Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca²⁺ concentration ([Ca²⁺]_c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10^–6, 10^–5, and 10^–3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10^–6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10^–3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca²⁺]_c in isolated alveoli. The morphological reactions and changes in [Ca²⁺]_c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca²⁺]_c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca²⁺]_c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca²⁺]_c. The dynamics of [Ca²⁺]_c of the gland alveoli are mostly dependent on extracellular Ca²⁺.     

Evidence for linkage between K88ab, K88ac intestinal receptors to Escherichia coli and transferrin loci in pigs

Segregation at the loci coding for the K88ab and K88ac small intestinal receptors to E. coli adhesins (K88abR, K88acR) and at the transferrin (TF) locus was studied in 38 pig families including 273 piglets. The TF locus showed a segregation deviation towards the B variant while each of the K88 receptors behaved as a single autosomal dominant gene. Recombinants between K88abR and K88acR provide evidence that they are under the control of two different loci. Thirty-two triple backcross families were selected to test linkage and estimate recombination rates (θ). Our results demonstrate that the two K88 receptor loci are closely linked (θ= 0.02) with a maximum lod score value (Z_m) of 46.0. In addition, they are linked to the TF locus, θ= 0.14, Z_m= 19.6 for the K88abR locus and θ= 0.16, Z_m= 17.9 for the K88acR locus. The estimated recombination rates, smaller in males than in females, are consistent with the order TF-K88abR-K88acR. This linkage thus localizes the K88 loci, as the TF locus, on chromosome 13.     

UreB immunodominant epitope-specific CD8+ T-cell responses were beneficial in reducing gastric symptoms in Helicobacter pylori-infected individuals

Background and aims

Although Helicobacter pylori is recognized as an extracellular infection bacterium, it can lead to an increase in the number of CD8⁺ T cells after infection. At present, the characteristics of H. pylori antigen-specific CD8⁺ T cells and the epitope response have not been elucidated. This study was focused on putative protective antigen UreB to detect specific CD8⁺ T-cell responses in vitro and screen for predominant response epitopes.

Methods

The PBMCs collected from H. pylori-infected individuals were stimulated by UreB peptide pools in vitro to identify the immunodominant CD8⁺ T-cell epitopes. Furthermore, their HLA restriction characteristics were detected accordingly by NGS. Finally, the relationship between immunodominant responses and appearance of gastric symptoms after H. pylori infection was conducted.

Results

UreB-specific CD8⁺ T-cell responses were detected in H. pylori-infected individuals. Three of UreB dominant epitopes (A-2 (UreB_443–451: GVKPNMIIK), B-4 (UreB_420–428: SEYVGSVEV), and C-1 (UreB_5–13: SRKEYVSMY)) were firstly identified and mainly presented by HLA-A*1101, HLA-B*4001 and HLA-C*0702 alleles, respectively. C-1 responses were mostly occurred in H. pylori-infected subjects without gastric symptoms and may alleviate the degree of gastric inflammation.

Conclusions

The UreB dominant epitope-specific CD8⁺ T-cell response was closely related to the gastric symptoms after H. pylori infection, and the C-1 (UreB_5-13) dominant peptides may be protective epitopes.     

动物胃粘膜提取物对双歧杆菌的促生长作用

本文阐述了不同浓度的动物胃粘膜提取物对双歧杆菌生长繁殖的影响。试验结果表明,胃粘膜提取物对双歧杆菌有明显的促进作用,而且随着浓度增加为1％时,其菌数为4.45亿/ml,而不加提取物的菌数为2.53亿/ml,加胃粘膜提取物的比不加的菌数增长175.9％,促生长作用明显。证明胃粘膜提取物中含有促进双歧杆菌生长因子。     

Culturing the epiblast cells of the pig blastocyst

Summary Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.     

Vagal nerve and the gastric mucosal defense

An essential role for an intact vagal nerve has been proven in the development of gastric mucosal cyto- and general protection. On the other hand, chemically-induced (ethanol, HCl, indomethacin) gastric mucosal damage is enhanced after acute surgical vagotomy. The aims of this paper were to study the possible mechanisms of the vagal nerve in the development of gastric mucosal defense. The following questions were addressed: 1) effect of surgical vagotomy on the development of ethanol- (ETOH), HCl-, and indomethacin (IND)-induced gastric mucosal damage; 2) changes in the gastric mucosal defense by scavengers, prostacyclin and other compounds (small doses of atropine and cimetidine: 3) changes in the gastric mucosal vascular permeability due to chemicals; 4) effect of indomethacin in the ETOH and HCl models with and without surgical vagotomy; 5) changes in the gastric mucosal content of prostacyclin and PGE₂ in the ETOH and HCl models after surgical vagotomy; and 6) changes in the role of SH-groups in gastric mucosal defense after surgical vagotomy. It was found that: 1) the gastric mucosal damage produced by chemicals (ETOH, HCl, and indomethacin) was enhanced after surgical vagotomy; 2) the cyto- and general gastric protective effects of β-carotene, prostacyclin, and small doses of atropine and cimetidine disappeared after surgical vagotomy; 3) the vascular permeability due to chemicals (ETOH, HCl, indomethacin) significantly increased after surgical vagotomy in association with an increase in both number and severity of gastric mucosal lesions; 4) IND alone (in animals with an intact vagus) did not produce gastric mucosal lesions (in 1-h experiments), but it aggravated ETOH-induced gastric mucosal damage (both its number and severity); 5) the gastric mucosal levels of prostacyclin and PGE₂ decreased after surgical vagotomy; 6) IND application (after surgical vagotomy) decreased further the tissue levels of prostacyclin and PGE₂ in association with an increase of gastric mucosal damage; and 7) the gastric mucosal protective effects of SH-groups were abolished by surgical vagotomy.     

