A Screen for Spore Wall Permeability Mutants Identifies a Secreted Protease Required for Proper Spore Wall Assembly

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.     

添加谷氨酰胺的肠内营养对急性重症胰腺炎大鼠肠上皮细胞间紧密结合蛋白的影响

目的探讨早期肠内营养中应用谷氨酰胺(glutine,Gln)对急性重症胰腺炎(severe acute pancreatitis,SAP)大鼠肠上皮细胞间紧密结合蛋白的作用。方法:雄性SD大鼠89只,随机分成对照组,SAP+PN(肠外营养)组,SAP+EN(肠内营养)组,SAP+EN+Gln,各组又分为4 d喂养组和7 d喂养组。分别在第4 d、第7 d剖杀大鼠检测各组各项指标。免疫组化法检测小肠组织中occludin蛋白的表达。结果:(1)空肠黏膜蛋白质含量测定:各EN组粘膜蛋白质含量显著高于PN组(p<0.01),EN+Gln组的4 d、7 d分别高于EN组的4 d、7 d(p<0.01)。(2)小肠粘膜occludin蛋白表达及含量测定:PN7 d、4 d组的平均灰度值均显著高于EN、EN+Gln组(p<0.01),EN4 d、7 d组平均灰度均显著高于EN+Gln组(p<0.05)。结论:肠内营养中应用谷氨酰胺能更有效增加occludin蛋白表达,改善肠上皮紧密连接,维护肠上皮屏障完整性。     

AICAR Attenuates Organ Injury and Inflammatory Response after Intestinal Ischemia and Reperfusion

Intestinal ischemia and reperfusion (I/R) is encountered in various clinical conditions and contributes to multiorgan failure and mortality as high as 60% to 80%. Intestinal I/R not only injures the intestine, but affects remote organs such as the lung leading to acute lung injury. The development of novel and effective therapies for intestinal I/R are critical for the improvement of patient outcome. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) is a cell-permeable compound that has been shown to possess antiinflammatory effects. The objective is to determine that treatment with AICAR attenuates intestinal I/R injury and subsequent acute lung injury (ALI). Male Sprague Dawley rats (275 to 325 g) underwent intestinal I/R injury with blockage of the superior mesenteric artery for 90 min and subsequent reperfusion. At the initiation of reperfusion, vehicle or AICAR (30 mg/kg BW) was given intravenously (IV) for 30 min. At 4 h after reperfusion, blood and tissues were collected for further analyses. Treatment with AICAR significantly decreased the gut damage score and the water content, indicating improvement in histological integrity. The treatment also attenuated tissue injury and proinflammatory cytokines, and reduced bacterial translocation to the gut. AICAR administration after intestinal I/R maintained lung integrity, attenuated neutrophil chemotaxis and infiltration to the lungs and decreased lung levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Inflammatory mediators, lung-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, were decreased in the lungs and lung apoptosis was significantly reduced after AICAR treatment. These data indicate that AICAR could be developed as an effective and novel therapeutic for intestinal I/R and subsequent ALI.     

早期肠内营养在重症急性胰腺炎中的应用

目的:探讨早期肠内营养在重症急性胰腺炎治疗中的应用价值。方法:将重症急性胰腺炎患者30例分为实验组和对照组,对照组予以常规治疗,实验组在常规治疗的基础上通过鼻空肠管予以早期肠内营养,记录患者WBC、CRP、血淀粉酶、尿淀粉酶恢复时间、白蛋白变化情况、感染率、病死率、并发症发生率、住院时间、住院费用等。结果:实验组WBC、CRP、血淀粉酶、尿淀粉酶恢复时间较对照组明显低,有显著性差异(P<0.05);实验组血清白蛋白升高较对照组明显高,有显著性差异(P<0.05);实验组感染率、病死率、并发症发生率较对照组明显低,但无显著差别(P>0.05);实验组住院时间、住院费用较对照组低,有显著性差异(P<0.05)。结论:早期肠内营养可以改善急性重症胰腺炎营养状况,缩短病程、减低感染率、病死率、并发症发生率、住院时间及住院费用。     

