Microbial regulation of host hydrogen sulfide bioavailability and metabolism

Hydrogen sulfide (H₂S), generated through various endogenous enzymatic and nonenzymatic pathways, is emerging as a regulator of physiological and pathological events throughout the body. Bacteria in the gastrointestinal tract also produce significant amounts of H₂S that regulates microflora growth and virulence responses. However, the impact of the microbiota on host global H₂S bioavailability and metabolism remains unknown. To address this question, we examined H₂S bioavailability in its various forms (free, acid labile, or bound sulfane sulfur), cystathionine γ-lyase (CSE) activity, and cysteine levels in tissues from germ-free versus conventionally housed mice. Free H₂S levels were significantly reduced in plasma and gastrointestinal tissues of germ-free mice. Bound sulfane sulfur levels were decreased by 50–80% in germ-free mouse plasma and adipose and lung tissues. Tissue CSE activity was significantly reduced in many organs from germ-free mice, whereas tissue cysteine levels were significantly elevated compared to conventional mice. These data reveal that the microbiota profoundly regulates systemic bioavailability and metabolism of H₂S.     

Metabolism of 2,4',5-trichlorobiphenyl: role of the intestinal microflora in the formation of bronchial-seeking methyl sulphone metabolites in mice

Groups of germ-free and conventional mice were treated with 2,4',5-trichlorobiphenyl (triCB) and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. Conventional mice accumulated significantly more extractable radioactivity both in lung and kidney tissues when compared to germ-free mice. The extracted radioactivity in lung and kidney tissues was shown to be due to the accumulation of methyl-[35S]sulphonyl-triCB. The low radioactivity in lungs of the germ-free mice was also shown to be due to the accumulation of small amounts of the sulphones. The results indicate an involvement of the intestinal flora in the formation of methyl sulphone metabolites of triCB.     

Enteric microflora contribute to constitutive ICAM-1 expression on vascular endothelial cells

Quantitative estimates of endothelial cell adhesion molecule expression have revealed that some adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1)] are abundantly expressed in different vascular beds under normal conditions. The objective of this study was to determine whether the enteric microflora contribute to the constitutive expression of ICAM-1 and other endothelial cell adhesion molecules in the gastrointestinal tract and other regional vascular beds. The dual radiolabeled monoclonal antibody technique was used to measure endothelial expression of ICAM-1, ICAM-2, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in conventional, germ-free mice and germ-free mice receiving the cecal contents of conventional mice to reestablish the enteric microflora (total association). Constitutive ICAM-1 expression was significantly lower in the splanchnic organs (pancreas, stomach, small and large intestine, mesentery, and liver), kidneys, skeletal muscle, and skin of germ-free mice compared with their conventional counterparts. These differences were abolished after total association of germ-free mice with the indigenous gastrointestinal flora. The expression of ICAM-2, VCAM-1, and E-selectin in the various tissues studied did not differ between conventional and germ-free mice. These findings indicate that the indigenous gastrointestinal microflora are responsible for a significant proportion of the basal ICAM-1 expression detected in both intestinal and extraintestinal tissues.     

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

Microbial-host interactions specifically control the glycosylation pattern in intestinal mouse mucosa

The glycosylation of the intestinal cell layer is thought to control several key functions of the gut such as vectorial transports, defence against microbial agents or immunological processes. It has been assumed that the gut microflora may modulate the glycosylation pattern of the intestinal cell layer. However, there is no direct evidence for this regulatory process. The first goal of this work was to establish the germ-free mice intestinal glycosylation baseline using a histochemical approach and a panel of ten lectins with defined glycan specificities to tissue sections prepared from various cellular compartments of the small and large intestine. Using this baseline, we have studied the contribution of the gut microflora on the carbohydrate composition of glycoconjugates of intestinal cells by comparing the germ-free and conventional mice glycosylation patterns. Analysis of the germ-free mice intestinal glycosylation baseline revealed that the expression of glycans depends on the proximodistal gradient (small to large intestine) and on the cell lineage (absorptive, goblet, crypt, and Paneth cells), indicating that mice are able to create and maintain a strict topological and cell lineage-specific regulation of glycosyltransferase expression. By comparing germ-free and conventional mice, we find that the gut microflora specifically modulates the gut glycosylation pattern, quantitatively as well as qualitatively by changing the cellular and subcellular distribution of glycans. This is the first report in mice to directly demonstrate the critical contribution of microflora to intestinal glycosylation, a key characteristic of the gut.     

