Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae

The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX₂C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX₂CX₈₁CX₂C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.     

Expression of the α-thionin gene from barley in tobacco confers enhanced resistance to bacterial pathogens

Thionins are cysteine-rich, 5 kDa polypeptides which are toxic to plant pathogens in vitro. Expression of the gene encoding α-thionin from barley endosperm, under the 35S promoter from cauliflower mosaic virus, conferred to transgenic tobacco enhanced resistance to the bacterial plant pathogens Pseudomonas syringae pv. tabaci 153 and P. syringae pv. syringae. The barley α-thionin gene, which has two introns, was correctly spliced in tobacco. The α-thionin in transgenic plants had the expected mobility in the gradient, when separated by high-performance liquid chromatography, reacted with monospecific antibodies and showed the expected antibiotic properties in vitro.     

番木瓜果糖-2,6-二磷酸酶CpF2KP基因克隆和蛋白表达纯化

番木瓜是岭南四大名果之一,在我国东南部地区广泛种植,因其具有食用和药用双重价值,因此深受人们的青睐。果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酯酶(fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase,F2KP)是一个独特的双功能酶,具有激酶功能域和酯酶功能域,能催化生物体内糖代谢的重要调节物果糖-2,6-二磷酸(Fru-2,6-P_(2))的合成和降解。为了研究番木瓜中编码该酶的基因CpF2KP的功能,得到目的蛋白尤为重要。本研究从番木瓜基因组中提取到CpF2KP基因的编码序列(coding sequence,CDS)序列,该基因CDS全长2274 bp。将该基因CDS全长扩增之后选用pGEX-4T-1载体进行原核表达。对载体pGEX-4T-1用EcoRⅠ和BamHⅠ进行双酶切,利用基因重组的方式将扩增序列构建到原核表达载体上。经过诱导条件探索,SDS-PAGE结果显示GST-CpF2KP重组蛋白的大小约为110 kDa,诱导CpF2KP蛋白表达的最适条件为:异丙基β-D-硫代半乳糖苷(isopropyl beta-D-thiogalactopyranoside,IPTG)浓度为0.5 mmol/L,温度28℃。对诱导后的CpF2KP蛋白进行纯化,得到了纯化的单一目的蛋白。此外,检测了该基因的组织表达特性发现该基因在种子中表达量最高,在果肉中表达量最低。该研究为进一步深入揭示番木瓜CpF2KP蛋白的功能及研究该基因参与的生物学过程提供了重要基础。     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Smart hydrogels based on semi-interpenetrating polymeric networks of collagen-polyurethane-alginate for soft/hard tissue healing,drug delivery devices,and anticancer therapies

In this work, hydrogels based on semi-interpenetrating polymeric networks (semi-IPN) based on collagen-polyurethane-alginate were studied physicochemically and from different approaches for biomedical application. It was determined that the matrices in the hydrogel state are crosslinked by the formation of urea and amide bonds between the biopolymer chains and the polyurethane crosslinker. The increment in alginate content (0–40 wt%) significantly increases the swelling capacity, generating semi-crystalline granular structures with improved storage modulus and resistance to thermal, hydrolytic, and proteolytic degradation. The in vitro bioactivity results indicated that the composition of these novel hydrogels stimulates the metabolic activity of monocytes and fibroblasts, benefiting their proliferation; while in cancer cell lines, it was determined that the composition of these biomaterials decreases the metabolic activity of breast cancer cells after 48 h of stimulation, and for colon cancer cells their metabolic activity decreases after 72 h of contact for the hydrogel with 40 wt% alginate. The matrices show a behavior of multidose release of ketorolac, and a higher concentration of analgesic is released in the semi-IPN matrix. The inhibition capacity of Escherichia coli is higher if the polysaccharide concentration is low (10 wt%). The in vitro wound closure test (scratch test) results indicate that the hydrogel with 20 wt% alginate shows an improvement in wound closure at 15 days of contact. Finally, the bioactivity of mineralization was evaluated to demonstrate that these hydrogels can induce the formation of carbonated apatite on their surface. The engineered hydrogels show biomedical multifunctionality and they could be applied in soft and hard tissue healing strategies, anticancer therapies, and drug release devices.     

Population structure of mud flounder Paralichthys orbignyanus from the south-western Atlantic Ocean

The mud flounder Paralichthys orbignyanus (Pleuronectiformes, Paralichthyidae) inhabits shallow waters of low salinities and mud bottoms in the temperate marine coastal regions of the Bonaerensean Ecoregion of the Argentinean Biogeographic Province in the south-western Atlantic Ocean. Specimens of P. orbignyanus were collected from Lagoa dos Patos (LDP) (southern Brazil), Mar Chiquita (MCH) and Marisol (MAR) both located in Buenos Aires (Argentina), and San Antonio Oeste (SAO) in the San Matías Gulf, Rio Negro (Argentina). A fragment of the mitochondrial DNA of the Control Region and seven microsatellite loci were characterized. In the Control Region, P. orbignyanus showed high variability, low nucleotide diversity, mild population expansion and a coalescence time of 35,000 years before the present. Flounders provided evidence of a genetic structure between the sampling sites LDP, MCH, MAR vs. SAO. On the other hand, P. orbignyanus displayed a lower to moderate contemporary genetic structure among all samples except between LDP and MCH. With no evidence of isolation by distance, this analysis supports a model of limited gene flow that is likely to be associated with a consistent larvae retention in all sampling sites. In addition, the present connectivity is ascribed to a lower migration process from SAO in the San Matías Gulf congruent with the prevailing littoral drift.     

Same species,different population dynamics: Spatio-temporal differences of Undaria pinnatifida (Ochrophyta,Phaeophyceae) in the intertidal of North Patagonia,Argentina

Population dynamics can be influenced by physical and biological factors, particularly in stressful environments. Introduced species usually have great physiological plasticity, resulting in populations with different traits. Undaria pinnatifida, a macroalga originally described from northeast Asia, was introduced in Northern Patagonia, Argentina (San Matías Gulf) around 2010. To describe the spatio-temporal variability in population structure and morphometry of U. pinnatifida, we conducted monthly field samplings for 2 years at the intertidal area of two contrasting sites in the San Matías Gulf. Individuals of U. pinnatifida were classified by developmental stage, and their morpho-gravimetric variables were measured. In both intertidal sites juveniles were found in higher proportion during austral autumn and grew and matured during the autumn-winter months (from May onwards), and individuals senesced during early austral summer (December and January). Conversely, density and biomass were largely different between sites, and individuals showed slight morphological variability between sites. Environmental (e.g., nutrient concentration, available substrate) and biological factors (e.g., facilitation, competition) may explain the observed differences. Since there is not a macroalga with U. pinnatifida morphometrical characteristics in the intertidal environments of San Matías Gulf, studying this recent introduction gives us a better understanding of its potential ecological effects.