The swelling-activated chloride channel ClC-2, the chloride channel ClC-3, and ClC-5, a chloride channel mutated in kidney stone disease, are expressed in distinct subpopulations of renal epithelial cells.

The mammalian genome encodes at least nine different members of the ClC family of chloride channels. So far only two of them could be localized on a cellular level in the kidney. We now report on the precise intrarenal localization of the mRNAs coding for the chloride channels ClC-2, ClC-3 and ClC-5. Expression of ClC-2 mRNA, encoding a swelling-activated chloride channel, could be demonstrated in the S3 segment of the proximal tubule. The chloride channel ClC-3 mRNA and ClC-5 mRNA, coding for a chloride channel mutated in kidney stone disease, were both expressed in intercalated cells of the connecting tubule and collecting duct. Whereas ClC-3 mRNA expression was most prominent in the cortex of rat kidneys, ClC-5 mRNA was expressed from the cortex through the upper portion of the inner medulla. A detailed analysis revealed that ClC-3 was expressed by type B intercalated cells, whereas ClC-5 was expressed by type A intercalated cells. These findings have important implications for the pathogenesis of hereditary kidney stone disease caused by mutations in the CLCN5 gene.     

Identification and characterization of functional rat arylamine N-acetyltransferase 3: comparisons with rat arylamine N-acetyltransferases 1 and 2

Arylamine N-acetyltransferases (NATs; EC 2.3.1.5) catalyze both the N-acetylation and O-acetylation of arylamines and N-hydroxyarylamines. Humans possess two functional N-acetyltransferase genes, NAT1 and NAT2, as well as a nonfunctional pseudogene, NATP. Previous studies have identified Nat1 and Nat2 genes in the rat. In this study, we identified and characterized a third rat N-acetyltransferase gene (Nat3) consisting of a single open reading frame of 870 base pairs encoding a 290-amino acid protein, analogous to the previously identified human and rat N-acetyltransferase genes. Rat Nat3 nucleotide sequence was 77.2 and 75.9% identical to human NAT1 and NAT2, respectively. Rat Nat3 amino acid sequence was 68.6 and 67.2% identical to human NAT1 and NAT2, respectively. Rat Nat1, Nat2, and Nat3 were each cloned and recombinantly expressed in Escherichia coli. Recombinant rat Nat3 exhibited thermostability intermediate between recombinant rat Nat1 and Nat2. Recombinant rat Nat3 was functional and catalyzed the N-acetylation of several arylamine substrates, including 3-ethylaniline, 3,5-dimethylaniline, 5-aminosalicylic acid, 4-aminobiphenyl, 4,4'-methylenedianiline, 4,4'-methylenebis(2-chloroaniline), and 2-aminofluorene, and the O-acetylation of N-hydroxy-4-aminobiphenyl. The relative affinities of arylamine carcinogens such as 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and 2-aminofluorene for N- and O-acetylation via recombinant rat Nat3 were comparable with recombinant rat Nat1 and higher than for recombinant rat Nat2. This study is the first to report a third arylamine N-acetyltransferase isozyme with significant functional capacity.     

Localization of NBC1 variants in rat kidney

Na+-HCO3- cotransporter (NBC1) plays a major role in bicarbonate reabsorption from proximal tubules. In a previous immunohistochemical study on human kidney, we showed that the kidney-type transporter (kNBC1) was abundantly expressed in the basolateral membranes of proximal tubules while the expression of pancreatic-type transporter (pNBC1) was undetectable. In the present study we tried to determine the localization of NBC1 variants in rat kidney using the antibodies against the unique N-terminal regions of kNBC1 and pNBC1. In Western blot analysis on the membrane-enriched fraction from rat kidney both anti-kNBC1 and anti-pNBC1 antibodies yielded a approximately 130 kDa band. In immunohistochemical analysis with confocal microscopy the anti-kNBC1 antibody produced a strong and exclusively basolateral labeling in proximal tubules. On the other hand, the occasional pNBC1 labeling was detected in the apical membranes of proximal tubules. The electron microscopic observation further supported the basolateral localization of kNBC1 as well as the localization of pNBC1 on the basis of the brush border. Acute metabolic acidosis did not change the protein expression levels as well as the intracellular distribution of both NBC1 variants in rat kidney. These results are consistent with a view that kNBC1 is the dominant variant that mediates bicarbonate reabsorption from rat renal proximal tubules. They also indicate that species difference may exist regarding the distribution of NBC1 variants in kidney.     

