Protection of thymosin beta-4 on corneal endothelial cells from UVB-induced apoptosis

Cornea absorbs most of daily ultraviolet (UV) light. An excess of UV damages results in not only keratopathy and cataract but also maculopathy. It has been reported that thymosin beta-4 (Tbeta4) promotes wound healing, decreases inflammatory response and prevents apoptosis of corneal epithelial cells. However, it is not clear whether Tbeta4 protects UVB-induced corneal injury, particularly in corneal endothelial cells because of its non-proliferation in nature. The purpose of this study is to compare the protective effects of Tbeta4 on bovine corneal endothelial (BCE) cells from low- and high-dose UVB damage. In this study, 1 microg/ml of Tbeta4 was added to BCE cells 2 h before low (12.5 mj/cm2) or high dosage (100 mj/cm2) UVB exposure. Using a fluorogenic substrate cleavage assay, we found that Tbeta4 diminished the reactive oxygen species level in BCE cells elicited by UVB. However, the protection of viability by Tbeta4 could only be detected under low-dose UVB exposure. Moreover, both caspase-9 activity and annexin V/propidium iodine staining demonstrated that Tbeta4 only protected BCE cells from low-dose UVB-induced apoptosis but not high-dose UVB-induced necrosis. Together, Tbeta4 protected corneal endothelial cells from UVB-induced oxidative stress and apoptosis after low-dose UVB exposure. The results support further investigation towards topical use or anterior chamber injection of this small hydrophilic peptide in treating and preventing UVB-induced corneal endothelial damage.     

Identity-by-Descent Matrix Decomposition Using Latent Ancestral Allele Models

Genetic linkage and association studies are empowered by proper modeling of relatedness among individuals. Such relatedness can be inferred from marker and/or pedigree information. In this study, the genetic relatedness among n inbred individuals at a particular locus is expressed as an n × n square matrix Q. The elements of Q are identity-by-descent probabilities, that is, probabilities that two individuals share an allele descended from a common ancestor. In this representation the definition of the ancestral alleles and their number remains implicit. For human inspection and further analysis, an explicit representation in terms of the ancestral allele origin and the number of alleles is desirable. To this purpose, we decompose the matrix Q by a latent class model with K classes (latent ancestral alleles). Let P be an n × K matrix with assignment probabilities of n individuals to K classes constrained such that every element is nonnegative and each row sums to 1. The problem then amounts to approximating Q by PP^T, while disregarding the diagonal elements. This is not an eigenvalue problem because of the constraints on P. An efficient algorithm for calculating P is provided. We indicate the potential utility of the latent ancestral allele model. For representative locus-specific Q matrices constructed for a set of maize inbreds, the proposed model recovered the known ancestry.HIGH-THROUGHPUT techniques allow extensive genotyping of individuals for thousands of SNP markers (Gibbs et al. 2003) and thereby provide accurate information about the genetic diversity within a population at many chromosomal loci. If two individuals within this population carry the same DNA sequence at a locus, and this sequence can be traced to the same common ancestor, the individuals are said to be identical by descent (IBD) for this segment (Chapman and Thompson 2003). Quite often, however, the ancestral source of a chromosomal segment is ambiguous and thus IBD relationships between haplotypes are given as probabilities. Various methods have been described to estimate the IBD probability of pairs of chromosomal segments (Meuwissen and Goddard 2001; Leutenegger et al. 2003). When pedigree relationships are known, these can be included to estimate IBD probabilities (Wang et al. 1995; Heath 1997; George et al. 2000; Meuwissen and Goddard 2000; Besnier and Carlborg 2007).In quantitative genetic analysis we seek to find and characterize associations between the large number of SNPs that are now available for many organisms and phenotypic variation for traits of interest (e.g., grain yield and time to flowering). Many current methods developed for this purpose make use of IBD information. For example, a locus-specific matrix of IBD probabilities can be incorporated into restricted maximum-likelihood (REML) procedures for fine mapping quantitative trait loci (Bink and Meuwissen 2004) as well as for marker-based genetic evaluation (Fernando and Grossman 1989) using mixed models. The IBD matrix takes the role of a covariance matrix in the REML procedure.Other approaches, however, require that chromosome segments (also referred to here as haplotypes or alleles) are assigned to independent ancestors. These approaches include regression approaches with genetic predictors (Malosetti et al. 2006) and Bayesian oligo-allelic approaches that sample the ancestral origin of each chromosomal segment (Heath 1997; Uimari and Sillanpaa 2001; Bink et al. 2008a). In the IBD matrix representation the ancestral alleles and their number remain implicit. For these approaches, the locus-specific matrix of IBD probabilities must therefore be decomposed into a matrix that links the chromosomal segments to independent ancestral alleles. This decomposition is addressed in this article.The individuals that we consider in this article are inbred. For n inbred individuals the IBD matrix at a given chromosomal position is thus n × n, because there is no need to distinguish between identical chromosomes. In diploid, outbred populations, each individual would be represented by two haplotypes (alleles) and the matrix would be 2n × 2n (Fernando and Grossman 1989). This is feasible if any phase ambiguity can be resolved. From now on, the term “individual” thus means chromosomal segment or haplotype. Analogously, ancestor will be shorthand for ancestral allele (ancestral haplotype).We propose two models of IBD matrix decomposition, a simple threshold model (TIBD) and a more sophisticated latent ancestral allele model (LAAM), that provide (1) an estimate of the number of independent ancestral alleles, (2) a concise, easy-to-interpret, summary of the relatedness, (3) an explicit (probabilistic) representation of the descent of alleles, and (4) the ability to sample alleles for each individual from a set of ancestral alleles in such a way that the probability that a pair of individuals shares the same allele corresponds to their IBD probability.The last two features of the model are essential for its use in Bayesian oligo-allelic approaches to quantitative trait locus (QTL) analysis (Uimari and Sillanpaa 2001; Bink et al. 2008a).     

A caged nicotine with nanosecond range kinetics and visible light sensitivity

We report the synthesis, characterization and applications of a ruthenium-bipyridine based caged nicotine. The complex [Ru(bpy)₂(nic)₂]²⁺ (where bpy = 2,2′ bipyridine and nic = nicotine (3-[(2S)-1-methylpyrrolidin-2-yl] pyridine)) releases nicotine with a quantum yield ? = 0.23 upon irradiation with biologically harmless, blue (473 nm) or green (532 nm) light. The photolysis reaction is clean and very fast, with a time constant of 17 ns. The synthesis is simple and the obtained compound is characterized by NMR, UV-Vis spectroscopy and cyclic voltametry. We find that this compound is active in biological systems, being able to elicit action potentials in leech neurons.     

The Intra-Hippocampal Leucine Administration Impairs Memory Consolidation and LTP Generation in Rats

Leucine accumulates in fluids and tissues of patients affected by maple syrup urine disease, an inherited metabolic disorder, predominantly characterized by neurological dysfunction. Although, a variable degree of cognition/psychomotor delay/mental retardation is found in a considerable number of individuals affected by this deficieny, the mechanisms underlying the neuropathology of these alterations are still not defined. Therefore, the aim of this study was to investigate the effect of acute intra-hippocampal leucine administration in the step-down test in rats. In addition, the leucine effects on the electrophysiological parameter, long-term potentiation generation, and on the activities of the respiratory chain were also investigated. Male Wistar rats were bilaterally administrated with leucine (80 nmol/hippocampus; 160 nmol/rat) or artificial cerebrospinal fluid (controls) into the hippocampus immediately post-training in the behavioral task. Twenty-four hours after training in the step-down test, the latency time was evaluated and afterwards animals were sacrificed for assessing the ex vivo biochemical measurements. Leucine-treated animals showed impairment in memory consolidation and a complete inhibition of long-term potentiation generation at supramaximal stimulation. In addition, a significant increment in complex IV activity was observed in hippocampus from leucine-administered rats. These data strongly indicate that leucine compromise memory consolidation, and that impairment of long-term potentiation generation and unbalance of the respiratory chain may be plausible mechanisms underlying the deleterious leucine effect on cognition.     

Identification of chalcones as in vivo liver monofunctional phase II enzymes inducers

Cancer preventive agents (CPA) are drugs able to suppress the carcinogen metabolic activation or block the formation of ultimate carcinogens. CPA could act through various molecular mechanisms, for example by interfering with the action of procarcinogen. This could be attained by increasing the phase II enzymes levels of quinone reductase (QR) and glutathione S-transferase (GST). New flavonoids, especially chalcones, have been identified as in vivo monofunctional phase II enzymes inducers. Oral administration of chalcone, 4, and both p-methoxy-substituted chalcones, 6 and 14, increased hepatic QR activity with concomitant decrease in CYP1A1 activity, a member of the most important group of phase I enzymes cytochrome P450. Among them, 4 also increased GST activity. While p-bromo-substituted chalcone 8 was the best inducer of QR it decreased hepatic GST expression and cytochrome P450, being the most effective decreasing cytochrome P450-expression. Thienyl-chalcone 20 being the bioisostere of chalcone 4 did not display the same in vivo profile in the phase I level modification. As chalcone 4 its bioisostere, chalcone 20, displayed low DNA strand breakage and absence of mutagenicity. Also, in our preliminary in vivo tumourigenesis/chemopreventive and acute-toxicity studies, chalcones 4, 6 and 8 showed the best behaviours as CPA justifying additional studies that are ongoing.     

Fluoroquinolone resistance in clinical and environmental isolates of Escherichia coli in Mexico City

Aims:  To assess the different phenotypes and mechanisms of fluoroquinolone (FQ) resistance in clinical and environmental isolates of Escherichia coli .
Methods and Results:  We compared FQ-resistant E. coli isolates, measuring minimal inhibitory concentrations (MIC) of ciprofloxacin, along with susceptibility to other antibiotics. We also searched for the presence of efflux pumps, using efflux inhibitors, and for plasmid-borne FQ-resistance by PCR. We found that, aside from the higher FQ-resistance prevalence among clinical strains, environmental ones resist much lower concentrations of ciprofloxacin. Efflux pumps mediate fluoroquinolone resistance as frequently among environmental isolates than in clinical strains. Plasmid-borne qnrA genes were not detected in any resistant strain.
Conclusions:  Environmental FQ-resistant strains may have a nonclinical origin and/or a selective pressure different from the clinical use of FQs.
Significance and Impact of the Study:  The identification of the source of low-level FQ-resistant strains (ciprofloxacin MIC c . 8 μg ml⁻¹) in the environment could be important to curb the rapid emergence and spread of FQ-resistance in clinical settings, as these strains can easily become fully resistant to FQ concentrations achievable in fluids and tissues during therapy.     

Enzyme replacement therapy for Morquio A: an active recombinant <Emphasis Type="Italic">N</Emphasis>-acetylgalactosamine-6-sulfate sulfatase produced in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2–4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.