燕麦米汤对严重烧伤患者休克期肠道复苏的影响

目的：研究严重烧伤患者休克期经肠道给予燕麦米汤对肠道复苏的影响。方法：选取烧伤面积≥30%TBSA的患者42例,并随机分为：燕麦米汤组,伤后24h内开始经鼻肠管给予燕麦米汤;对照组,伤后24h内开始经鼻肠管给予50g/L葡萄糖盐水,连续4d,每组21例。在伤后1、2、3、4 d分别检测其血清二氨氧化酶（DAO）值及动脉血乳酸（LAC）含量,观察两组患者腹胀缓解时间、肠鸣音恢复时间等临床指标。结果：两组患者伤后血清二氨氧化酶及动脉血乳酸含量均呈下降趋势,燕麦米汤组血清二氨氧化酶在伤后2、3、4d显著低于对照组（P〈0.01）,而脉血乳酸含量在伤后2、3d显著低于对照组（P〈0.05或0.01）,治疗过程中燕麦米汤组其腹胀缓解时间、肠鸣音恢复时间、排气时间、开始完全肠内营养时间、继发感染例数均优于对照组（P〈0.01）。结论：在严重烧伤休克期尽早鼻饲燕麦米汤能较好地促进肠道复苏。     

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na⁺,K⁺-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na⁺,K⁺-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na⁺,K⁺-ATPase activity afforded by preconditioning be related to cellular neuroprotection.     

Fecal Protease Activity Is Associated with Compositional Alterations in the Intestinal Microbiota

Objective

Intestinal proteases carry out a variety of functions in the gastrointestinal (GI) tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial) of elevated proteases in patients with GI disease is unclear.

Aim

The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples.

Design

In order to capture a wide range of fecal protease (FP) activity stool samples were collected from 30 IBS patients and 24 healthy controls. The intestinal microbiota was characterized using 454 high throughput pyro-sequencing of the 16S rRNA gene. The composition and diversity of microbial communities were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. FP activity levels were determined using an ELISA-based method. FP activity was ranked and top and bottom quartiles (n=13 per quartile) were identified as having high and low FP activity, respectively.

Results

The overall diversity of the intestinal microbiota displayed significant clustering separation (p = 0.001) between samples with high vs. low FP activity. The Lactobacillales, Lachnospiraceae, and Streptococcaceae groups were positively associated with FP activity across the entire study population, whilst the Ruminococcaceae family and an unclassified Coriobacteriales family were negatively associated with FP activity.

Conclusions

These data demonstrate significant associations between specific intestinal bacterial groups and fecal protease activity and provide a basis for further causative studies investigating the role of enteric microbes and GI diseases.     

A Protease that Participates in Yeast Cell Wall Lysis during Zymolyase Digestion

Zymolyase B decreased the turbidity of a yeast cell wall suspension by about 50% and caused release of peptide-mannan from the cell walls. However cell walls treated with the enzyme still maintained the cell shape. The effect of the enzyme on the cell walls was inhibited by yeast mannan and completely counteracted by treatment of the enzyme with DFP. The activity was not affected by pH, but was considerably reduced by incubation of the enzyme at 55°C for 15 min, a treatment that did not affect the proteolytic activity. Heat-treatment decreased the molecular weight of the enzyme from 29,000 to 22,500 and its sensitivity to yeast mannan. Yeast mannan caused noncompetitive inhibition of the proteolytic activity of the native enzyme and competitive inhibition of that of the heat-treated enzyme. Modification of tryptophan residues of Zymolyase B resulted in decreased sensitivity to yeast mannan and a decrease in the activity of the enzyme on yeast cell walls as well as heat-treatment. On the basis of these results, it is hypothesized that Zymolyase B binds to the cell wall mannans and changes their conformation, making the attached proteins susceptible to proteolysis, and then releases peptide-mannan from the cell walls.     

Sex Differences in Ischemia/Reperfusion Injury: The Role of Mitochondrial Permeability Transition

Neurochemical Research - Brain and heart ischemia are among the leading causes of death and disability in both men and women, but there are significant sex differences in the incidence and severity...     

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using "intestinal sacs" are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (P_app)of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.     

Probiotic Bacteria Regulate Intestinal Epithelial Permeability in Experimental Ileitis by a TNF-Dependent Mechanism

Background

We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the “paradoxical” effects of TNF in preventing SAMP ileitis.

Methods

Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice.

Results

Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice.

Conclusions

Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.     

