Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure–activity relationships and a proposed mechanism of action for this novel inhibitor chemotype.     

Glucose-stimulated translation regulation of insulin by the 5' UTR-binding proteins

Insulin is the key regulator of glucose homeostasis in mammals, and glucose-stimulated insulin biosynthesis is essential for maintaining glucose levels in a narrow range in mammals. Glucose specifically promotes the translation of insulin in pancreatic β-islet, and the untranslated regions of insulin mRNA play a role in such regulation. Specific factors in the β-islets bind to the insulin 5' UTR and regulate its translation. In the present study we identify protein-disulfide isomerase (PDI) as a key regulator of glucose-stimulated insulin biosynthesis. We show that both in vitro and in vivo PDI can specifically associate with the 5' UTR of insulin mRNA. Immunodepletion of PDI from the islet extract results in loss of glucose-stimulated translation indicating a critical role for PDI in insulin biosynthesis. Similarly, transient overexpression of PDI resulted in specific translation activation by glucose. We show that the RNA binding activity of PDI is mediated through PABP. PDI catalyzes the reduction of the PABP disulfide bond resulting in specific binding of PABP to the insulin 5' UTR. We also show that glucose stimulation of the islets results in activation of a specific kinase that can phosphorylate PDI. These findings identify PDI and PABP as important players in glucose homeostasis.     

pH dependence of folding of iso-2-cytochrome c

Starting from a standard unfolded state (3.0 M guanidine hydrochloride, pH 7.2), the kinetics of refolding of iso-2-cytochrome c have been investigated as a function of final pH between pH 3 and pH 10. Absorbance in the ultraviolet and visible spectral regions and tryptophan fluorescence are used to monitor folding. Over most of the pH range, fast and slow folding phases are detected by both fluorescence and absorbance probes. Near neutral pH, the rate of fast folding appears to be the same when monitored by absorbance and fluorescence probes. At higher and lower pH, there are two fast folding reactions, with absorbance-detected fast folding occurring in a slightly faster time range than fluorescence-detected fast folding. The rates of both fast folding reactions pass through broad minima near neutral pH, indicating involvement of ionizable groups in rate-limiting steps. The rates of slow folding also depend on the final pH. At acid pH, there appears to be a single slow folding phase for both fluorescence and absorbance probes. At neutral pH, the absorbance-detected and fluorescence-detected slow folding phases separate into distinct kinetic processes which differ in rate and relative amplitude. At high pH, absorbance-detected slow folding is no longer observed, while fluorescence-detected slow folding is decreased in amplitude. In contrast, the equilibrium and kinetic properties of proline imide bond isomerization, believed to be involved in the slow folding reactions, are largely independent of pH. The results suggest that the pH dependence of slow folding involves coupling of pH-sensitive structure to proline imide bond isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parental Age Affects Somatic Mutation Rates in the Progeny of Flowering Plants

In humans, it is well known that the parental reproductive age has a strong influence on mutations transmitted to their progeny. Meiotic nondisjunction is known to increase in older mothers, and base substitutions tend to go up with paternal reproductive age. Hence, it is clear that the germinal mutation rates are a function of both maternal and paternal ages in humans. In contrast, it is unknown whether the parental reproductive age has an effect on somatic mutation rates in the progeny, because these are rare and difficult to detect. To address this question, we took advantage of the plant model system Arabidopsis (Arabidopsis thaliana), where mutation detector lines allow for an easy quantitation of somatic mutations, to test the effect of parental age on somatic mutation rates in the progeny. Although we found no significant effect of parental age on base substitutions, we found that frameshift mutations and transposition events increased in the progeny of older parents, an effect that is stronger through the maternal line. In contrast, intrachromosomal recombination events in the progeny decrease with the age of the parents in a parent-of-origin-dependent manner. Our results clearly show that parental reproductive age affects somatic mutation rates in the progeny and, thus, that some form of age-dependent information, which affects the frequency of double-strand breaks and possibly other processes involved in maintaining genome integrity, is transmitted through the gametes.In humans, it has long been recognized that the reproductive age of the parents has an influence on the health of their progeny. An older reproductive age of the mother is known to increase the fraction of aneuploid gamete formation (Hurles, 2012). For instance, the risk for a trisomy increases from 2% to 3% for mothers in their 20s to more than 30% for mothers in their 40s (Hassold and Hunt, 2009). The age of the father also has an effect on the frequency of spontaneous congenital disorders and common complex diseases, such as autism and some cancers (Goriely and Wilkie, 2012). Indeed, sperm from 36- to 57-year-old men have more double-strand breaks (DSBs) than those of 20- to 35-year-old individuals (Singh et al., 2003). Similarly, the efficiency of DSB repair was reported to decrease with age in vegetative tissues of the plant model system Arabidopsis (Arabidopsis thaliana; Boyko et al., 2006).Owing to the continuous divisions of spermatogonial stem cells, the male germline of humans is thought to be more mutagenic than the female germline. Indeed, it was shown that the paternal germline is more mutagenic than the maternal one with respect to base substitutions (Kong et al., 2012) and replication slippage errors at microsatellites (Sun et al., 2012). It is also known that carriers of germline mutations in mismatch repair (MMR) genes in humans are prone to get colorectal cancer and that the risk depends on the parent-of-origin of the mutation (van Vliet et al., 2011). The molecular basis of these parental effects is not entirely clear but is likely to involve higher rates of nondisjunction during female meiosis, higher mutation rates during spermatogenesis, and probably additional effects of aging.In contrast to the effect of parental age on germline mutations, not much is known about potential effects of parental reproductive age on somatic mutation rates in the offspring. However, it has been shown in animal studies that radiation of males can lead to somatic mutations in their progeny—and subsequent generations—that cannot be attributed to mutations in the paternal germline (for review, see Little et al., 2013). Moreover, several recent studies have illustrated the existence of complex parental and transgenerational effects in humans, although their molecular basis is not clear (Grossniklaus et al., 2013). These effects can be of either genetic nature (but the effect is seen even in offspring that did not inherit the genetic variant from their parents; for review, see Nadeau, 2009) or epigenetic nature (where environmental influences can possibly exert effects on subsequent generations; for review, see Pembrey et al., 2006; Pembrey, 2010; Curley et al., 2011). It is currently not known whether such parental effects affect the somatic mutation rates in the offspring or whether the effects are modulated by parental age.Taking advantage of the plant model system Arabidopsis, in which various somatic mutation rates can readily be assessed (Bashir et al., 2014), we investigated the effects of parental reproductive age on somatic mutation rates in the progeny. We report that there is a pronounced effect of parental age on somatic mutation rates in their offspring in a parent-of-origin-dependent fashion. Thus, some form of parental information, which is inherited through the gametes to the next generation, seems to alter the somatic mutation rates in the progeny and changes with parental reproductive age.     

