Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.     

Nitricoxide synthase-induced oxidative stress in prolonged alcoholic myopathies of rats

Previous studies showed that nitricoxide synthase (NOS) and oxidative stress can induce skeletal muscle atrophy in the muscular dystrophy and inclusion-body myopathy. There is a correlation between NOS and oxidative stress. However, it is not clear, whether there are some changes of the NOS activity in prolonged alcoholic myopathy (PAM), and whether NOS activity has relation to amyotrophy of PAM. We established experimental alcoholic myopathy model of rats by prolonged alcohol intake. We found that there is a reduction in GSH-px (P < 0.05) and an increase of SOD (P < 0.05), MDA (P < 0.05) and iNOS (P < 0.05) in the plantaris of the experimental group by spectrophotometer. In the soleus of the experimental group, except for MDA showed an increase (P < 0.05), the other enzymes showed no obvious difference (P > 0.05). The immunohistochemistry results showed that there was obvious expression of iNOS in the cytoplasm of plantaris in the experimental group and there was no expression of iNOS in the control group. There was a decrease of nNOS expression on the membranes of the plantaris cells in the experimental group by immunofluorescence. Meanwhile, we found the expression of nNOS in some cytoplasm. Our results suggested that NOS might be an important factor during the development of PAM. We could infer that there are some disturbances with regard to output and scavenging of free radical in PAM. Alcohol can induce the oxidative stress reaction and further result in imbalance of the oxidant-antioxidant status in the organism. Haiying Chu is the co-first author.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.     

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP. The refolding kinetics was determined in real time. Both the recovering yield and refolding rate of denatured EGFP in the presence of imidazole were increased. The studies on elucidating the mechanism show that imidazole may catalyze the prolyl cis/trans isomerization and the possible mechanism was discussed. To our knowledge, there are no data on the effect of imidazole on protein folding. Considering the prolyl isomerization is the rate-limited step for refolding of most proteins and aggregation is a universal serious problem for biotechnology, imidazole thus represents a previous unknown type of protein-folding catalyst.     

Comparison of C40/82A and P27A C40/82A barstar mutants using 19F NMR

Barstar, an inhibitor of the enzyme barnase, contains two phenylalanine residues, three tryptophan residues, and two proline residues. After incorporating either 2-19F-Phe, 4-19F-Phe, or 6-19F-Trp, the structural, dynamic, and folding properties of two mutants (C40/82A, a double mutant, and P27A C40/82A, a triple mutant) were studied by 19F NMR. Experiments were performed as a function of temperature and urea with the two mutants. We show that the consequences of the P27A mutation are extensive. The effect of the mutation is transmitted to distant residues (Phe56 and Trp53) as well as to a residue deeply buried in the hydrophobic core (Phe74). By incorporating 2-19F-Phe, it is shown that Phe56 undergoes a slow ring flipping on the NMR time scale in the triple mutant that is not observed in the double mutant. On the other hand, incorporating 4-19F-Phe shows that the P27A mutation has little effect along the Cbeta-Cgamma axis of Phe56. Labeling with 4-19F-Phe shows, from line broadening, that Phe74 experiences more dynamic motion than does Phe56 in both the double and triple mutant. After incorporating 6-19F-Trp, it is found that, in the triple mutant, Trp53 shows conformational heterogeneity at low temperature while Trp44, which is close to the P27A mutation, does not. At 20 degrees C, residual native-like structure was detected around Trp53 at high concentrations of denaturant. Barstar is cold denatured in the presence of urea. For the double mutant at temperatures below 15 degrees C, and in the presence of 2.5-3.5 M urea, the resonance for Phe74 broadens, and two peaks are observed at 5 degrees C indicative of an exchange process. From line-shape analysis, assuming a two-site conformational exchange, the rate constants as a function of temperature can be extracted. An Eyring plot is linear at 0 M urea but deviates from linearity below 20 degrees C in the presence of 2.5 or 3.5 M urea. The data as a function of urea suggest sequential events in the unfolding process.     

Tetranuclear manganese(III) clusters containing both carboxylate and phosphonate bridging ligands

This paper reports two tetranuclear manganese clusters, namely, [Mn₄O₂(O₂CCH₃)₄(O₃PC₆H₁₁)₂(phen)₂] (1) and [Mn₄O₂(O₂CPh)₄ (O₃PC₆H₁₁)₂(bpy)₂] (2). Both contain a butterfly-like [Mn₄(μ₃-O)₂]⁸⁺ core. The neighboring Mn atoms within the core are bridged by the carboxylate groups, forming approximately a plane. The phosphonate ligands locate above and below the plane, and cap on top of the Mn₃O triangles by using its three phosphonate oxygen atoms. The magnetic measurements of complexes 1 and 2 reveal that dominant antiferromagnetic interactions are propagated between the magnetic centers.     

滇东南马关古林箐热带雨林望天树群落的研究

云南东南部马关县古林箐的望天树群落以龙脑香科植物望天树为乔木层优势种,无患子科植物番龙眼为亚优势种,外貌以常绿大、中高位芽植物组成为特征,林内板根和茎花现象普遍,层间木质藤本和维管附生植物丰富,属于一种热带季节性雨林群落类型。在植物区系组成上,该群落以樟科、番荔枝科、楝科、大戟科、桑科、无患子科、橄榄科、柿树科等为主要组成科,在重要值统计上,以大戟科、无患子科、柿树科、龙脑香科、番荔枝科、樟科、楝科、橄榄科、桑科等为较优势的科,与东南亚热带雨林很接近,故为东南亚热带雨林的北缘类型。     

黄土高原马栏林区辽东栎更新特性研究

研究了黄土高原马栏林区主要植被类型中的辽东栎幼苗的数量特征,更新层幼苗、幼树的实生和萌生特性及其辽东栎在垂直结构上的数量分布。结果表明:(1)辽东栎幼苗在黄土高原马栏林区分布广泛且数量充足。不同的植被类型中辽东栎种群表现出不同的大小级结构,在油松—辽东栎混交林、油松林和辽东栎林中辽东栎种群的幼苗、幼树和成树均占一定的比例,而在油松—白桦混交林、白桦林、山杨林和山杨—白桦混交林中辽东栎种群则以幼苗和幼树为主。表明辽东栎种群在该地区植被的发展过程中将产生重要的作用。(2)辽东栎在这一地区是由实生和萌生的个体混合组成的。在各种植被类型中实生植株的密度都高于萌生植株,辽东栎种群的更新在该地区可能主要是通过实生植株来完成的,即辽东栎实生植株在更新过程中起重要的作用。但萌生植株作为辽东栎顺利通过瓶颈的一种手段,作为辽东栎种群繁衍和稳定的一种途径,在辽东栎种群的更新中也起着一定的作用。     

Correlation of carboxylic acid pKa to protein binding and antibacterial activity of a novel class of bacterial translation inhibitors

Previously we reported the discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding. This report describes efforts directed toward understanding the relationship of the acidity of the carboxylic acid with the extent of protein binding. The pK(a) of the acid was modified via the synthesis of a number of anthranilic acid analogs which vary the aromatic ring substituent at the 4-position. The pK(a) and HSA binding constants have been determined for each of the analogs. Our results indicate a correlation between pK(a) and HSA K(d). The physical properties and antibacterial activities will be discussed as well as how these results help address the protein binding issue with this series of compounds.