昆虫TMED基因进化及家蚕TMED基因表达模式分析

跨膜p24 (transmembrane emp24 domain, TMED)基因与哺乳动物的免疫反应、信号传导、生长发育和疾病发展等密切相关。然而,昆虫中仅有果蝇TMED的报道。本研究从基因组鉴定了家蚕、赤拟谷盗、烟草天蛾和意大利蜂的TMED家族基因,并发现1个α类、1个β类、1个δ类和多个γ类的TMED家族基因成员构成模式产生于膜翅目分化前昆虫的共同祖先,而果蝇类TMED家族成员构成在进化中形成了独特模式。昆虫TMED家族γ类基因进化速度较快,分化成了TMED6-like、TMED5-like和TMED3-like这3个独立的亚类。TMED5-like基因在膜翅目昆虫发生了丢失,在鳞翅目昆虫祖先中发生了复制,在果蝇类发生了重复。昆虫TMED蛋白除具有典型的TMED结构特征外,还有明显的信号肽。家蚕7个TMED基因分布在6条染色体上,1个基因为单外显子,6个基因为多外显子。从幼虫组织克隆了家蚕7个TMED基因的开放阅读框(open reading frame, ORF)全序列并登录到GenBank数据库。BmTMED1、BmTMED2和BmTMED6在家蚕各个时期和组织中均表达,所...     

Psychedelic-inspired drug discovery using an engineered biosensor

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Effect of IPTG amount on apo- and holo- forms of glycerophosphate oxidase expressed in Escherichia coli

Escherichia coli has proved to be a successful host for the expression of many heterologous proteins, and much efforts have been made toward improving recombinant protein expression including the usage of strong promoters and co-expression with chaperones. But little attention was paid on the relation between expression level and function of the target protein. Glycerophosphate oxidase (GPO) is a protein with FAD cofactor (without free cysteine and disulfide bonds).It was observed that the specific activity of GPO dramatically decreased with the increase of inducer IPTG. In addition, the stability of it decreased correspondingly. The structural difference of samples expressed under varying IPTG was investigated using size-exclusion and reverse-phase high performance liquid chromatography, together with CD spectrum. It was found that the conformation of peptide and organization of subunits were not affected. The loss of specific activity and stability were correlated to incomplete attachment of FAD onto GPO. These results revealed that synthesis speed should be controlled either by reduction of IPTG amount or using weak promoters in the production of GPO.     

Mechanisms of Nerve Capping Technique in Prevention of Painful Neuroma Formation

Nerve capping techniques have been introduced as a promising treatment modality for the treatment of painful neuroma with varied outcomes; however, its exact mechanism is still unknown. RhoA is one of the members of the RAS superfamily of GTPases that operate as molecular switches and plays an important role in peripheral nerve regeneration. Our aim was to investigate the structural and morphologic mechanisms by which the nerve capping technique prevents the formation of painful neuromas after neuroectomy. We also hoped to provide a theoretical basis for this treatment approach. An aligned nanofiber conduit was used for the capping procedure and the sciatic nerve of Sprague-Dawley rats was selected as the animal model. Behavioral analysis, extent of neuroma formation, histological assessment, expressions of pain markers of substance P and c-fos, molecular biological changes as well as ultrastructural features were investigated and compared with the findings in a no-capping control group. The formation of traumatic neuromas was significantly inhibited in the capping group with relatively “normal” structural and morphological features and no occurrence of autotomy and significantly lower expression of pain markers compared to the no-capping group. The gene expression of RhoA was consistently in a higher level in the capping group within 8 weeks after surgery. This study shows that capping technique will alter the regeneration state of transected nerves and reduce painful neuroma formation, indicating a promising approach for the treatment of painful neuroma. The initiation of the “regenerative brake” induced by structural as well as morphological improvements in the severed nerve is theorized to be most likely a key mechanism for the capping technique in the prevention of painful neuroma formation.     

Growth and physiological responses to drought and elevated ultraviolet-B in two contrasting populations of Hippophae rhamnoides

In the southeast of the Qinghai-Tibetan Plateau of China, sea buckthorn ( Hippophae rhamnoides L.), which is a thorny nitrogen-fixing deciduously perennial shrub, has been widely used in forest restoration as the pioneer species. In our study, two contrasting populations from the low and high altitudinal regions were employed to investigate the effects of drought, ultraviolet-B (UV-B) and their combination on sea buckthorn. The experimental design included two watering regimes (well watered and drought stressed) and two levels of UV-B (with and without UV-B supplementation). Drought significantly decreased total biomass, total leaf area and specific leaf area (SLA), and increased root/shoot ratio, fine root/coarse root ratio and abscisic acid content (ABA) in both populations. However, the high altitudinal population was more responsive to drought than the low altitudinal population. On the other hand, elevated UV-B induced increase in anthocyanins in both populations, whereas the accumulation of UV-absorbing compounds occurred only in the low altitudinal population. The drought-induced enhancement of ABA in the high altitudinal population was significantly suppressed in the combination of drought and elevated UV-B. Moreover, significant drought × UV-B interaction was detected on total biomass in both populations, total leaf area and fine root/coarse root in the low altitudinal population, and SLA in the high altitudinal population. These results demonstrated that there were different adaptive responses between two contrasting populations, the high altitudinal population exhibited higher tolerance to drought and UV-B than the low altitudinal population.     

The phenyl linker markedly increases the immunogenicity of the pneumococcal polysaccharide conjugate vaccine

Objective

Capsular polysaccharide (PS) of Streptococcus pneumoniae is a key virulence factor and typically conjugated with a carrier protein. It is necessary to improve the immunogenicity of the conjugate vaccine against S. pneumoniae.

Results

A phenyl linker between tetanus toxoid (TT) and S. pneumoniae Type 14 PS was used to improve the PS-specific immunogenicity of the conjugate vaccine. As compared with the one with the amyl linker (PS-TT), the conjugate with the phenyl linker (PS-phe-TT) decreased the TT-specific IgG titers and significantly increased the PS-specific IgG titers and the IL-5 level.

Conclusion

The phenyl linker could potentiate a robust humoral immune response to PS by decreasing the carrier-induced epitopic suppression effect. PS-phe-TT was expected to act as an effective vaccine against S. pneumoniae.

     

Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute

Disuccinimidyl suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCl2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the twoα subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pI of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.     

Attenuation of Acute Phase Injury in Rat Intracranial Hemorrhage by Cerebrolysin that Inhibits Brain Edema and Inflammatory Response

The outcome of intracerebral hemorrhage (ICH) is mainly determined by the volume of the hemorrhage core and the secondary brain damage to penumbral tissues due to brain swelling, microcirculation disturbance and inflammation. The present study aims to investigate the protective effects of cerebrolysin on brain edema and inhibition of the inflammation response surrounding the hematoma core in the acute stage after ICH. The ICH model was induced by administration of type VII bacterial collagenase into the stratum of adult rats, which were then randomly divided into three groups: ICH + saline; ICH + Cerebrolysin (5 ml/kg) and sham. Cerebrolysin or saline was administered intraperitoneally 1 h post surgery. Neurological scores, extent of brain edema content and Evans blue dye extravasation were recorded. The levels of pro-inflammatory factors (IL-1β, TNF-α and IL-6) were assayed by Real-time PCR and Elisa kits. Aquaporin-4 (AQP4) and tight junction proteins (TJPs; claudin-5, occludin and zonula occluden-1) expression were measured at multiple time points. The morphological and intercellular changes were characterized by Electron microscopy. It is found that cerebrolysin (5 ml/kg) improved the neurological behavior and reduced the ipsilateral brain water content and Evans blue dye extravasation. After cerebrolysin treated, the levels of pro-inflammatory factors and AQP4 in the peri-hematomal areas were markedly reduced and were accompanied with higher expression of TJPs. Electron microscopy showed the astrocytic swelling and concentrated chromatin in the ICH group and confirmed the cell junction changes. Thus, early cerebrolysin treatment ameliorates secondary injury after ICH and promotes behavioral performance during the acute phase by reducing brain edema, inflammatory response, and blood–brain barrier permeability.