Membrane insertion and topology of the p7B movement protein of Melon Necrotic Spot Virus (MNSV)

Cell-to-cell movement of the Melon Necrotic Spot Virus (MNSV) is controlled by two small proteins working in trans, an RNA-binding protein (p7A) and an integral membrane protein (p7B) separated by an amber stop codon. p7B contains a single hydrophobic region. Membrane integration of this region was observed when inserted into model proteins in the presence of microsomal membranes. Furthermore, we explored the topology and targeting mechanisms of full-length p7B. Here we present evidence that p7B integrates in vitro into the ER membrane cotranslationally and with an Nt-cytoplasmic/Ct-luminal orientation. The observed topology was monitored in vivo by fusing GFP to the Ct of p7B, enabling the overexpression in Escherichia coli cultures. Finally, the topology of a putative p14 movement protein was established by replacing the amber stop codon located between p7A and p7B.     

Leptin and leptin receptor genes in Atlantic salmon: Cloning, phylogeny, tissue distribution and expression correlated to long-term feeding status

The present study reports the complete coding sequences for two paralogues for leptin (sLepA1 and sLepA2) and leptin receptor (sLepR) in Atlantic salmon. The deduced 171-amino acid (aa) sequence of sLepA1 and 175 aa sequence for sLepA2 shows 71.6% identity to each other and clusters phylogenetically with teleost Lep type A, with 22.4% and 24.1% identity to human Lep. Both sLep proteins are predicted to consist of four helixes showing strong conservation of tertiary structure with other vertebrates. The highest mRNA levels for sLepA1 in fed fish (satiation ration = 100%) were observed in the brain, white muscle, liver, and ovaries. In most tissues sLepA2 generally had a lower expression than sLepA1 except for the gastrointestinal tract (stomach and mid-gut) and kidney. Only one leptin receptor ortholog was identified and it shares 24.2% aa sequence similarity with human LepR, with stretches of highest sequence similarity corresponding to domains considered important for LepR signaling. The sLepR was abundantly expressed in the ovary, and was also high in the brain, pituitary, eye, gill, skin, visceral adipose tissue, belly flap, red muscle, kidney, and testis. Fish reared on a rationed feeding regime (60% of satiation) for 10 months grew less than control (100%) and tended to have a lower sLepA1 mRNA expression in the fat-depositing tissues visceral adipose tissue (p < 0.05) and white muscle (n.s.). sLepA2 mRNA levels was very low in these tissues and feeding regime tended to affect its expression in an opposite manner. Expression in liver differed from that of the other tissues with a higher sLepA2 mRNA in the feed-rationed group (p < 0.01). Plasma levels of sLep did not differ between fish fed restricted and full feeding regimes. No difference in brain sLepR mRNA levels was observed between fish fed reduced and full feeding regimes. This study in part supports that sLepA1 is involved in signaling the energy status in fat-depositing tissues in line with the mammalian model, whereas sLepA2 may possibly play important roles in the digestive tract and liver. At present, data on Lep in teleosts are too scarce to allow generalization about how the Lep system is influenced by tissue-specific energy status and, in turn, may regulate functions related to feed intake, growth, and adiposity in fish. In tetraploid species like Atlantic salmon, different Lep paralogues seems to serve different physiological roles.     

赖型钩端螺旋体OmpA膜蛋白Loa22基因的克隆分析及蛋白表达

目的分析赖型钩端螺旋体OmpA膜蛋白Loa22的结构特征,并对其进行克隆表达。方法分别以问号状赖型钩体017株、56601株及双曲状钩体PatocI株基因组为模板PCR扩增目的基因。构建Loa22基因与质粒pGEX-4T-1的重组原核表达质粒,克隆筛选并测序。利用Bioedit、Dnaman、PSIPRED、NCBI及SignaIP等对其结构进行分析。最后在大肠杆菌JM109中诱导表达目的蛋白。结果不同毒力赖型钩体均能扩增出约600bp的片段,而PatocI株则未能扩增出目的片段;PCR、双酶切及测序证实pGEX-Loa22构建成功:分析显示不同赖型钩体的Loa22的同源性很高,C末端都具有同OmpA一致的保守序列及肽聚糖相关基序,都有信号肽序列;表达产物经SDS-PAGE检测证明是目的蛋白。结论赖型钩体具有编码OmpA膜蛋白的Loa22基因,可能与赖型钩体毒力和免疫原性存在某种相关性。     

The hypoglycemic activity of Lithocarpus polystachyus Rehd. leaves in the experimental hyperglycemic rats

Ethnopharmacological relevance

Leaves of Lithocarpus polystachyus Rehd. are used for the treatment of disorders such as diabetes, hypertension, and epilepsy in folk medicine of South China. The possible antidiabetic effects of the leaves were investigated in experimental type 2 and type 1 diabetic rats.

