Intrinsically disordered regions in the rodent hepevirus proteome

Hepatitis E virus (HEV) is the causative agent of Hepatitis E infections across the world. Intrinsically disordered protein regions (IDPRs) or intrinsically disordered proteins (IDPs) are regions or proteins that are characterized by lack of definite structure. These IDPRs or IDPs play significant roles in a wide range of biological processes, such as cell cycle regulation, control of signaling pathways, etc. IDPR/IDP in proteins is associated with the virus''s pathogenicity and infectivity. The prevalence of IDPR/IDP in rat HEV proteome remains undetermined. Hence, we examined the unstructured/disordered regions of the open reading frame (ORF) encoded proteins of rat HEV by analyzing the prevalence of intrinsic disorder. The intrinsic disorder propensity analysis showed that the different ORF proteins consisted of varying fraction of intrinsic disorder. The protein ORF3 was identified with maximum propensity for intrinsic disorder while the ORF6 protein had the least fraction of intrinsic disorder. The analysis revealed ORF6 as a structured protein (ORDP); ORF1 and ORF4 as moderately disordered proteins (IDPRs); and ORF3 and ORF5 as highly disordered proteins (IDPs). The protein ORF2 was found to be moderately as well as highly disordered using different predictors, thus, was categorized into both IDPR and IDP. Such disordered regions have important roles in pathogenesis and replication of viruses.     

The proteome of gastric lymph in normal and nematode infected sheep

Lymph node cannulation allows the collection of lymph draining from a defined anatomical region. Proteomic analysis of that lymph offers a potentially valuable insight into the immunoinflammatory response of that particular region. In this study, ovine gastric lymph has been used to monitor the proteomic changes occurring in the tissue fluid of the abomasum, in response to infection with the parasitic nematode, Teladorsagia circumcincta. Lymph, collected temporally over an experimental infection period, was analysed by means of 2-DE and subsequent gel analysis using densitometry software. In addition, the composition of the lymphatic proteome was further explored by means of MALDI-TOF and MS/MS analyses. The concentration of gelsolin, alpha-1 beta glycoprotein and haemopexin were altered significantly (p<0.05) with infection.     

The origin of cholesterol in the mesenteric lymph of the rat

These studies were performed to quantitate the amounts of newly synthesized cholesterol secreted in the mesenteric lymph of the rat and to define the origin of this cholesterol. In control animals receiving no dietary fat, the amount of newly synthesized sterol entering the lymph increased linearly with respect to time over 24 hr. When a continuous intravenous infusion of chylomicrons was given or when the animals were prefed a diet containing 2.0% cholesterol to inhibit hepatic, but not intestinal or peripheral, cholesterol synthesis, the secretion of newly synthesized sterol in lymph was markedly suppressed, suggesting that the liver was its ultimate site of origin. When the animals were subjected to either blockade of intestinal cholesterol absorption or biliary diversion, there was a decrease in both the newly synthesized and total mass of cholesterol in lymph by approximately 60%, indicating that the majority was normally derived from the absorption of luminal (primarily biliary) sterol. In the absence of dietary cholesterol, the remainder was probably derived from plasma lipoproteins that were filtered through the intestinal capillaries into the lymph. In contrast, when lymph was collected during active fat absorption, the intestine was found to secrete sterol newly synthesized by the epithelium. Such newly synthesized cholesterol was found predominantly in the unesterified fraction and accounted for approximately 27% of the total sterol found in lymph at the end of the experiment. From these studies it was concluded that in the absence of fat absorption, sterol synthesized in the intestinal mucosa was incorporated predominantly into cell membranes and did not enter intestinal lymph to any significant degree. However, during fat absorption, a fraction of this newly synthesized sterol pool was incorporated into lipoproteins and so was delivered through the intestinal lymph to the body pools of cholesterol.     

Morphology of mesenteric lymph nodes after balneologic procedures

Strong and weak sulfate baths produce certain increase in amount and in area of the lymphoid noduli with the germinative center, in the deep cortex and a decrease in the relative area of the medullary substance, while iodobromine baths facilitate to an outgrowth of the medullary cords and to ectasia of sinus lumens. After the balneoprocedures lymphoid noduli appear in the deep cortex; amount of destructive cells in the medullary sinuses, in the medullary cords and in the intermedullary zone decreases; amount of mast cells, eosinophiles and neutrophiles increases (slightly). Blast transformation of small lymphocytes and increase in number of middle lymphocytes are observed in the germinative centers.     

Fructose-2,6-bisphosphate in rat mesenteric lymph nodes

1. The fructose-2,6-bisphosphate (Fru-2,6-P2) content of mesenteric lymph nodes was measured in rats. 2. The effects of Fru-2,6-P2 on the activity of 6-phosphofructo-1-kinase (PFK-1) from rat mesenteric lymph nodes were also studied. 3. The affinity of the enzyme for fructose-6-phosphate was increased by Fru-2,6-P2 whereas the inhibition of the enzyme with high concentrations of ATP was released by Fru-2,6-P2. 4. The activity of lymphocyte PFK-1 was highly stimulated in a simultaneous presence of low concentrations of AMP and Fru-2,6-P2. 5. These results show that rat lymphocyte PFK-1 is highly regulated with Fru-2,6-P2 which means that glycolysis in rat lymphocytes is controlled by Fru-2,6-P2.     

Antioxidant defenses in rat intestine and mesenteric lymph

Abstract

Dietary oxysterols can reach the circulation and this may contribute to atherosclerosis, where lipid oxidation is thought to be important. There is also evidence that, in rats,peroxidized lipids are absorbed and transported into lymph [Aw TY, Williams MW, Gray L. Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH. Am J Physiol 1992; 262: G99–G106], although the method used to detect lipid peroxides lacked specificity. We tested whether intragastric administration of vegetable oils containing triglyceride hydroperoxides (TG-OOH) to rats resulted in detectable lipid hydroperoxides in mesenteric lymph. Using sensitive HPLC with postcolumn chemiluminescence detection, we were unable to detect hydroperoxides of triglycerides, cholesterylesters or phospholipids during the course of lipid absorption, and lymph levels of ascorbate, urate, α-tocopherol and ubiquinol-9 did not change significantly. By contrast, we observed a striking reducing activity judged by the efficient reduction of administered ubiquinones-9 and -10 to the corresponding ubiquinols. Exposure of rat lymph and isolated chylomicrons to aqueous peroxyl radicals revealed patterns of antioxidant consumption and lipid hydroperoxide formation similar to those described previously for human extravascular fluids and isolated lipoproteins, respectively. In particular, rates of TG-OOH formation in lymph and chylomicrons were very low to undetectable as long as ascorbate and/or ubiquinols were present, but subsequently proceeded in a chain reaction despite the presence of α-tocopherol. These studies demonstrate that rat intestine and mesenteric lymph possess efficient antioxidant defenses against preformed lipid hydroperoxides and (peroxyl) radical mediated lipid oxidation. We conclude that dietary lipid hydroperoxides or postprandial oxidation of lipids are not likely to contribute to these particular forms of oxidized lipids in circulation and aortic tissue.     

Ultrastructural study of mesenteric lymph nodes in old rats

The large mononuclear cells showed the features of mononuclear phagocytes and were mainly characterised by a large number of vacuolar structures. The results suggest that there is storage of lipids and mucosubstances, probably in enlarged lysosomes.     

Comparison of lung proteome profiles in two rodent models of pulmonary arterial hypertension

We studied the lung proteome changes in two widely used models of pulmonary arterial hypertension (PAH): monocrotaline (MCT) injection and chronic hypoxia (CH); untreated rats were used as controls (n = 6/group). After 28 days, invasive right ventricular systolic pressure (RVSP) was measured. Lungs were immunostained for alpha-smooth muscle actin (alphaSMA). 2-DE (n = 4/group) followed by nano-LC-MS/MS was applied for protein identification. Western blotting was used additionally if possible. RVSP was significantly increased in MCT- and CH-rats (MCT 62.5 +/- 4.4 mmHg, CH 62.2 +/- 4.1 mmHg, control 25.0 +/- 1.7 mmHg, p<0.001). This was associated with an increase of alphaSMA positive vessels. In both groups, there was a significantly increased expression of proteins associated with the contractile apparatus (diphosphoHsp27 (p<0.001), Septin2 (p<0.001), F-actin capping protein (p<0.01), and tropomyosin beta (p<0.02)). In CH, proteins of the nitric oxide (Hsc70; p = 0.002), carbon monoxide (biliverdin reductase; p = 0.005), and vascular endothelial growth factor (VEGF) pathway (annexin 3; p<0.001) were significantly increased. In MCT, proteins involved in serotonin synthesis (14-3-3; p = 0.02), the enhanced unfolded protein response (ERp57; p = 0.02), and intracellular chloride channels (CLIC 1; p = 0.002) were significantly elevated. Therefore, MCT- and CH-induced vasoconstriction and remodeling seemed to be mediated via different signaling pathways. These differences should be considered in future studies using either PAH model.     

Microscopic structure of the mesenteric lymph nodes of monkeys]

The structure and cytoarchitectonics of mesenterial lymph nodes were studied in thin histological sections by different methods. Under study were 5 rhesus monkeys and 3 hamadryas baboons. The square surface of different structural elements of the nodes (the cortical and medullary substances, follicles, the connective tissue framework, sinuses) occupied by them in sections was calculated and the amount of cellular elements was determined (%). Comparison of the data obtained in rhesus monkeys and baboons speaks of certain species-specific features of the organs under study. The morphological picture of lymph node sections in baboons (as compared with rhesus monkeys) might suggest earlier ageing of nodes in baboons.     

肠淋巴液引流对失血性休克大鼠红细胞流变性的影响

目的:观察肠淋巴液引流对失血性休克大鼠红细胞流变性指标以及血液黏度的作用。方法:Wistar雄性大鼠均分为假休克组、休克组(复制失血性休克模型)、引流组(复制失血性休克模型,自低血压1 h引流休克肠淋巴液)。在低血压3 h或相应时间,经腹主动脉取血,检测红细胞参数、红细胞电泳、红细胞沉降率(ESR)以及血液黏度,计算红细胞聚集指数、红细胞变形指数。结果:与假休克组比较,休克组红细胞数量、红细胞比积(HCT)、血红蛋白(Hb)、平均红细胞血红蛋白浓度(MCHC)、红细胞电泳率与迁移率、红细胞变形指数、全血黏度、全血低切与高切相对黏度和还原黏度显著降低,休克组平均红细胞体积、红细胞电泳时间、ESR、血沉方程K值与校正K值、红细胞聚集性指数、血浆黏度显著升高;引流组MCHC、红细胞电泳率与迁移率、全血黏度、全血低切与高切还原黏度均显著降低,引流组红细胞体积分布宽度(RDW-SD)显著增加。同时,引流组HCT、RDW-SD、红细胞变形指数、全血黏度、全血低切与高切相对黏度显著高于休克组;ESR、血沉方程K值与校正K值、红细胞聚集性指数、血浆黏度显著低于休克组。结论:休克肠淋巴液引流可改善失血性休克大鼠红细胞流变行为,从而改善血液流变性。     

Scanning electron microscope observation of the dog mesenteric lymph node

Summary The perfused large mesenteric lymph node of the dog was observed under the scanning electron microscope.The lymph sinus contains reticulum cells which mostly are two-dimensionally formed stellate plates oriented in a uniform direction. Large round macrophages are loosely fixed by the reticulum cell processes. No intermediate type between both cells has been observed. Macrophages having a few long tentacle-like projections are densely covered by clubbed cytoplasmic processes. Smaller round cells, probably plasma cells and lymphocytes also remained in the sinus.The pulp of the node is built up by reticulum cells, much smaller than those in the sinus, and by densely packed round cells including a few macrophages.The trabeculae and the reticulum of the nodal parenchyme form a continuous structure.Cordial thanks are expressed to Dr. Toshihiro Ishii, Professor of Anatomy of the Tohoku University Medical School, for his valuable advice. Thanks are also due to the kind cooperation of Mr. Akira Kubotsu of the Central Research Laboratories, Kuraray Co. Ltd.     