Anatomical and functional analysis of aquaporin 1, a water channel in primary afferent neurons

Aquaporin 1 (AQP1) is the archetypal member of a family of water channel proteins that contribute to water homeostasis in kidney, lung, and other tissues. Although there is limited evidence that aquaporins are expressed in the nervous system, AQP4 is expressed in glia and AQP9 is present on some neuronal and glial mitochondria. In the present study, we used immunohistochemistry to show that AQP1 is heavily expressed in a population of small diameter primary sensory neurons of dorsal root, trigeminal, and nodose ganglia. AQP1 immunoreactivity is abundant in DRG cell bodies and in both the peripheral and central branches of primary afferent neurons, and colocalizes with markers of nociceptors, notably substance P and IB4. AQP1 expression in DRG is first detectable at embryonic day 15.5, which corresponds to the developmental stage when the majority of fine cutaneous afferents penetrate the dorsal horn. Electron microscopy revealed dense membrane labeling of unmyelinated axons, a few fine diameter myelinated axons, and synaptic terminals in the superficial dorsal horn. Because this restricted and dense expression suggested that AQP1 contributes to nociceptive processing, we studied behavioral responses of wildtype and AQP1 -/- mice in a comprehensive battery of acute and persistent pain tests. We also used in vivo electrophysiology in wildtype and mutant mice to measure the responses of wide dynamic range neurons in lamina V of the dorsal horn to thermal stimulation before and after noxious stimulus-induced sensitization. To date we have not detected a differential phenotype suggestive of a functional contribution of AQP1 to nociceptive processing.     

Cytolytic T cell granules. Isolation, structural, biochemical, and functional characterization

The cytoplasmic, dense granules of cloned T cell lines were isolated and analyzed for their functional and biochemical properties. Isolated granules of approximately 90% homogeneity, in the presence of Ca, effect strong tumoricidal and hemolytic activity. Tumor cell lysis is complete in less than 30 min, with less than 2 micrograms granule protein corresponding to a killer/target ratio of 3-6:1 by assuming 50% yield for granule isolation. The granules contain a set of unique proteins, responsible for cytolytic activity and designated K1 to K6, in the molecular weight range of 14,000 to 75,000, as defined by sodium dodecyl sulfate (SDS) polyacrylamide slab gel analysis under reducing and nonreducing conditions. Cytolysis mediated by isolated granules is accompanied by the assembly of tubular complexes of 160 A (poly P1) and of approximately 70 A width (poly P2) that are inserted into membranes and form ultrastructural membrane lesions. As shown by immunofluorescence and by Percoll gradient fractionation, cytolytic granules are detected in cells of cytolytic T cell lineage and not in the T cell lymphomas E14 and S194. Poly perforin 1 assembled by CTLL-2 upon stimulation with concanavalin A (Con A) and phorbol myristate acetate (PMA) was isolated by detergent extraction and gel filtration. Poly P1 is composed of disulfide-linked subunits that, after reduction, co-migrate with certain granule proteins. The results are compatible with the hypothesis that the dense granules of cytolytic T cells contain cytolytic proteins that polymerize to disulfide-linked tubular poly perforins in a Ca-dependent reaction and may cause cytolysis by membrane insertion and transmembrane channel formation.     

Cloning, expression, and functional characterization of human cyclooxygenase-1 splicing variants: evidence for intron 1 retention

Recently, a splicing variant of cyclooxygenase (COX)-1, arising via the retention of its intron 1, was identified in canine. It was called COX-3 and was reported to be differentially sensitive to inhibition by various nonsteroidal anti-inflammatory drugs (NSAIDs) as well as acetaminophen (Chandrasekharan et al., 2002). However, the existence of an orthologous splicing variant in human tissues has been questioned due to a reading frame shift and premature termination. In this study, we first confirmed the existence of intron 1-retained COX-1 in certain human tissues at both the mRNA and protein levels. Molecular biology studies revealed that three distinct COX-1 splicing variants exist in human tissues. The most prevalent of these variants, called COX-1b1, arises via retention of the entire 94 base pair (bp) of intron 1, leading to a shift in the reading frame and termination at bp 249. However, the other two variant types, called COX-1b2 and COX-1b3, retain entire intron 1, but they are missing a nucleotide in one of two different positions, thereby encoding predicted full-length and likely COX-active proteins. Functional studies revealed that the COX-1b2 is able to catalyze the synthesis of prostaglandin F2alpha from arachidonic acid with Km and Vmax values of 0.54 microM and 3.07 pmol/mg/min, respectively. However, no significant differential selectivity for inhibition by selected NSAIDs was observed. Accordingly, we conclude that intron 1-retained human COX-1 is not likely to be the therapeutic target of acetaminophen or a candidate of COX-3.     

Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.

Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.     

间接免疫荧光法观察内向整流性钾通道Kir2.1在大鼠睫状体的分布

目的:Kir2.1通道存在角膜的内皮细胞,参与膜静息膜电位的产生和跨膜转运,睫状体对房水的生成过程中K 的转运起到关键性的作用.文章在蛋白水平探讨Kir2.1在大鼠睫状体的分布情况.方法:实验于2005-03/2006-10在中南大学湘雅医学院人体解剖学与神经生物学系完成.①实验材料:清洁级成年Wistar大鼠8只,雌雄不拘,体质量150～180g,眼睛活动正常,外观正常,角膜透明、无损伤.②实验过程及评估:取正常Wistar大鼠眼球冰冻包埋切片,行苏木精-伊红染色,普通光镜下观察大鼠睫状体的组织结构;在睫状体组织切片加入多克隆兔抗kit2.1一抗,再加Alexa Fluor羊抗兔IgG 555荧光二抗,用常规间接免疫荧光组织化学方法检测2.1在大鼠睫状体的分布情况.结果:①睫状体组织结构从外向内可分为7层,即睫状体上腔、睫状体肌层、基质层、玻璃膜、色素睫状上皮层、无色素睫状上皮层和内界膜层.②Kir2.1免疫活性分布在睫状体无色素上皮细胞近房水侧的基底面的胞膜和胞浆中,睫状体的色素上皮细胞无免疫活性.结论:Kir2.1分布于大鼠睫状体的无色素上皮的基底面的胞膜和胞浆中,可能与房水的生成有重要关系.     

Cloning, expression and functional characterization of the full-length murine ADAMTS13.

Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.     

Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel.

Cystic fibrosis transmembrane conductance regulator (CFTR) generates cAMP-regulated Cl- channels; mutations in CFTR cause defective Cl- channel function in cystic fibrosis epithelia. We used the patch-clamp technique to determine the single channel properties of Cl- channels in cell expressing recombinant CFTR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl- channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical Cl- solutions, the channel had a channel conductance of 10.4 +/- 0.2 (n = 7) pS and a linear current-voltage relation. The channel was more permeable to Cl- than to I- and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of Cl- channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFTR-generated channels allows further studies of their regulation and the mechanism of ion permeation.     

Localization of a proton-pumping ATPase in rat kidney.

The distribution of vacuolar H+ATPase in rat kidney was examined by immunocytochemistry using affinity-purified antibodies against the 31-, 56-, and 70-kD subunits of the bovine kidney proton pump. Proximal convoluted tubules were labeled over apical plasma membrane invaginations, and in the initial part of the thin descending limb, apical and basolateral plasma membranes were moderately stained. Thick ascending limbs and distal convoluted tubules were apically stained although the intensity was greater in the distal convoluted tubule. Collecting duct principal cells were virtually unlabeled, but intercalated cells had intense staining with an apical, basolateral or diffuse pattern in the cortex, and exclusively apical staining in the medulla. These results (a) show the presence of an H+ATPase in the apical plasma membrane of the proximal tubule that may contribute to H+ transport in this segment; (b) provide direct evidence that the intercalated cell contains most of the H+ATPase detectable in the collecting duct, supporting its proposed role in H+ transport; (c) demonstrate that subpopulations of cortical intercalated cells have opposite polarities of an H+ATPase, consistent with the presence of both proton- and bicarbonate-secreting cells; and (d) suggest a role for the H+ATPase in acid/base regulation or H+ transport in segments other than the collecting duct and the proximal tubule.     

Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.

A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.     

Detection, isolation, and functional characterization of two human T-cell subclasses bearing unique differentiation antigens.

A heterologous antihuman T-cell serum (anti-TH1), raised against purified peripheral T cells, and absorbed with an autologous Ig+ line, was shown to bind specifically to T- but not to B-lymphoid cells by both a complement-dependent cytotoxic assay and indirect immunofluorescence. Whereas 90% fetal thymocytes and thymocytes were killed by anti-TH1 and complement, a consistently restricted population (50-60%) of peripheral T cells from several normal donors were lysed, indicating that anti-TH1 is directed against one or more thymus-specific antigens which are lost or reduced on a subpopulation of human T cells in the periphery. Functional analysis of the unreactive (TH1-) and reactive (TH1+) T-cell subclasses demonstrated that TH1- cells mounted a good proliferative response to a battery of specific soluble antigens (mumps, PPD, tetanus toxoid) but neither responded in MLC, nor elaborated LMF in response to tetanus toxoid. In contrast TH1+ cells proliferated in MLC and elaborated LMF but did not respond by 3H-incorporation to soluble antigens. The relevance of these findings to human T-cell functions in vivo and to previously described functional subclasses of murine T cells is discussed.     

Regulation of GABAA receptor trafficking, channel activity, and functional plasticity of inhibitory synapses

Neural inhibition in the brain is mainly mediated by ionotropic gamma-aminobutyric acid type A (GABA(A)) receptors. Different subtypes of these receptors, distinguished by their subunit composition, are either concentrated at postsynaptic sites where they mediate phasic inhibition or found at perisynaptic and extrasynaptic locations where they prolong phasic inhibition and mediate tonic inhibition, respectively. Of special interest are mechanisms that modulate the stability and function of postsynaptic GABA(A) receptor subtypes and that are implicated in functional plasticity of inhibitory transmission in the brain. We will summarize recent progress on the classification of synaptic versus extrasynaptic receptors, the molecular composition of the postsynaptic cytoskeleton, the function of receptor-associated proteins in trafficking of GABA(A) receptors to and from synapses, and their role in post-translational signaling mechanisms that modulate the stability, density, and function of GABA(A) receptors in the postsynaptic membrane.     

Isolation and functional characterization of human intestinal mucosal lymphoid cells.

Viable suspensions of human colonic mucosal lymphoid cells have been prepared by sequential treatment of tissue with dithiothreitol, EDTA in calcium- and magnesium-free salt solutions, and purified collagenase. The intestinal lymphocyte population, in comparison with that of peripheral blood, had greater numbers of bone marrow-derived cells, particularly cells bearing membrane IgA; showed spontaneous association with macrophages; underwent rapid rosette formation with sheep erythrocytes; and demonstrated increased in vitro synthesis of immunoglobulin. Total thymus-derived cells were equal in the two populations. Decreases were found in "null" cell numbers, in cells bearing membrane IgD and IgM, and in responsiveness to phytohemagglutinin. Macrophage/monocytes in the intestinal population were increased in size, granularity, motility, sustained glass adherence, and phagocytic activity. Human intestinal lymphoid cells appear to constitute a cell population that is more "mature" and/or "activated", in comparison with the lymphoid cells of peripheral blood. The method of preparation should lend itself to the study of inflammatory bowel disease, gastrointestinal cancer, and the intestinal secretory immune system.     

Precursors of murine B lymphocytes. Physical and functional characterization, and distinctions from myeloid stem cells

The emergence of functional B cells was monitored in irradiated or unirradiated CBA/N recipients of either adult bone marrow or fetal liver from CBA/HT6T6 donors. The cells that are primarily responsible for the generation of B lymphocytes, at least during the first 6 wk, are rapidly sedimenting (4.5-6 mm/h), lack surface immunoglobulin, and are found in both the adult bone marrow and the fetal liver from day 12 onward. These pre-B cells are distinct from the colony-forming unit spleen (CFU-s) as demonstrated by the following criteria: (a) absence from yolk sac (19), (b) lack of correlation between CFU-s number and the ability to generate B cells in fetal liver populations of different ages of gestation, and (c) hybridoma antibodies that significantly inhibited B cell reconstitution but have no effect on CFU-s numbers. The antigen detected by this antiserum is present on both the fetal liver and bone marrow B cell progenitor, although its expression is not restricted to the B lineage. The pre-B cells that we monitor are not homogeneous, however, as both physical and functional differences are found. These observations reinforce our thesis that committed progenitor cells for the humoral immune system are formed early in development and thereafter constitute the major precursor pool for the generation of B lymphocytes.     

Localization of cerebral functional deficits in treatment-naive,first-episode schizophrenia using resting-state fMRI

BackgroundSpontaneous low-frequency fluctuations (LFF) in the blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) signal have been shown to reflect cerebral spontaneous neural activity, and the present study attempts to explore the functional changes in the regional brain in patients with schizophrenia using the amplitude of the BOLD signals.MethodsA total of 66 treatment-naïve, first-episode schizophrenia (FES) patients and 66 normal age- and sex-matched controls were recruited. Resting-state fMRIs were obtained using a gradient-echo echo-planar imaging sequence. The amplitude of LFF (ALFF) was calculated using REST software. Voxel-based analysis of the ALFF maps between control and patient groups was performed with twos-sample t-tests using SPM2.ResultsCompared to the controls, the FES group showed significantly decreased ALFF in the medial prefrontal lobe (MPFC) and significant increases in the ALFF in the left and right putamen. Significant positive correlations were observed between ALFF values in the bilateral putamen in both the patient and control groups.ConclusionsAlterations of the ALFF in the MPFC and putamen in FES observed in the present study suggest that the functional abnormalities of those areas are at an early stage of the disease.     

Localization and rapid regulation of Na+/myo-inositol cotransporter in rat kidney.

myo-inositol, a major compatible osmolyte in renal medulla, is accumulated in several kinds of cells under hypertonic conditions via Na+/myo-inositol cotransporter (SMIT). To investigate the physiological role of the SMIT, we sought to determine its localization by in situ hybridization and its acute regulation by NaCl and furosemide administration. Northern analysis demonstrated that SMIT is strongly expressed in the medulla and at low levels in the cortex of kidney. Intraperitoneal injection of NaCl rapidly induced SMIT mRNA in both the cortex and medulla, and furosemide completely abolished this induction. In situ hybridization revealed that SMIT it predominantly present in the medullary and cortical thick ascending limbs of Henle's loop (TALH) and macula densa cells. Less intense signals were seen in the inner medullary collecting ducts (IMCD). NaCl loading increased the signals throughout the TALH, and furosemide reduced the signals. SMIT in the IMCD is less sensitive to these kinds of acute regulation. Thus, the distribution pattern of SMIT does not correspond to the corticomedullary osmotic gradient, and SMIT in the TALH and macula densa cells is regulated very rapidly. These results suggest that SMIT expression in TALH may be regulated by intracellular and/or peritubular tonicity close to the basolateral membrane, which is supposed to be proportional to the magnitude of NaCl reabsorption.