the initial rate of response than adding blue lightat equivalent irradiances, whereas added blue light was moreeffective in increasing the steady-state. In continuous bluelight the initial (maximal) angular velocity of laminar reorientation,as well as the eventual steady-state of the response increasedlinearly with log PFD (up to 800 µmol m^–2s^–2).Laminar reorientation also took place in continuous red lightby itself, and the angular velocity of the response was initiallyhigh, then became considerably slower. The initial phase wasapparently independent of irradiance up to PFD 100 µmolm^–2 s^–1 but increased progressively with log PFDat higher irradiances. During the second phase, the rate increasedlinearly with irradiance, becoming saturated at PFD 200 µmolm^–2 s^–1. Key words: Phaseolus, phototropism, pulvinule, spectral dependence, trifoliate leaf movements     

Pathogenesis of acute viral disease induced in fish by carp interstitial nephritis and gill necrosis virus

A lethal disease of koi and common carp (species Cyprinus carpio) has afflicted many fish farms worldwide since 1998, causing severe financial losses. Morbidity and mortality are restricted to common carp and koi and appear in spring and autumn, when water temperatures are 18 to 28 degrees C. We have isolated the virus causing the disease from sick fish, propagated it in koi fin cell culture, and shown that virus from a single clone causes lethal disease in carp and koi upon infection. Intraperitoneal virus injection or bathing the fish in virus-containing water kills 85 to 100% of the fish within 7 to 21 days. This virus is similar to the previously reported koi herpesvirus; however, it has characteristics inconsistent with the herpesvirus family, and thus we have called it carp interstitial nephritis and gill necrosis virus. We examined the pathobiology of this disease in carp by using immunohistochemistry and PCR. We found large amounts of the virus in the kidneys of sick fish and smaller amounts in liver and brain. A rapid increase in the viral load in the kidneys was detected by using both immunofluorescence and semiquantitative PCR. Histological analyses of fish at various times after infection revealed signs of interstitial nephritis as early as 2 days postinfection, which increased in severity up to 10 days postinfection. There was severe gill disease evidenced by loss of villi with accompanying inflammation in the gill rakers. Minimal focal inflammation was noted in livers and brains. This report describes the etiology and pathology of a recently described viral agent in fish.     

Lipid peroxidation in the presence of albumin, inhibitory and prooxidative effects

Oxidative modifications of LDL are involved in atherogenesis. Previously we have developed a simple assay to evaluate the susceptibility of lipids to copper-induced peroxidation in the relatively natural milieu of unfractionated serum in the presence of excess citrate. Based on our previous results we have proposed that the inducer of peroxidation in our optimized assay is a copper-citrate complex. Recent investigations indicate that under certain conditions a copper-albumin complex may induce peroxidation of ascorbate. Two different complexes may be formed in albumin-containing systems (e.g. serum) namely 1:1 and 2:1 copper-albumin complexes. The aim of the present work was to evaluate the possibility that at least one of these complexes may be responsible for the induction of peroxidation of lipids in lipidic systems containing copper and albumin, including our optimized assay. Towards this end, we have investigated the dependence of copper-induced peroxidation on the concentration of added albumin in lipidic systems in the absence and presence of citrate. In all the systems investigated in this study (PLPC liposomes, LDL, HDL and mixtures of HDL and LDL) we found that at low concentrations of free copper (e.g. in the presence of excess citrate) the 2:1 copper-albumin complex is redox-active and that this complex is the major contributor to the initiation of lipid peroxidation in these systems and in our optimized assay. The possible relevance of the induction of peroxidation in vivo by the latter complex has yet to be studied. *This work was performed in partial fulfillment of the requirements for a Ph.D. degree of Dorit Samocha-Bonet, Sackler Faculty of Medicine, Tel-Aviv University, Israel.     

Increased T-type Ca2+ channel activity as a determinant of cellular toxicity in neuronal cell lines expressing polyglutamine-expanded human androgen receptors

We have analyzed Ca²⁺ currents in two neuroblastoma-motor neuron hybrid cell lines that expressed normal or glutamine-expanded human androgen receptors (polyGln-expanded AR) either transiently or stably. The cell lines express a unique, low-threshold, transient type of Ca²⁺ current that is not affected by L-type Ca²⁺ channel blocker (PN 200-110), N-type Ca²⁺ channel blocker (-conotoxin GVIA) or P-type Ca²⁺ channel blocker (Agatoxin IVA) but is blocked by either Cd²⁺ or Ni²⁺. This pharmacological profile most closely resembles that of T-type Ca²⁺ channels [1-3]. Exposure to androgen had no effect on control cell lines or cells transfected with normal AR but significantly changed the steady-state activation in cells transfected with expanded AR. The observed negative shift in steady-state activation results in a large increase in the T-type Ca²⁺ channel window current. We suggest that Ca²⁺ overload due to abnormal voltage-dependence of transient Ca²⁺ channel activation may contribute to motor neuron toxicity in spinobulbar muscular atrophy (SBMA). This hypothesis is supported by the additional finding that, at concentrations that selectively block T-type Ca²⁺ channel currents, Ni²⁺ significantly reduced cell death in cell lines transfected with polyGln-expanded AR.     