Mitogen responsiveness and inhibitory activity of mesenteric lymph node cells

Summary Blood-, lymph node-, and tumour-infiltrating lymphocytes (PBL, LNC, and TIL, respectively) from patients with colonic neoplasms were tested for responsiveness to phytohaemagglutinin (PHA). All populations responded, with LNC and PBL showing comparable reactivities while TIL were less reactive as assessed by incorporation of ³H-thymidine. Increased mitogen responsiveness was observed for T cells enriched by SRBC rosette formation or passage through nylon columns. Mitomycin C-treated LNC and TIL inhibited PHA induced ³H-thymidine incorporation of admixed autologous PBL, suggesting the presence of suppressor cells. Suppressor activity resided primarily in the SRBC rosetting population and was dose-dependent, with increasing numbers of LNC giving greater diminution of PHA response. Suppression by LNC was apparent only when they were added to PBL responders within 6 h of the initiation of stimulation assays, in common with the effects of Concanavalin A (Con A)-induced suppressors on PBL phytomitogen responsiveness. Con A-induced and LNC-suppressor activity could be reversed by addition of lymphocyte-conditioned medium (CM) containing T cell growth factor (TCGF; interleukin IL-2). These data provide further evidence that the suppressor phenomena observed in this system are a function of activated T cells present both in drainage lymph nodes and at the tumour site.     

Three-dimensional analysis of erythrophagosomes in rat mesenteric lymph node macrophages

Erythrocytes extravasated into the sinuses of the rat mesenteric lymph nodes as a result of short-term clamping of the portal vein were, although autologous, phagocytized markedly by the lymph node macrophages at 1 hr after reopening of the vein. The erythrophagosomes formed in the macrophages were exposed three-dimensionally by the cellular matrix maceration method and observed with a high-resolution scanning electron microscope. These results were compared to those obtained by conventional transmission electron microscopy. The process of degradation of an erythrocyte took about 6 hr. Coated pits were formed on the erythrophagosomal membrane at the early stage, and the erythrophagosomes were degraded by two different pathways: 1) the degradative pathway by invaginations of the phagosomal membrane, through which the erythrophagosome shrank and broke into secondary lysosomes, and 2) the hemolytic degradative pathway, by which it lost its content and formed a ghost.     

Defining the regulated secreted proteome of rodent adipocytes upon the induction of insulin resistance

Insulin resistance defines the metabolic syndrome and precedes, as well is the hallmark of, type II diabetes. Adipocytes, besides being a major site for energy storage, are endocrine in nature and secrete a variety of proteins, adipocytokines (adipokines), that can modulate insulin sensitivity, inflammation, obesity, hypertension, food intake (anorexigenic and orexigenic), and general energy homeostasis. Recent data demonstrates that increased intracellular glycosylation of proteins via O-GlcNAc can induce insulin resistance and that a rodent model with genetically elevated O-GlcNAc levels in muscle and fat displays hyperleptinemia. The link between O-GlcNAc levels, insulin resistance, and adipocytokine secretion is further explored here. First, with the use of immortalized and primary rodent adipocytes, the secreted proteome of differentiated adipocytes is more fully elucidated by the identification of 97 and 203 secreted proteins, respectively. Mapping of more than 80 N-linked glycosylation sites on adipocytokines from the cell lines further defines this proteome. Importantly, adipocytokines that are modulated when cells are shifted from insulin responsive to insulin resistant conditions are determined. By the use of two protocols for inducing insulin resistance, classical hyperglycemia with chronic insulin exposure and pharmacological elevation of O-GlcNAc levels, several proteins are identified that are regulated in a similar fashion under both conditions including HCNP, Quiescin Q6, Angiotensin, lipoprotein lipase, matrix metalloproteinase 2, and slit homologue 3. Detection of these potential prognostic/diagnostic biomarkers for metabolic syndrome, type II diabetes, and the resulting complications of both diseases further establishes the central role of the O-GlcNAc modification of intracellular proteins in the pathophysiology of these conditions.     

Basic mechanisms controlling lymph transport in the mesenteric lymphatic net

This minireview summarizes an oral presentation given at the National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health workshop "Lymphatics in the Digestive System: Physiology, Health, and Disease" in Bethesda, Maryland on November 3-4, 2009. The concepts of extrinsic and intrinsic pumps, as well as intrinsic and extrinsic flows, are discussed in relation to the lymph transport in mesenteric lymphatic vessels. Age-related alterations in the structure and regulatory mechanisms of lymph flow in mesenteric lymphatic vessels may provide the basis for their diminished ability to work during the periods of increased functional loads in them. The recent development of modern experimental tools provides the opportunity to extend the knowledge on lymph transport function of lymphatic vessels that is absolutely necessary to maintain fluid and macromolecular homeostasis and to provide a transportation route for lipids adsorbed in gut and to immune cells.