Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw

The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg²⁺ and Ca²⁺ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by CelA. Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998     

Cafeteria diet-induced obesity plus chronic stress alter serum leptin levels

Obesity is a disease that has become a serious public health issue worldwide, and chronic stressors, which are a problem for modern society, cause neuroendocrine changes with alterations in food intake. Obesity and chronic stress are associated with the development of cardiovascular diseases and metabolic disorders. In this study, a rat model was used to evaluate the effects of a hypercaloric diet plus chronic restraint stress on the serum leptin and lipids levels and on the weight of specific adipose tissue (mesenteric, MAT; subcutaneous, SAT and visceral, VAT). Wistar rats were divided into the following 4 groups: standard chow (C), hypercaloric diet (HD), stress plus standard chow (S), and stress plus hypercaloric diet (SHD). The animals in the stress groups were subjected to chronic stress (placed inside a 25 cm × 7 cm plastic tube for 1 h per day, 5 days per week for 6 weeks). The following parameters were evaluated: the weight of the liver, adrenal glands and specific adipose tissue; the delta weight; the Lee index; and the serum levels of leptin, corticosterone, glucose, total cholesterol, and triglycerides. The hypercaloric diet induced obesity in rats, increasing the Lee index, weight, leptin, triglycerides, and cholesterol levels. The stress decreased weight gain even in animals fed a hypercaloric diet but did not prevent a significant increase in the Lee index. However, an interaction between the independent factors (hypercaloric diet and stress) was observed, which is demonstrated by the increased serum leptin levels in the animals exposed to both protocols.     

The Diamine Oxidase Gene Is Associated with Hypersensitivity Response to Non-Steroidal Anti-Inflammatory Drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) are the drugs most frequently involved in hypersensitivity drug reactions. Histamine is released in the allergic response to NSAIDs and is responsible for some of the clinical symptoms. The aim of this study is to analyze clinical association of functional polymorphisms in the genes coding for enzymes involved in histamine homeostasis with hypersensitivity response to NSAIDs. We studied a cohort of 442 unrelated Caucasian patients with hypersensitivity to NSAIDs. Patients who experienced three or more episodes with two or more different NSAIDs were included. If this requirement was not met diagnosis was established by challenge. A total of 414 healthy unrelated controls ethnically matched with patients and from the same geographic area were recruited. Analyses of the SNPs rs17740607, rs2073440, rs1801105, rs2052129, rs10156191, rs1049742 and rs1049793 in the HDC, HNMT and DAO genes were carried out by means of TaqMan assays. The detrimental DAO 16 Met allele (rs10156191), which causes decreased metabolic capacity, is overrepresented among patients with crossed-hypersensitivity to NSAIDs with an OR  = 1.7 (95% CI  = 1.3–2.1; Pc  = 0.0003) with a gene-dose effect (P = 0.0001). The association was replicated in two populations from different geographic areas (Pc  = 0.008 and Pc  = 0.004, respectively).

Conclusions and implications

The DAO polymorphism rs10156191 which causes impaired metabolism of circulating histamine is associated with the clinical response in crossed-hypersensitivity to NSAIDs and could be used as a biomarker of response.     

Post‐fire recovery occurs overwhelmingly by resprouting in the Chaco Serrano forest of Central Argentina

In fire‐prone landscapes, differences in post‐fire regeneration by resprouting between species or sites could be far more important in explaining vegetation physiognomy and composition than seed regeneration. This is the first study exploring the relative contribution of tree resprouts and seeds to post‐fire crown volume in the Chaco Serrano forest of South America. Additionally, we compare the resprouting response among species and quantify post‐fire changes in tree composition among sites. We established 290 permanent plots distributed in three sites affected by wildfires in 2005. For all tree species in all plots we recorded survival of every individual 1 year after the fire; at the plot level, we recorded the above‐ground tree volume before and 3 years after the fire. Resprouting from the base was the main resprouting type. Survival varied between species from 73% to 100% for native species and from 7% to 100% for non‐natives. Before the fire, crown volume was similar in the three sites, and was completely lost after the fire in 92% of the plots. Three years after the fire, between 8% and 58% of crown volume was recovered. The ratio of crown recovery because of resprouts and seedlings was 1562:1. Tree composition exhibited few changes because of the high post‐fire survival of most native species. We conclude that in the semiarid Chaco Serrano ecosystem tree species regenerate mainly by resprouting. This regeneration mode should be taken into account to better understand post‐fire successional pathways of these forests, their management and the restoration of burnt forest areas.     

Development of a panel of genome-wide ancestry informative markers to study admixture throughout the Americas

Most individuals throughout the Americas are admixed descendants of Native American, European, and African ancestors. Complex historical factors have resulted in varying proportions of ancestral contributions between individuals within and among ethnic groups. We developed a panel of 446 ancestry informative markers (AIMs) optimized to estimate ancestral proportions in individuals and populations throughout Latin America. We used genome-wide data from 953 individuals from diverse African, European, and Native American populations to select AIMs optimized for each of the three main continental populations that form the basis of modern Latin American populations. We selected markers on the basis of locus-specific branch length to be informative, well distributed throughout the genome, capable of being genotyped on widely available commercial platforms, and applicable throughout the Americas by minimizing within-continent heterogeneity. We then validated the panel in samples from four admixed populations by comparing ancestry estimates based on the AIMs panel to estimates based on genome-wide association study (GWAS) data. The panel provided balanced discriminatory power among the three ancestral populations and accurate estimates of individual ancestry proportions (R2 > 0.9 for ancestral components with significant between-subject variance). Finally, we genotyped samples from 18 populations from Latin America using the AIMs panel and estimated variability in ancestry within and between these populations. This panel and its reference genotype information will be useful resources to explore population history of admixture in Latin America and to correct for the potential effects of population stratification in admixed samples in the region.     

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.     

The human spindle assembly checkpoint protein Bub3 is required for the establishment of efficient kinetochore-microtubule attachments

The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.     

Use of sulfhydryl reagents to investigate branched chain alpha-keto acid transport in mitochondria

The goal of this paper was to determine the contribution of the mitochondrial branched chain aminotransferase (BCATm) to branched chain alpha-keto acid transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data suggest that essential cysteine residues in BCATm are accessible from the cytosolic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimide (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in reconstituted mitochondrial detergent extracts by 70% and in the intact organelle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzyme. Additionally, the apparent mitochondrial equilibrium KIC concentration was decreased 50-60% after NEM labeling, and this difference could not be attributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix aspartate and glutamate levels revealed that the effects of NEM on the steady-state KIC concentration resulted from inhibition of BCATm catalyzed transamination of KIC with matrix glutamate to form leucine. Furthermore, circular dichroism spectra of recombinant human BCATm with liposomes showed that the commercial lipids used in the reconstituted transport assay contain BCAT amino acid substrates. Thus BCATm is distinct from the branched chain alpha-keto acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact and reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates.