Thalamic reticular nucleus parcellation delineated by VIP and TRH gene expression in the rat

The distribution of the mRNAs encoding VIP (vasoactive intestinal peptide) and TRH (thyrotropin releasing hormone) was examined in the thalamic reticular nucleus of the adult rat using hybridization histochemistry with S35-labeled oligoprobes. Low levels of TRH expression were found in a medial tier. High levels of VIP expression were found in neurons located in a lateral shell of the same portion. High levels of TRH expression were found in a tier located dorsally and in a tier located ventrally to the first one. In these regions no VIP expression could be detected. These data suggest a parcellation of this nucleus according to the differential expression patterns of TRH and VIP.     

Submolecular recognition profiles in two mouse strains of non-protective and protective antibodies against botulinum neurotoxin A

We have used a set of synthetic overlapping peptides encompassing the entire heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A) to map, in two mouse strains (BALB/c, H2^d, and SJL, H2^S), the regions on the H-chain recognized by Abs in the last bleed of non-protective anti-BoNT/A antisera and in the bleed of protective antisera immediately following it in the bleeding schedule. Although the protective antisera bound slightly higher amounts of total (IgG + IgM) Abs, non-protective and protective BALB/c antisera showed similar peptide-binding profiles involving peptides N6/N7, N25, C2/C3, C9/C10/C11, C15, C18, C24, C30, and C31 and, at lower amounts of bound Abs, peptides N19, C6/C7, and C28. IgG + IgM antibodies of the protective SJL antisera recognized peptides N5, N22, and C21, and these peptides were only slightly recognized (N22, C21) or unrecognized (N5) by the non-protective antisera. Additionally, peptides N7/N8, N25, C11, C15, and less so N27/N28 bound two-fold or more Abs from the SJL protective antisera than the non-protective antisera. The Abs bound to peptides C4 and C29 were of relatively lower affinity. Peptides C2/C3, C7, C18/C19, C24, C30, and C31 bound higher amounts of Abs in the SJL protective versus the non-protective antisera, but the differences were less than double. We also mapped the binding profiles of the IgG Abs in these sera. BALB/c and SJL had 13–36-fold higher of IgG Abs that bound to BoNT/A in the protective antisera relative to non-protective antisera. The IgG Abs in the protective antisera of each mouse haplotype bound to the same peptides that bound total Abs in the correlate antiserum. But in both mouse strains, the non-protective Abs showed little or no IgG Abs that bound to these peptides. In the SJL haplotype, the IgG response to peptide N5 was transient, appearing strongly in early protective Abs and disappearing by day 70. It is not clear whether the response to region N5 plays a role in initiating and contributing to the protective activity of the toxin in the SJL strain in the early stages but is not needed in later hyperimmune stages of the Ab response. It is concluded that the switch in BALB/c and SJL mice from non-protective to protective Abs is not associated with major changes in the epitope-recognition profiles. Although some slight differences between non-protective and protective antisera appeared in their levels of Abs that were bound by some peptides, these differences are not sufficient to explain differences in the protection properties. Protection was mostly associated with the immunoglobulin class of the antibodies. IgM antibodies were non-protective, while IgG Abs produced after the switch were protective.     

Identification of vestibular efferent neurons in the gerbil: histochemical and retrograde labelling

Summary The efferent neurons of the gerbil vestibular system were investigated by retrograde tracing techniques and cytochemical staining for acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and a number of peptides. The location, bilateral distribution, cell area and number of neurons in two identified groups of retrogradely labelled cells were described and quantified. The larger of the two groups was located dorsolateral to the facial nerve genu, ventral and medial to the vestibular nuclei. Unilateral tracer injection in the vestibular end organs labelled cells bilaterally in this and the smaller group, which was located immediately ventral to the genu. No cells were found that individually projected bilaterally to both labyrinths. After injections of horseradish peroxidase (HRP) in the utricle or saccule, significantly more cells were located on the contralateral side of the brainstem. The average (±SD) cross sectional area of labelled cell bodies associated with the otolith organs was 259.8 (±75.2) m². ChAT immuno-reactive and AChE positive cells were found in an area coextensive with the location of the dorsal efferent group. In double-labelling studies, cell bodies in the same group that had been retrogradely labelled with a utricular injection of HRP, were immunocytochemically stained for calcitonin generelated peptide and met-enkephalin. In contrast, the ventral group of efferents did not have cells that were cytochemically stained for either of the acetylcholine-related enzymes or either peptide. The significance of the existence of peptidergic vestibular efferent neurons is discussed.     

胰岛素自复乳化给药系统的制备与质量评价

目的 制备胰岛素自复乳化给药系统并评价其质量.方法 以自复乳的微观结构、体积平均粒径和自乳化速率为评价指标筛选较优处方,并考察其稳定性及体外释放特性.结果 优选的处方组成为内水相-中链甘油三酸酯-亲脂乳化剂-亲水乳化剂(21:24:6:9),平均粒径为6.74±0.55 μm,包裹进入复乳的胰岛素为86.8％±8.2％;于25℃静置30 d后,其显微形态及平均粒径等指标均未见明显变化;8h后胰岛素在体外的累计释放量达约80％,具一定缓释作用.结论 成功制备了胰岛素自复乳化给药系统,可为蛋白多肽类药物的口服给药提供新的方法.     

消斑肽加速氧化低密度脂蛋白诱导的血管平滑肌细胞凋亡

目的：我们已经发现消斑肽能逆转平滑肌源性的泡沫细胞,阻抑C57BL/6J小鼠动脉粥样硬化斑块的形成,对已形成的斑块有消退作用。本文主要探讨消斑肽的作用机制。方法：体外培养的猪主动脉平滑肌细胞,先与15 mg/L的氧化低密度脂蛋白孵育72 h,再与0.1 mg/L消斑肽孵育24 h,应用荧光染色技术、激光共聚焦显微技术和流式细胞术,观察消斑肽对平滑肌细胞的作用。结果：氧化低密度脂蛋白能诱导血管平滑肌细胞发生凋亡;消斑肽能加速氧化低密度脂蛋白诱导的细胞凋亡。结论：综合以前的实验,消斑肽可能是通过加速斑块中细胞的凋亡发挥抗动脉粥样硬化效果。     

Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tethered-ligand

Thrombin is the most potent agonist of human platelets and its effects are primarily mediated through the protease-activated receptors (PARs)-1 and -4. Although PAR-1 has higher affinity for thrombin than PAR-4, both receptors contribute to thrombin-mediated actions on platelets. Recently, a potent and selective PAR-1 antagonist (vorapaxar) was approved for clinical use in selected patients. In contrast, despite the fact that several PAR-4 antagonists have been developed, few of them have been tested in clinical trials.

The aim of the present study was to elucidate the molecular requirements involving the PAR-4 mechanism of activation by peptide analogues of its tethered-ligand.

Eight synthetic PAR-4 tethered-ligand peptide analogues were synthesized and studied for their agonistic/antagonistic potency and selectivity toward human washed platelet aggregation, using light transmittance aggregometry. In addition, in silico studies were conducted to describe the receptor–peptide interactions that are developed following PAR-4 exposure to the above analogues. To provide a first structure-activity relationship rationale on the bioactivity profiles recorded for the studied analogues, molecular docking was applied in a homology model of PAR-4, derived using the crystal structure of PAR-1.

The following peptide analogues were synthesized: AYPGKF-NH₂ (1), GYPGKF-NH₂ (2), Ac-AYPGKF-NH₂ (3), trans-cinnamoyl-AYPGKF-NH₂ (4), YPGKF-NH₂ (5), Ac-YPGKF-NH₂ (6), trans-cinnamoyl-YPGKF-NH₂ (7), and caffeoyl-YPGKF-NH₂ (8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC₅₀ value of 26.2 μM. Substitution of Ala-1 with Gly-1 resulted in peptide (2), which significantly reduces the agonistic potency of peptide (1) by 25-fold. Importantly, substitution of Ala-1 with trans-cinnamoyl-1 resulted in peptide (7), which completely abolishes the agonistic activity of peptide (1) and renders it with a potent antagonistic activity toward peptide (1)-induced platelet aggregation. All other peptides tested were inactive. Tyr-2, residue, along with its neighboring environment was a key determinant in the PAR-4 recognition mode. When the neighboring residues to Tyr-2 provided an optimum spatial ability for the ligand to enter into the binding site of the transmembrane receptor, a biological response was propagated. These results were compared with the predicted binding poses of small molecule antagonists of PAR-4, denoted as YD-3, ML-354, and BMS-986120. π–π stacking interaction with Tyr-183 appears to be critical and common for both small molecules antagonists and the peptide trans-cinnamoyl-YPGKF-NH₂.

Conclusively, the lipophilicity, size, and aromatic nature of the residue preceding Tyr-2 are determining factors on whether a human platelet PAR-4 tethered-ligand peptide analogue will exert an agonistic or antagonistic activity.     

In vivo analysis of intestinal permeability following hemorrhagic shock

AIM: To determine the time course of intestinal permeability changes to proteolytically-derived bowel peptides in experimental hemorrhagic shock. METHODS: We injected fluorescently-conjugated casein protein into the small bowel of anesthetized Wistar rats prior to induction of experimental hemorrhagic shock. These molecules, which fluoresce when proteolytically cleaved, were used as markers for the ability of proteolytically cleaved intestinal products to access the central circulation. Blood was serially sampled to quantify the relative change in concentration of proteolytically-cleaved particles in the systemic circulation. To provide spatial resolution of their location, particles in the mesenteric microvasculature were imaged using in vivo intravital fluorescent microscopy. The experiments were then repeated using an alternate measurement technique, fluorescein isothiocyanate(FITC)-labeled dextrans 20, to semi-quantitatively verify the ability of bowel-derived low-molecular weight molecules( 20 k D) to access the central circulation.RESULTS: Results demonstrate a significant increase in systemic permeability to gut-derived peptides within 20 min after induction of hemorrhage(1.11 ± 0.19 vs 0.86 ± 0.07, P 0.05) compared to control animals. Reperfusion resulted in a second, sustained increase in systemic permeability to gut-derived peptides in hemorrhaged animals compared to controls(1.2 ± 0.18 vs 0.97 ± 0.1, P 0.05). Intravital microscopy of the mesentery also showed marked accumulation of fluorescent particles in the microcirculation of hemorrhaged animals compared to controls. These results were replicated using FITC dextrans 20 [10.85 ± 6.52 vs 3.38 ± 1.11 fluorescent intensity units(× 105, P 0.05, hemorrhagic shock vs controls)], confirming that small bowel ischemia in response to experimental hemorrhagic shock results in marked and early increases in gut membrane permeability. CONCLUSION: Increased small bowel permeability in hemorrhagic shock may allow for systemic absorption of otherwise retained proteolytically-generated peptides, with consequent hemodynamic instability and remote organ failure.     

两株新种放线菌次生代谢产物中的环二肽(英文)

目的:研究两株新种放线菌次生代谢产物。方法:采用柱层析方法分离纯化,根据理化性质和光谱数据鉴定化合物的结构。结果:从白色球孢囊菌分离鉴定了4个环二肽为环(L-脯氨酸-L-天冬酰氨)(1)、环(L-脯氨酸-L-丝氨酸)(2)、环(L-脯氨酸-L-丙氨酸)(3)、环(L-脯氨酸-L-苏氨酸)(4)。从云南韩国生工菌分离鉴定了3个环二肽为环(L-脯氨酸-L-丝氨酸)(2)、环(L-脯氨酸-L-苏氨酸)(4)、cyclo-di-Nδ-acetyl-L-ornithyl(5)。结论:化合物5为新天然产物。首次对化合物1,5的碳氢谱数据进行了归属。所有化合物均首次从该两种放线菌中分离得到。其中化合物1对慢性粒细胞白血病细胞株有一定的细胞毒活性。     

运用基质辅助激光解吸电离飞行时间质谱技术建立鼻咽癌特异血清多肽谱诊断模型

目的 探讨鼻咽癌患者与正常健康者血清蛋白谱的差异,建立特异的鼻咽癌血清多肽谱诊断模型.方法 收集治疗前的鼻咽癌患者和正常健康对照者的血清标本,通过特异的液体磁珠分选后,进行基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)分析,得到鼻咽癌患者的血清多肽谱.采用专用的生物信息学分析软件ClinProtTM进行差异分析,运用遗传算法,建立鼻咽癌的血清多肽谱诊断模型.通过盲法检验模型的灵敏度和特异度.结果 检测出鼻咽癌患者与正常健康者的差异蛋白峰有99个,选取两组间差异有统计学意义的、相对分子质量为808.99、834.61、3954.82和8141.88的蛋白峰,建立鼻咽癌诊断模型,该模型鼻咽癌的识别率为90.0%,预测能力为84.3%,盲法榆验模型的灵敏度为80.0%,特异度为64.0%.结论 鼻咽癌患者与正常对照者的血清间存在蛋白表达的差异,建立的特异的血清多肽谱诊断模型在鼻咽癌的诊断中有一定的应用前景,为寻找特异的鼻咽癌血清肿瘤标记物提供了一定的依据.

Abstract：

Objective To determine the specific serum peptide profile by comparing the serum differences between nasopharyngeal carcinoma patients (NPC) and normal control subjects, and to provide a diagnostic model of nasopharyngeal carcinoma. Methods Pre-treatment serum samples of NPC and normal control subjects were collected and assayed by MALDI-TOF MS analysis. The peptides were extracted with magnetic beads coated with WCX. Mass spectrographic data were analyzed with ClinProtTM software. The specific serum peptide model of NPC was established by using genetic algorithms. The sensitivity and specificity of model were tested by blind testing. Results The serum peptidome patterns of nasopharyngeal carcinoma was obtained. Differential expression of 99 peptide peaks was deteced, and the 808.99 Da,834.61 Da, 3954.82 Da, 8141.88 Da peptide peaks showing statistically significant differences between the two groups, were used to establish the diagnostic model for nasopharyngeal cancer. The recognition rate and predictive power of the model were 90.0% and 84.3%, respectively. The sensitivity and specificity of the model were 80. 0% and 64. 0% determined by blind testing, respectively. Conclusions Significant differences of serum peptide peaks are detected between NPC and normal control groups. The established specific serum peptide model may have certain application in the diagnosis of nasopharyngeal carcinoma, and provides the basis for discovering specific tumor markers of nasopharyngeal carcinoma.     

精子抗原肽P10G和YLP12的合成及在ELISA检测中的意义

目的 人工合成精子肽P10G和YLP12,以此为抗原建立检测抗精子抗体(AsAb)的酶联免疫吸附测定法(ELISA).方法 用PS3全自动合成仪合成精子肽P10G和YLP12,经反相高效液相色谱纯化和质谱鉴定.分别包板制备ELISA试剂盒,用以检测血清中的AsAb.结果 成功合成两种目的肽,质谱结果显示其相对分子质量均与理论相对分子质量相吻合,高效液谱法(HPLC)结果显示其纯度为97.5％和97.8％.以该两种合成肽为抗原,建立AsAb的ELISA,与商品化试剂盒比较均显示极好的一致性(Kappa值为0.77和0.79,＞0.75).结论 全自动固相多肽合成技术可用于合成高纯度的精子抗原肽,所合成的P10G和YLP12均具有较好的抗原活性,可作为包被抗原用于AsAb的ELISA检测.