Inhibition of JNK Aggravates the Recovery of Rat Hearts after Global Ischemia: The Role of Mitochondrial JNK

c-Jun N-terminal kinase (JNK), a stress-activated MAPK, is activated during cardiac ischemia-reperfusion (IR). The role of JNK inhibitors in cardioprotection against IR still remains controversial, in part, due to spill-over effects of non-specific inhibitors. In the present study, we sought to examine whether inhibition of JNK by SU3327, a specific JNK inhibitor that inhibits upstream JNK signaling rather than the kinase activity of JNK, improves cardiac function and reduces heart damage during IR. Hearts of male Sprague-Dawley rats perfused by Langendorff were subjected to 25 min of global ischemia followed by 30 min reperfusion in the presence or absence of SU3327. Cardiac function was monitored throughout the perfusion period. Myocardial damage was extrapolated from LDH activity in the coronary effluent. At the end of reperfusion, mitochondria were isolated and used to measure respiration rates and mitochondrial permeability transition pore opening. Protein analysis of mitochondria predictably revealed that SU3327 inhibited JNK phosphorylation. Although SU3327 significantly reduced cell damage during the first minutes of reperfusion, it did not improve cardiac function and, furthermore, reduced the mitochondrial respiratory control index. Interestingly, SU3327 activated the other stress-related MAPK, p38, and greatly increased its translocation to mitochondria. Mitochondrial P-JNK and P-p38 were co-immunoprecipitated with complex III of the electron transfer chain. Thus, JNK plays an essential role in cardiac signaling under both physiological and pathological conditions. Its inhibition by SU3327 during IR aggravates cardiac function. The detrimental effects of JNK inhibition are associated with reciprocal p38 activation and mitochondrial dysfunction.     

Guanylyl Cyclase-G Modulates Jejunal Apoptosis and Inflammation in Mice with Intestinal Ischemia and Reperfusion

Background

Membrane bound guanylyl cyclase-G (mGC-G), a novel form of GC mediates ischemia and reperfusion (IR)-induced renal injury. We investigated the roles of mGC-G in intestinal IR-induced jejunal damage, inflammation, and apoptosis.

Materials and methods

Male C57BL/6 wild-type (WT) and mGC-G gene knockout (KO) mice were treated with a sham operation or 45 min of superior mesenteric arterial obstruction followed by 3, 6, 12, or 24 h of reperfusion.

Results

Sham-operated KO mice had significantly lower plasma nitrate and nitrite (NOx) levels and jejunal villus height, crypt depth, and protein expression of phosphorylated-nuclear factor-kappa-B (NF-κB), phosphorylated-c-Jun N-terminal kinases (JNK) 2/3, phosphorylated-p38, and B-cell lymphoma-2 (Bcl-2). They had significantly greater jejunal interleukin-6 mRNA, cytochrome c protein, and apoptotic index compared with sham-operated WT mice. Intestinal IR significantly decreased plasma NOx in WT mice and increased plasma NOx in KO mice. The jejunal apoptotic index and caspase 3 activities were significantly increased, and nuclear phosphorylated-NF-κB and phosphorylated-p38 protein were significantly decreased in WT, but not KO mice with intestinal IR. After reperfusion, KO mice had an earlier decrease in jejunal cyclic GMP, and WT mice had an earlier increase in jejunal proliferation and a later increase in cytosol inhibitor of kappa-B-alpha. Intestinal IR induced greater increases in plasma and jejunal interleukin-6 protein in WT mice and a greater increase in jejunal interleukin-6 mRNA in KO mice.

Conclusions

mGC-G is involved in the maintenance of jejunal integrity and intestinal IR-induced inflammation and apoptosis. These results suggest that targeting cGMP pathway might be a potential strategy to alleviate IR-induced jejunal damages.     

Noninvasive Monitoring of Small Intestinal Oxygen in a Rat Model of Chronic Mesenteric Ischemia

