Characterization of Upstream Sequences of the <Emphasis Type="Italic">LOJ</Emphasis> Gene Leads to Identification of a Novel Enhancer Element Conferring Lateral Organ Junction-Specific Expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering.     

Metastatic mouse melanoma cells release collagen-gelatin degrading metalloproteinases as components of shed membrane vesicles

The purpose of this study has been to compare collagen-gelatin degrading enzymes isolated from cancer cell organelles and cytosol to the metalloproteinases released by cancer cells. To this end, metastatic mouse melanoma cell organelles were isolated by sucrose density gradient centrifugation and metalloproteinases were assayed using native and denatured [methyl-3H]collagen substrates. Solubilized proteinases were purified by ammonium sulfate precipitation, anion exchange, concanavalin A affinity and gel-filtration column chromatographic procedures and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conclusions were as follows: malignant melanoma cells have a metalloproteinase (Mr = 59,000) which is shed from cells into conditioned medium as a component of intact membrane vesicles rather than as a soluble enzyme; storage of tumor-conditioned medium leads to the generation of autoactivated soluble metalloproteinases of lower molecular weight; purification of these metalloproteinase species yielded variant collagenases that have considerable gelatinolytic activity and a cleavage preference site for the Gly-Ile bond in a collagen-like synthetic octapeptide substrate which is typical for collagenase-type metalloproteinases. It is proposed that localization of potent proteinases to the surface of cancer cells facilitates the local breakdown of connective tissues during the invasive process.     

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.     

Bacterial Factors Associated with Lethal Outcome of Enteropathogenic Escherichia coli Infection: Genomic Case-Control Studies

Background

Typical enteropathogenic Escherichia coli (tEPEC) strains were associated with mortality in the Global Enteric Multicenter Study (GEMS). Genetic differences in tEPEC strains could underlie some of the variability in clinical outcome.

Methods

We produced draft genome sequences of all available tEPEC strains from GEMS lethal infections (LIs) and of closely matched EPEC strains from GEMS subjects with non-lethal symptomatic infections (NSIs) and asymptomatic infections (AIs) to identify gene clusters (potential protein encoding sequences sharing ≥90% nucleotide sequence identity) associated with lethality.

Results

Among 14,412 gene clusters identified, the presence or absence of 392 was associated with clinical outcome. As expected, more gene clusters were associated with LI versus AI than LI versus NSI. The gene clusters more prevalent in strains from LI than those from NSI and AI included those encoding proteins involved in O-antigen biogenesis, while clusters encoding type 3 secretion effectors EspJ and OspB were among those more prevalent in strains from non-lethal infections. One gene cluster encoding a variant of an NleG ubiquitin ligase was associated with LI versus AI, while two other nleG clusters had the opposite association. Similar associations were found for two nleG gene clusters in an additional, larger sample of NSI and AI GEMS strains.

Conclusions

Particular genes are associated with lethal tEPEC infections. Further study of these factors holds potential to unravel the mechanisms underlying severe disease and to prevent adverse outcomes.     

The effect of experimental diabetes on the molecular characteristics of soluble rat-tail tendon collagen

Acid soluble rat-tail tendon collagen was prepared from animals rendered diabetic by treatment with either streptozotocin or alloxan and from matched controls. In comparison to the normal, the diabetic collagens consistently demonstrated decreased solubility of reconstituted fibrils, marked increase in intrinsic viscosity and a decreased ratio of alpha to beta components. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed a marked decrease in migration of alpha1, alpha2, and beta components from both types of diabetic collagen. These data indicate that diabetic collagens are larger than normal and are capable of higher degrees of polymerization due to increased intra- and inter-molecular interactions. These changes could explain, in part, the altered response of diabetic connective tissues to inflammation and trauma.     

Molecularly imprinted polyaniline molecular receptor–based chemical sensor for the electrochemical determination of melamine

Molecularly imprinted polymer‐modified glassy carbon electrode (GCE)‐based electrochemical sensor is prepared using the electropolymerization of aniline in the presence of melamine (MA) as a template. In this work, the advantages of molecularly imprinted conducting polymers (MICPs) and electroanalytical methods were combined to obtain an electronic device with better performances. The sensor performance was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) with the linear range of 0.6‐16 × 10^?9M, quantification limit of 14.9 × 10^?10M, and detection limit of 4.47 × 10^?10M (S/N = 3). The selectivity of the sensor was tested in the presence of acetoguanamine (AGA), diaminomethylatrazine (DMT), casein, histidine, and glycine interfering molecules taken at the triple concentration with MA that demonstrated too small current response compared with that of the analyte indicating high specificity of the sensor towards the template. The sensor was successfully applied to determine MA in infant formula samples with significant recovery greater than 96% and relative standard deviation (RSD) less than 4.8%. Moreover, the good repeatability, recyclability, and stability make this sensor device promising for the real‐time monitoring of MA in different food stuffs.     

Stress response, cell death and signalling: the many faces of reactive oxygen species

Plants respond to pathogens and abiotic stresses by transient increases in the production of reactive oxygen species (ROS) and ion fluxes, which activate both local programmed cell death and systemic increases in stress- and pathogen-resistance. The present essay explores the emerging complexity of the multiple roles that ROS play in intra- and intercellular communication in both stressed and unstressed organisms.     

Synthesis and Biological Activity of a Bis-Substituted 3′-Deoxyadenosine Analog of 2–5A

Abstract

The 5′-monophosphate, p5′(3′dA)2′p5′A2′5′(3′dA), was synthesized and found to bind to the 2–5A-dependent endonuclease of mouse L cells only two-three times less effectively than the parent p5′A2′p5′A2′p5′A. When evaluated for its ability to activate the 2–5A-dependent endonuclease, ppp5′(3′dA)2′p5′A2′p5′(3′dA) was found to be fifty times more effective than ppp5′A2′p5′(3′dA)2′p5′A and ten times less effective than 2–5A as an endonuclease activator     

Recent Progress in Oligoribonucleotide Synthesis

Abstract

The usefulness of the p-nitrophenylethylsulfonyl (NPES) group for 2′-OH protection in oligoribonucleotide synthesis is further investigated. The difficulties of uridine protection are discussed and the p-cyanophenylethyl (CPE) group introduced as a versatile new 0⁴-blocking group.     

Mechanism of photoisomerization of optically pure trans-2,3-diphenylcyclopropane-1-carboxylic acid derivatives.

The photochemistry of optically pure isomers of alpha-methylbenzylamide of trans-2,3-diphenylcyclopropane-1-carboxylic acid has been examined in isotropic solution and within zeolites. The results suggest that these isomerize through cleavage of C2-C3 bond. The direct excitation in solution leads to non-equilibrating 1,3-singlet diradical intermediates whereas triplet sensitization results in equilibrating 1,3-triplet diradical intermediates. The direct excitation within NaY zeolite seems to result in equilibrating zwitterionic intermediates. Studies on the optically pure trans isomers allow one to understand the mechanism of chiral induction during the photoisomerization of mesocis-2,3-diphenylcyclopropane-1-carboxylic acid. The current study has clarified the nature of the excited states involved during the classic (R)-N-acetyl-1-naphthylethylamine sensitized isomerization of 1,2-diphenylcyclopropane.