Aedes aegypti TMOF modulates ecdysteroid production by prothoracic glands of the gypsy moth, Lymantria dispar

Trypsin modulating oostatic factor (TMOF) is a decapeptide that inhibits the biosynthesis of trypsin-like enzymes in the midgut of several insect species and, as such, serves as a dipteran oostatic hormone. In vitro incubation of lepidopteran prothoracic glands with Aedes aegypti TMOF revealed that this decapeptide, in the presence of brain extract, modulates ecdysteroid production. The modulatory effect was highly dependent on both the concentration of TMOF and brain extract. Typically, TMOF was stimulatory in the presence of lower concentrations of Lymantria dispar brain extract (0.01 and 0. 025 brain equivalent), and either neutral or inhibitory at higher concentrations (0.25, 0.5, and 1.0 brain equivalent) of extract. In the presence of European corn borer (Ostrinia nubilalis) brain extract, TMOF also exhibited modulatory effects, effects that again were dependent on the concentrations of both brain extract and TMOF present in the incubation medium. At 1.5 brain equivalents, TMOF was inhibitory at all but the highest concentration tested (5x10(-6) M), at 1.0 brain equivalent, TMOF was stimulatory at 10(-6) M and at 0. 5 brain equivalents, TMOF did not significantly affect PTG synthesis of ecdysteroids. Results suggest the presence of a modulatory peptide(s), which fine tunes the synthesis and release of ecdysteroids by PTGs in accordance with the insect's developmental/physiological requirements.     

TLR-3 is Present in Human Adipocytes,but Its Signalling is Not Required for Obesity-Induced Inflammation in Adipose Tissue In Vivo

Innate immunity plays a pivotal role in obesity-induced low-grade inflammation originating from adipose tissue. Key receptors of the innate immune system including Toll-like receptors-2 and -4 (TLRs) are triggered by nutrient excess to promote inflammation. The role of other TLRs in this process is largely unknown. In addition to double-stranded viral mRNA, TLR-3 can also recognize mRNA from dying endogenous cells, a process that is frequently observed within obese adipose tissue. Here, we identified profound expression of TLR-3 in adipocytes and investigated its role during diet-induced obesity. Human adipose tissue biopsies (n=80) and an adipocyte cell-line were used to study TLR-3 expression and function. TLR-3-/- and WT animals were exposed to a high-fat diet (HFD) for 16 weeks to induce obesity. Expression of TLR-3 was significantly higher in human adipocytes compared to the non-adipocyte cells part of the adipose tissue. In vitro, TLR-3 expression was induced during differentiation of adipocytes and stimulation of the receptor led to elevated expression of pro-inflammatory cytokines. In vivo, TLR-3 deficiency did not significantly influence HFD-induced obesity, insulin sensitivity or inflammation. In humans, TLR-3 expression in adipose tissue did not correlate with BMI or insulin sensitivity (HOMA-IR). Together, our results demonstrate that TLR-3 is highly expressed in adipocytes and functionally active. However, TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance.     

Dysregulated miRNA biogenesis downstream of cellular stress and ALS‐causing mutations: a new mechanism for ALS

Interest in RNA dysfunction in amyotrophic lateral sclerosis (ALS) recently aroused upon discovering causative mutations in RNA‐binding protein genes. Here, we show that extensive down‐regulation of miRNA levels is a common molecular denominator for multiple forms of human ALS. We further demonstrate that pathogenic ALS‐causing mutations are sufficient to inhibit miRNA biogenesis at the Dicing step. Abnormalities of the stress response are involved in the pathogenesis of neurodegeneration, including ALS. Accordingly, we describe a novel mechanism for modulating microRNA biogenesis under stress, involving stress granule formation and re‐organization of DICER and AGO2 protein interactions with their partners. In line with this observation, enhancing DICER activity by a small molecule, enoxacin, is beneficial for neuromuscular function in two independent ALS mouse models. Characterizing miRNA biogenesis downstream of the stress response ties seemingly disparate pathways in neurodegeneration and further suggests that DICER and miRNAs affect neuronal integrity and are possible therapeutic targets.