We noninvasively monitored the partial pressure of oxygen (pO₂) in rat’s small intestine using a model of chronic mesenteric ischemia by electron paramagnetic resonance oximetry over a 7-day period. The particulate probe lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) was embedded into the oxygen permeable material polydimethyl siloxane by cast-molding and polymerization (Oxy-Chip). A one-time surgical procedure was performed to place the Oxy-Chip on the outer wall of the small intestine (SI). The superior mesenteric artery (SMA) was banded to ~30 % of blood flow for experimental rats. Noninvasive measurement of pO₂ was performed at the baseline for control rats or immediate post-banding and on days 1, 3, and 7. The SI pO₂ for control rats remained stable over the 7-day period. The pO₂ on day-7 was 54.5 ± 0.9 mmHg (mean ± SE). SMA-banded rats were significantly different from controls with a noted reduction in pO₂ post banding with a progressive decline to a final pO₂ of 20.9 ± 4.5 mmHg (mean ± SE; p = 0.02). All SMA-banded rats developed adhesions around the Oxy-Chip, yet remained asymptomatic. The hypoxia marker Hypoxyprobe? was used to validate the low tissue pO₂. Brown cytoplasmic staining was consistent with hypoxia. Mild brown staining was noted predominantly on the villus tips in control animals. SMA-banded rats had an extended region of hypoxic involvement in the villus with a higher intensity of cytoplasmic staining. Deep brown stainings of the enteric nervous system neurons and connective tissue both within layers and in the mesentery were noted. SMA-banded rats with lower pO₂ values had a higher intensity of staining. Thus, monitoring SI pO₂ using the probe Oxy-Chip provides a valid measure of tissue oxygenation. Tracking pO₂ in conditions that produce chronic mesenteric ischemia will contribute to our understanding of intestinal tissue oxygenation and how changes impact symptom evolution and the trajectory of chronic disease.     

Liquid Chromatography-Tandem Mass Spectrometry for Analysis of Intestinal Permeability of Loperamide in Physiological Buffer

Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC–MS/MS for the determination of loperamide in HEPES-buffered Ringer''s solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d₃) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer''s solution as a matrix compound.     

腹腔感染大鼠肠粘膜通透性改变与I-FABP表达关系

目的:观察腹腔感染大鼠肠屏障损伤后肠道通透性的改变与I-FABP表达的关系.方法:清洁级健康的成年雄性Wistar大鼠40只,随机分为空白对照组(仅行简单的剖腹手术)和腹腔感染(采用CLP盲肠结扎穿孔法制作腹腔感染模型)术后12h、24 h、36h、48 h组,每组8只,相应时间点处死大鼠后,测定血浆D-乳酸和I-FABP含量.结果:腹腔感染后12h,大鼠血浆D-乳酸含量开始增加(P＜0.05),血浆I-FABP含量也开始增加(P＜0.01);血浆D-乳酸含量在24 h达到最高值(P＜0.01),血浆I-FABP含量在36h达到最高值(P＜0.01).大鼠血浆D-乳酸水平与I-FABP具有显著相关性(r=0.626,P＜0.01).结论:腹腔感染状态下,肠屏障严重受损,肠粘膜通透性的改变与血浆I-FABP水平具有显著的相关性.     

危重症患者肠功能障碍相关临床因素分析

目的:研究危重症患者肠功能障碍的相关临床因素,为有效防治肠功能障碍提供理论依据.方法:选取31例合并肠功能障碍的危重症患者为研究对象,以无肠功能障碍的30例危重症患者为对照.研究平均动脉压(MPA)、动脉血氧分压(PaO2),血白细胞计数、钾离子、GPT/GST、白蛋白等指标与肠功能衰竭发生的相关性;分析肠功能衰竭与脏器损伤个数及死亡率的关系.结果:伴有肠功能障碍的危重症患者MPA、PaO2和血清白蛋白水平低于对照组(P<0.05);血白细胞计数和GPT含量明显高于无肠功能障碍组(P<0.05);血钾离子浓度及GST两组无差异(P>0.05);发展至肠功能障碍的患者平均脏器损伤个数多于对照组,并且有着较高的死亡率(P<0.05).结论:伴有肠功能障碍的危重症患者早期就表现出严重的低血压,低血氧,低白蛋白血症,肝功能的损害,对于这些方面的监测既可以帮助我们早期发现肠功能障碍的存在,也有助于指导早期干预.     

小鼠脑缺血神经功能障碍行为学评价的研究进展

在脑缺血及脑缺血-再灌注损伤的研究中,小鼠的可逆性大脑中动脉阻塞（middle cerebral artery occlusion,MCAO）模型应用广泛。除了测量脑梗死体积外,神经功能缺损的行为学评价也是判定脑缺血严重程度的重要指标。其评价指标大多移植于大鼠的评价体系,方法较多,没有统一的评价标准,本文对小鼠脑缺血后神经功能损伤的行为学评价方法及各自的优缺点及侧重点做一综述。