Sources of nonlinearity in cDNA microarray expression measurements

Background  

A key assumption in the analysis of microarray data is that the quantified signal intensities are linearly related to the expression levels of the corresponding genes. To test this assumption, we experimentally examined the relationship between signal and expression for the two types of microarrays we most commonly encounter: radioactively labeled cDNAs on nylon membranes and fluorescently labeled cDNAs on glass slides.     

Deregulation of Beclin 1 in patients with tobacco-related oral squamous cell carcinoma

Autophagy is a physiologically regulated and evolutionary conserved process that plays a critical role in degradation of cytoplasmic proteins and other macromolecules within the lysosomes. Beclin-1, the mammalian orthologue of yeast Atg6, is an important mediator of autophagy that has been studied in many human cancers. However, the expression of Beclin-1 has not yet been investigated in oral cancer. We for the first time investigated the expression of Beclin-1 in serum and tissues and correlated it with the clinic-pathological features of oral cancer patients. m-RNA expression of Beclin-1 was evaluated in tumor and normal areas of surgical specimens from 10 oral cancer patients by real-time PCR. Approximately, 8-fold lower expression (p<0.001) of Beclin-1 mRNA was observed in tumor tissue as compared to the normal tissue. Serum levels of Beclin-1 were evaluated by SPR and ELISA. No significant difference was observed in serum Beclin-1 levels in patients as compared to healthy subjects, similarly no correlation was found between serum levels and clinic-pathological parameters such as stage, lymph node involvement and tumor size. Our results demonstrate that down-regulation of Beclin-1 may play an important role in the development and progression of oral cancer possibly by dysregulation of autophagy in tumor cells.     

Induction of replication protein A in bystander cells

The bystander effect is a biological phenomenon whereby cells not directly targeted by DNA-damaging agents elicit a response similar to that of targeted cells. Understanding the mechanisms underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for radiotherapy of tumors. Identification of DNA repair and signal transduction proteins that are induced specifically in bystander cells may help in deducing the molecular mechanism(s) responsible for this complex phenomenon. With this objective, we have studied the expression of replication protein A (RPA), which is involved in various DNA metabolic activities such as replication, repair and recombination. We analyzed RPA expression by immunofluorescence and Western blot techniques in both gamma-irradiated primary human fibroblast cells and bystander cells that were recipients of conditioned growth medium harvested from gamma-irradiated cell cultures. A two- to threefold induction of RPA was observed in bystander MRC5 cells treated with conditioned medium collected from gamma-irradiated WI38 or MRC5 cells. Lack of induction of RPA in sham-manipulated MRC5 cells treated with irradiated medium alone (without cells) indicates that the signal elicited from the irradiated cells is responsible for induction of RPA in bystander cells. RPA was induced more effectively in bystander cells than in irradiated cells at the earliest time analyzed (30 min), and the RPA level declined to that of sham-treated control cells by 24 h after treatment. In addition to RPA, apurinic/apyrimidinic endonuclease (APE, a key enzyme of the base excision repair pathway) also showed enhanced expression in bystander cells. Our findings suggest that the induction of RPA and APE is due to a combination of DNA strand breaks and oxidized base lesions in the genomic DNA of bystander cells.