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.     

Morphology of germ-free piglets

The postnatal ontogeny of primary (thymus) and secondary (spleen, lymph node, lingual tonsil) lymphoid tissues was studied in germ-free colostrum-deprived piglets up to age of 68 days. The thymus, which is morphologically fully developed by the end of gestation, showed no significant differences in the germ-free and conventional state. In germ-free piglets, slow development of periarteriolarly organized lymph follicles occurred in the spleen up to the end of the observation period. As distinct from the conditions in the spleen of conventional animals, the presence of a large number of pyroninophilic cells was not observed in germ-free piglets and no germinal centres were found. A similar situation was seen in the mesenteric lymph nodes, in which, in conventional piglets, cells belonging to the plasmacyte series, as well as the germinal centres, proliferate by the 13th day. Differences were also found in the organization of the follicular lymphoid tissue in the wall of the terminal ileum. In germ-free piglets, the lymph follicles increased only very slowly in size during the observation period and germinal centres were absent, while in conventional piglots germinal centres were present from the 12th day. The view is expressed that the intestinal lymphoid tissue ought rather to be classified as peripheral lymphoid tissue.     

Postnatal changes in the expression of genes for cryptdins 1-6 and the role of luminal bacteria in cryptdin gene expression in mouse small intestine.

Although there have been many fascinating studies on cryptdins, the information for each cryptdin isoform was not completely provided. In this study, the postnatal changes in the gene expression of cryptdin 1-6 were evaluated, and the patterns of change were compared between conventional and germ-free mice. Two patterns of postnatal change were observed: gene expression of cryptdins 1, 3 and 6 increased gradually, and that of cryptdins 2 and 5 increased rapidly. Gene expression of cryptdin 4 increased gradually in the ileum but rapidly in the jejunum. Conventional mice showed significantly higher gene expression for all isoforms than germ-free mice. Interestingly, the difference in the gene expression for cryptdin 2, 4 and 5 between the jejunum and ileum seemed to be increased by the presence of the luminal bacteria. The results indicate that cryptdin isoforms develop differently depending on the isoform type, and that the gene expression of all cryptdin isoforms was affected by the presence of the luminal bacteria.     

High expression of the mRNA of cytochrome P450 and phase II enzymes in the lung and kidney tissues of cattle

The objective of this study was to investigate the tissue-specific mRNA expression of different cytochrome P450 (CYP) isoforms, UDP glucuronsyl transferase 1A1 (UGT1A1) and glutathione-S-transferase (GSTA1) in the different tissues (liver, mammary gland, lungs, spleen, kidney cortex, heart, masseter muscle and tongue) of cattle, using quantitative real-time polymerase chain reaction (qPCR). CYP1A1-like mRNA was expressed in all of the tissues examined, including the liver, with the highest expression level in the kidney. CYP1A2-, 2E1- and 3A4-like mRNAs were only expressed hepatically. Interestingly, significant expression of CYP2B6-like mRNA was recorded in the lung tissue, while CYP2C9-like mRNA was expressed in the liver and kidney tissues of the cattle examined. UGT1A1- and GSTA1-like mRNAs were expressed in all of the examined tissues, except the mammary glands, and the highest expression levels were recorded in the kidney. The high expression of UGT1A1 in the lung tissue and GSTA1 in the liver tissue was unique to cattle; this has not been reported for rats or mice. The findings of this study strongly suggest that the liver, kidneys and lungs of cattle are the major organs contributing to xenobiotics metabolism.     