Dampening HOTAIR sensitizes the gastric cancer cells to oxaliplatin through miR-195-5p and ABCG2 pathway

Long non-coding RNAs (lncRNA) have an extensive role in the progression and chemoresistance of gastric cancer (GC). Deeply study the regulatory role of lncRNAs could provide potential therapeutic targets. The aim of this study is to explore the regulatory role of HOTAIR in the progression and oxaliplatin resistance of GC. The expression of HOTAIR in GC and cell lines were detected by using qRT-PCR. Cell proliferation and apoptosis were analysed by CCK-8, EdU incorporation and flow cytometry. Luciferase reporter assay was used to identify the interaction between HOTAIR and ABCG2 (ATP-binding cassette (ABC) superfamily G member 2, ABCG2) via miR-195-5p. The regulatory functions were verified by using molecular biology experiments. HOTAIR was significantly overexpressed in GC and associated with poor prognosis. Knock-down of HOTAIR inhibited the GC cells proliferation and oxaliplatin resistance, while overexpression of HOTAIR showed opposite functions. Further studies found that HOTAIR acted as a competing endogenous RNA (ceRNA) to absorb miR-195-5p and elevated the expression of ABCG2, which leads to resistance of GC cells to oxaliplatin. Taken together, our findings demonstrated that HOTAIR regulates ABCG2 induced resistance of GC to oxaliplatin through miR-195-5p signalling and illustrate the great potential of developing new therapeutic targets for GC patients.     

血清TAP、TFF3与晚期胃癌患者含奥沙利铂化疗方案敏感性和预后的关系

摘要 目的：探讨血清肿瘤异常蛋白（TAP）、三叶因子3（TFF3）与晚期胃癌患者应用含奥沙利铂化疗方案敏感性和预后的关系。方法：选择2017年1月至2020年1月河北大学附属医院收治的115例晚期胃癌患者,所有患者接受含奥沙利铂化疗方案治疗,根据疗效分为敏感组（47例）和耐药组（68例）。化疗前检测血清TAP、TFF3水平,受试者工作特征（ROC）曲线分析TAP、TFF3预测晚期胃癌患者接受含奥沙利铂化疗疗效的价值。治疗后随访,Wilcoxon检验不同血清TAP、TFF3表达下晚期胃癌患者中位OS时间差异。结果：耐药组血清TAP、TFF3水平高于敏感组（P＜0.05）。TAP、TFF3预测晚期胃癌患者含奥沙利铂化疗耐药的曲线下面积分别为0.717、0.690,联合TAP和TFF3预测晚期胃癌患者含奥沙利铂化疗耐药的曲线下面积为0.801,高于单独TAP、TFF3单独检测。随访期间失访2例,死亡54例,高水平TAP、高水平TFF3晚期胃癌患者中位OS时间短于低水平TAP、低水平TFF3晚期胃癌患者（P＜0.05）。结论：对含奥沙利铂化疗耐药的晚期胃癌患者血清TAP、TFF3水平显著增高,高水平TAP、TFF3晚期胃癌患者中位OS时间较短,联合检测血清TAP和TFF3可预测晚期胃癌患者化疗反应性和预后。     

Signal transduction pathways in guinea pig sperm

Trifluoperazine (TFP), the antagonist of calmodulin (CaM). significantly stimulated the capacitation and acrosome reaction of guinea pig spermatozoa at the concentration of 10-100μmol/L, independent of the external Ca2+. Forskolin, dbcAMP and caffeine evidently promoted the occurrence of acrosome reaction of spermatozoa at early capacitation stage (5 h) in nonsynchronous system but not in synchronous system. If the spermatozoa were capacitated for 15 h in synchronous system, the above three drugs significantly stimulated acrosome reaction in a Ca2+-independent manner. Protein kinase C activators, i.e. phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) did not influence the occurrence of acrosome reaction of spermatozoa at early capacitation stage, but significantly increased the acrosome reaction rate in capacitated spermatozoa in a Ca2+-independent manner. In contrast. PKC inhibitor staurosporine significantly inhibited the occurrence of acrosome reaction.     

Solid-phase synthesis of human salivary mucin-derived O-linked glycopeptides

Summary Short glycopeptides derived from salivary mucin have been synthesized in order to delineate the O-glycosylation pattern that is important in the biological activity of mucin. Two glycopoptides, APPETT*AAP-OMe and PAPPSS*SAP-OMe (*=-d-GalNAc), were prepared by solid-phase peptide synthesis integrating the Fmoc and Boc strategies. Since these peptides contain a C-terminal proline, we devised an efficient strategy using facile Boc chemistry, where the glycosylation at the desired position in the sequence was achieved using corresponding Fmoc-glycoamino acid esters A and B as building blocks. The transformation of the 2-azido group into the acetamido derivative was performed with thioacetic acid on the polymer-bound glycopeptides. Corresponding nonglycosylated peptides were also synthesized to study the influence of -d-GalNAc on peptide backbone conformation.