Materials and methods

Type 2 diabetic rats received orally three different extracts of Lithocarpus polystachyus Rehd. leaves for 4 weeks (aqueous extract [ST-1], ethanol extract [ST-2], flavonoid-rich fraction [ST-3]). At the end of the experiment biochemical parameters were tested and livers and pancreases were excised for histological study. After the comparison of the pharmacological test results of the three extracts, the one which showed the best bioactivity was further studied to confirm its antidiabetes effect on both type 2 and type 1 diabetic rats.

Results

Compared to ST-1 and ST-2, ST-3 had better effects on regulation of blood glucose, glycosylated serum protein, cholesterol, triglyceride, malondialdehyde, superoxide dismutase and attenuation of liver injury in type 2 diabetic rats (p < 0.01 or p < 0.05). ST-3 administration for four weeks also significantly reduced the fasting serum insulin and C-peptide level and improved the insulin tolerance (p < 0.05). In type 1 diabetic rats, ST-3 supplement for three weeks caused significant reduction in fasting blood glucose, total cholesterol, triglyceride, urea nitrogen, creatinine and liver mass, along with significantly inhibiting the decline of insulin level compared to diabetic control (p < 0.05 or p < 0.01).

Conclusion

The flavonoid-rich fraction of Lithocarpus polystachyus Rehd. leaves (ST-3) had better beneficial effect than that of the ethanol or aqueous extract in experimental diabetic rats, which means that the bioactivity of the herbal leaves is probably due to the presence of flavonoids. The results also strongly suggest that the antidiabetic effect of ST-3 was possibly through multiple mechanisms of action including blood lipid and antioxidant mediation. The results indicated that the aqueous flavonoid-rich fraction of Lithocarpus polystachyus Rehd. leaves possessed significant protective activity in type 2 and type 1 diabetes.     

支气管哮喘患儿治疗前后血清SOD、VIP、TNF-α和Lep水平变化的临床意义

目的:为了探讨支气管哮喘患儿治疗前后血清SOD、VIP、TNF-α和Lep水平变化的临床意义。方法:放射免疫分析和酶免疫分析测定了87例支气管哮喘患儿和60例正常儿的血清SOD、VIP、TNF-α和Lep水平并进行了比较性分析。结果:在治疗前,87例支气管哮喘患儿血清SOD和VIP水平较之60例正常儿明显降低(t SOD=3.018,t VIP=3.146,P均<0.01),而血清TNF-α和Lep水平明显增高(t TNF-α=4.637,P<0.001,t Lep=3.261,P<0.01)。在治疗后,87例支气管哮喘患儿血清SOD和VIP水平恢复至正常,较之正常儿无明显的差异(t SOD=1.586,t VIP=1.563,P均>0.05),而血清TNF-α和Lep水平明显降低,但相较之正常儿增高(t TNF-α=2.103,t Lep=2.243,Pall<0.05)。结论:SOD和VIP水平的测定是诊断支气管哮喘患儿的良好指标,而且在糖皮质激素综合治疗后,可以进行随访和疗效的考核。     

血清IL-6、hs—CRP、Lep、E2及神经功能相关指标在老年高血压脑出血患者中的检测价值

目的：研究观察血清白介素-6（IL-6）、超敏C反应蛋白（hs—CRP）、瘦素（Lep）、雌二醇（E2）及神经功能相关指标在老年高血压脑出血患者中的检测价值。方法：选取本院收治的54例高血压脑出血患者为观察组,并以54名同龄健康人员为对照组,然后将两组的血清IL-6、hs—CRP、Lep、E2及神经功能相关指标进行检测与比较,并比较轻度、中度及重度高血压脑出血患者的检测水平,同时以Logistic回归分析处理上述血清指标与疾病的关系。结果：观察组的血清IL-6、hs—CRP、Lep及神经功能相关指标均高于对照组,血清E2则低于对照组,同时重度患者的血清IL-6、hs—CRP、Lep及神经功能相关指标均高于轻度及中度患者,中度患者则高于轻度患者,重度患者的血清E2低于轻度及中度患者,中度患者低于轻度患者,差异具有统计学意义（P〈0．05）,且Logistic回归分析显示,上述血清指标均与老年高血压脑出血有密切的关系。结论：血清IL-6、hs—CRP、Lep、E2及神经功能相关指标在老年高血压脑出血患者中的检测价值较高,且对于疾病的严重程度也有积极的反应作用。     

T cell responses to recombinant isoforms, synthetic peptides and a mutant variant of Lep d 2, a major allergen from the dust mite Lepidoglyphus destructor

BACKGROUND: The dust mite Lepidoglyphus destructor is an important cause of allergic reactions to dust, especially in farming environments. Two isoforms, recombinant (r)Lep d 2.01 and rLep d 2.02, of the major allergen Lep d 2, have previously been expressed as recombinant proteins. These isoforms differ 10.4% at the amino acid level. Furthermore, a mutant form of Lep d 2.01 (rLep d 2.6Cys) with a highly reduced IgE reactivity, has also been produced. OBJECTIVE: To investigate the T cell responses to the recombinant isoforms of Lep d 2, the Lep d 2.6Cys mutant and peptides of Lep d 2, in allergic and non-allergic individuals. METHODS: Peripheral blood mononuclear cells from 18 allergic and 16 non-allergic individuals were stimulated with the different antigens and the proliferative responses were measured. The cytokine production (interleukin (IL)-4, IL-5 and interferon (IFN)-gamma) were measured by ELISA. RESULTS: Higher T cell proliferation was measured to isoform 01 than to 02 in 28/34 subjects. The responses to rLep d 2.6Cys were lower than to isoform 01 in most subjects, but higher than to Lep d 2.02. Two immuno-dominant peptides, corresponding to amino acid residue 11-25 and 61-75 were identified. The atopic subjects produced significantly lower IFN-gamma in response to Lep d 2.01 as compared to the non-atopics. CONCLUSIONS: There was a significant difference in T cell response between the two isoforms of rLep d 2. The hypoallergenic mutant rLep d 2.6Cys was able to evoke a T cell response with a magnitude which is between the two isoforms. Amino acid residue 11-25 and 61-75 are the most frequently recognized parts of Lep d 2 and are likely to contain the immuno-dominant T cell epitopes.     

Graves’病患者^131I治疗前后血清TNF-α和瘦素测定的临床意义

目的探讨Graves’病患者^131I治疗前后血清TNF．仅和瘦素（LEP）水平变化的临床意义。方法用放射免疫分析法对初诊32例Graves’病女性患者^131I治疗前后及30例健康女性血清TNF-α和LEP含量进行测定。结果32例Graves’病患者^131I治疗前血清LEP含量明显低于正常对照组（P〈0．01）;TNF-α含量显著高于正常对照组（P〈0．01）。经^131I治疗血清FT3,FT4和sTSH恢复正常后,其血清LEP含量较治疗前明显升高（P〈0．01）,但与对照组比较差异无显著性（P〉0．05）;TNF-α比治疗前明显降低（P〈0．01）,但仍高于正常对照组（P〈0．05）。结论本研究结果提示测定血清LEP可作为判断Graves’病患者^131I治疗效果的指标;在^131I治疗前后测定Graves’病患者TNF-α可反映其体内的免疫状态,可能与其发病机制和预后有关。     

Allergenic crossreactivity between Lepidoglyphus destructor and Blomia tropicalis

Background In general, the non-pyroglyphid mites Lepidoglyphus destructor and Blomia tropicalis show a different geographical distribution. Allergic sensitization to both species have been demonstrated in several investigations. However, whether this reflects crossreactivity or dual sensitization is so far not known. Objective The aim of the study was to investigate the allergenicity and allergenic crossreactivity of L destructor and B. tropicalis using sera from Sweden and Brazil. Objective Allergens in extracts of L. destructor and B. tropicalis were identified with SDS-PAGE and immunoblotting and the crossreactivity was studied by an immunoblot inhibition method. In addition to mite extracts, a recombinant major allergen of L destructor, Lep d 2, was used. Results The extract prepared from L. destructor contained 21 IgE-binding components when using the Swedish or the Brazilian sera. A 15 kDa allergen was recognized by 85% of the Swedish sera and 78% of the Brazilian. The B. tropicalis extract exposed 23 IgE-binding components when the Brazilian sera were used and 19 when the Swedish sera were used. A total of 83% of the Brazilian sera and 80% of the Swedish sera identified a 14.5 kDa allergen. The IgE response of the Swedish serum pool to 10 B. tropicalis allergens was inhibited by L. destructor extract. Likewise, the response of the Brazilian serum pool to four different L. destructor allergens was inhibited by B. tropicalis extract. The recombinant Lep d 2 allergen inhibited 33% of the IgE binding of the Swedish serum pool to the 14.5 kDa allergen in the B. tropicalis extract. Conclusion crossreactivity with several proteins from L. destructor and B. tropicalis was demonstrated. The results suggest that a B. tropicalis 14.5 kDa allergen is antigenically crossreactive with recombinant L. destructor allergen Lep d 2.