Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

The interaction between host and host plant influences the oviposition and performance of a generalist ectoparasitoid

The successful development of parasitoids of herbivores depends on the quality of their host, which is often affected by the host plant. Therefore, a parasitoid’s oviposition decisions will directly depend on the host, but also on plant quality. Here, we investigated the direct effects of host species and the indirect effects of the host’s food plant on the oviposition decisions and performance of the gregarious ectoparasitoid Euplectrus platyhypenae Howard (Hymenoptera: Eulophidae). With a series of no‐choice experiments, we determined the oviposition and performance of the parasitoid on: (1) two caterpillar species, fall armyworm, Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae), and velvet armyworm, Spodoptera latifascia Walker, reared on maize (Zea mays L., Poaceae), (2) the same caterpillars reared on maize, bean (Phaseolus vulgaris L., Fabaceae), or squash (Cucurbita pepo L., Cucurbitaceae) leaves, and (3) S. latifascia caterpillars reared on leaves of wild and cultivated lima bean, Phaseolus lunatus L. All these insects and plants originate from Mesoamerica where they have coexisted for thousands of years in the traditional agricultural system known as Milpa in which maize, beans, and squash are planted together. We found that the preferred and best combination of host and host plant for parasitoid performance was S. frugiperda on maize. Parasitoids laid larger clutches, had higher survival, and more females and larger adults emerged from S. frugiperda reared on maize. However, when both caterpillar species were reared on squash, S. latifascia was the better host. Contrary to the literature, S. frugiperda was not able to develop on bean plants. Results from the lima bean experiment showed that parasitoid performance was best when S. latifascia was reared on leaves of cultivated compared to wild lima bean. These findings are discussed in the context of mixed cropping in which the ability of generalist parasitoids to switch among hosts and host plant species could be advantageous for pest management.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Characterization of substrate specificity and inhibitory mechanism of bile salt hydrolase from Lactobacillus reuteri CRL 1098 using molecular docking analysis

Objectives

To elucidate the molecular mechanisms involved in the substrate interaction of the bile salt hydrolase of Lactobacillus reuteri CRL 1098 (LrBSH) with bile acids (BAs) and to evaluate potential enzyme inhibitors based on computer and in vitro modeling assays.

Results

Asp19, Asn79, and Asn171 participated in the LrBSH interaction with all BAs tested while Leu56 and Glu 222 played an important role in the interaction with glyco- and tauro-conjugated BAs, respectively. A great percentage of hydrophobic and polar interactions were responsible for the binding of LrBSH with glyco- and tauro-conjugated BAs, respectively. Remarkably, the four binding pocket loops participated in the substrate binding site of LrBSH unlike most of the reported BSHs. Inhibition assays showed that ascorbic acid, citric acid, penicillin G, and ciprofloxacin decreased LrBSH activity by 47.1%, 40.14%, 28.8%, and 9%, respectively. Docking analysis revealed that tetracycline and caffeic acid phenethyl ester had the low binding energy (?7.32 and ?7.19 kcal/mol, respectively) and resembled the interaction pattern of GDCA (?6.88 kcal/mol) while penicillin (?6.25 kcal/mol) and ascorbic acid (?5.98 kcal/mol) interacted at a longer distance.

Conclusion

This study helps to delve into the molecular mechanisms involved in the recognition of substrates and potential inhibitors of LrBSH.

     

Exome sequences and multi‐environment field trials elucidate the genetic basis of adaptation in barley

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi‐environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype‐by‐environment (G×E) modelling. Sub‐populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock‐related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large‐effect alleles. Our analysis supports a gene‐level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.     

Induction of TIMP-1 expression in rat hepatic stellate cells and hepatocytes: a new role for homocysteine in liver fibrosis.

Elevated plasma levels of homocysteine have been shown to interfere with normal cell function in a variety of tissues and organs, such as the vascular wall and the liver. However, the molecular mechanisms behind homocysteine effects are not completely understood. In order to better characterize the cellular effects of homocysteine, we have searched for changes in gene expression induced by this amino acid. Our results show that homocysteine is able to induce the expression and synthesis of the tissue inhibitor of metalloproteinases-1 (TIMP-1) in a variety of cell types ranging from vascular smooth muscle cells to hepatocytes, HepG2 cells and hepatic stellate cells. In this latter cell type, homocysteine also stimulated alpha 1(I) procollagen mRNA expression. TIMP-1 induction by homocysteine appears to be mediated by its thiol group. Additionally, we demonstrate that homocysteine is able to promote activating protein-1 (AP-1) binding activity, which has been shown to be critical for TIMP-1 induction. Our findings suggest that homocysteine may alter extracellular matrix homeostasis on diverse tissular backgrounds besides the vascular wall. The liver could be considered as another target for such action of homocysteine. Consequently, the elevated plasma levels of this amino acid found in different pathological or nutritional circumstances may cooperate with other agents, such as ethanol, in the onset of liver fibrosis.