基于免疫组化和癌症基因组图谱数据分析的脾酪氨酸激酶在宫颈癌中的表达研究

摘要 目的：探讨Syk 在宫颈癌中的表达及其临床意义。方法：应用免疫组化检测Syk在宫颈癌、癌前病变(CIN)和相应的正常宫颈组织中的表达。借助R2生物信息平台挖掘Syk在TCGA数据库305例宫颈鳞癌中的mRNA表达及其与预后的关系。结果：免疫组化结果显示,Syk在宫颈癌巢分化较好的中心区表达较强,在分化较低的癌巢周边区表达较弱。Syk 染色主要定位在宫颈癌和正常宫颈组织的细胞质和细胞膜,正常宫颈组织基底细胞无 Syk 表达,8例CIN组织细胞核中可见Syk表达, 但宫颈癌组织细胞核中未见Syk表达。Syk在宫颈癌、CIN和正常宫颈组织中的阳性率分别是76%、54%、40%,三组间的表达差异具有统计学意义（P=0.001）。Syk 在深度浸润和淋巴结转移中表达较强。数据挖掘结果显示,Syk mRNA在305例不同临床分期的宫颈癌中均表达,Syk mRNA高表达组219例,Syk mRNA低表达组73例,其中13例生存数据缺失,Syk高表达组的患者预后较差。结论：Syk在宫颈癌中的表达提示Syk在宫颈癌中具有致癌蛋白的作用,Syk在某些CIN中的核表达可能与更好的预后相关。     

The essential role of the intestinal microbiota in facilitating acute inflammatory responses

The restoration of blood flow, i.e., reperfusion, is the treatment of choice to save viable tissue following acute ischemia of a vascular territory. Nevertheless, reperfusion can be accompanied by significant inflammatory events that limit the beneficial effects of blood flow restoration. To evaluate the potential role of the intestinal microbiota in facilitating the development of tissue injury and systemic inflammation, germ-free and conventional mice were compared in their ability to respond to ischemia and reperfusion injury. In conventional mice, there was marked local (intestine) and remote (lung) edema formation, neutrophil influx, hemorrhage, and production of TNF-alpha, KC, MIP-2, and MCP-1. Moreover, there was an increase in the concentration of serum TNF-alpha and 100% lethality. In germ-free mice, there was no local, remote, or systemic inflammatory response or lethality after intestinal ischemia and reperfusion and, in contrast to conventional mice, germ-free animals produced greater amounts of IL-10. Similar results were obtained after administration of LPS, i.e., little production of TNF-alpha or lethality and production of IL-10 after LPS in germ-free mice. Blockade of IL-10 with Abs induced marked inflammation and lethality in germ-free mice after ischemia and reperfusion or LPS administration, demonstrating that the ability of these mice to produce IL-10 was largely responsible for their "no inflammation" phenotype. This was consistent with the prevention of reperfusion-associated injury by the exogenous administration of IL-10 to conventional mice. Thus, the lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.     

Isoforms,Expression, Glycosylation,and Tissue Distribution of CTL2/SLC44A2

Antibodies to the solute carrier protein, CTL2/SLC44A2, cause hearing loss in animals, are frequently found in autoimmune hearing loss patients, and are implicated in transfusion-related acute lung injury. We cloned a novel CTL2/SLC44A2 isoform (CTL2 P1) from inner ear and identified an alternate upstream promoter and exon 1a encoding a protein of 704 amino acids which differs in the first 10–12 amino acids from the known exon 1b isoform (CTL2 P2; 706 amino acids). The expression of these CTL2/SLC44A2 isoforms, their posttranslational modifications in tissues and their localization in HEK293 cells expressing rHuCTL2/SLC44A2 were assessed. P1 and P2 isoforms with differing glycosylation are variably expressed in cochlea, tongue, heart, colon, lung, kidney, liver and spleen suggesting tissue specific differences that may influence function in each tissue. Because antibodies to CTL2/SLC44A2 have serious pathologic consequences, it is important to understand its distribution and modifications. Heterologous expression in X. laevis oocytes shows that while human CTL2-P1 does not transport choline, human CTL2-P2 exhibits detectable choline transport activity.     

Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity

In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot analysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with beta-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney > spleen > lung > heart > brain > muscle. Measurement of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.     

汉坦病毒核酸疫苗在小鼠体内表达分布的初步研究

运用生物荧光技术,肌肉免疫接种小鼠含汉坦病毒S抗原基因片段的核酸疫苗,观察重组核酸疫苗在其体内的表达分布。进一步探讨汉坦病毒核酸疫苗的应用前景。扩增纯化已构建好的含有汉坦病毒S抗原基因片段和绿色荧光蛋白(GFP)基因的重组质粒pEGFP/S;免疫接种小鼠胫前肌,观察pEGFP/S在小鼠体内的表达分布。免疫接种3 d后在实验组小鼠肝、肾、脾、肌肉各组织均检测到较强的绿色荧光。重组质粒pEGFP/S能在小鼠的多个组织器官表达,为深入研究汉坦病毒核蛋白和有效的核酸疫苗奠定了基础。     

Peptidylarginine deiminase expression and activity in PAD2 knock-out and PAD4-low mice

Citrullination, the conversion of peptidylarginine to peptidylcitrulline is catalyzed by peptidylarginine deiminases (PAD). The expression of PAD isoforms displays great variation among different tissues as demonstrated by PAD mRNA analyses. Here we have analyzed the differential expression of PAD2, PAD4 and PAD6 in mouse tissues at the protein level and by enzymatic activity assays using PAD2 and PAD4 knock-out strains. As expected, no PAD2 expression was detected in the PAD2−/− mice. In contrast, the PAD4 protein was observed in several tissues of the PAD4 knock-out mice, albeit at reduced levels in most tissues, and are therefore referred to as PAD4-low mice. In material from PAD2−/− mice, except for leukocyte lysates, hardly any PAD activity was found and no citrullinated proteins were detected after incubation in the presence of calcium. PAD activity in the PAD4-low mice was similar to that in wild-type mice. In both PAD knock-out strains the expression of PAD6 appeared to be up-regulated in all tissues analyzed, with the exception of spleen and testis. Our data demonstrate that the PAD2 protein is expressed in brain, spinal cord, spleen, skeletal muscle and leukocytes, but not detectably in liver, lung, kidney and testis. PAD4 was detected in each of these tissues, although the expression levels varied. In all tissues where PAD2 was detected, except for blood cells, this PAD isoform appeared to be responsible for virtually all peptidylarginine deiminase activity.     

Constitutive expression of CXCL14 in healthy human and murine epithelial tissues

CXCL14 (BRAK) is an ill-described chemokine with unknown receptor selectivity. The human chemokine is constitutively expressed in epithelial tissues and is selective for dendritic cell precursors, indicating a possible function in the maintenance of epithelial DCs. Several studies have addressed the question of human CXCL14 expression in cancerous tissues; however, distribution in healthy tissues and, in particular, the cellular origin of this chemokine has not been thoroughly investigated. The expression pattern of murine CXCL14 is largely unknown. In agreement with the human chemokine, we demonstrated ubiquitous and constitutive expression of murine CXCL14 in various tissues, foremost in those of epithelial origin such as the skin and the gastrointestinal tract. In addition, we did not find any CXCL14 in lymphoid tissues. Interestingly and in contrast to humans, murine CXCL14 was strongly expressed in the lung. In the skin, CXCL14 was produced by keratinocytes and dermal macrophages in both mice and humans, whereas CXCL14-expressing mast cells could only be found in the human dermis. Therefore, despite the remarkable structural homology and the broad similarity in the tissue distribution of human and murine CXCL14, distinct differences point to diverse, species-specific needs for CXCL14 in epithelial immunity.