Proteinuria induces tubular cell turnover: A potential mechanism for tubular atrophy.
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BACKGROUND: Proteinuria and tubular atrophy have both been closely linked with progressive renal failure. We hypothesized that apoptosis may be induced by tubular cell exposure to heavy proteinuria, potentially leading to tubular atrophy. Apoptosis was studied in a rat model of "pure" proteinuria, which does not induce renal impairment, namely protein-overload proteinuria. METHODS: Adult female Lewis rats underwent intraperitoneal injection of 2 g of bovine serum albumin (BSA, N = 16) or sham saline injections (controls, N = 8) daily for seven days. Apoptosis was assessed at day 7 in tissue sections using in situ end labeling (ISEL) and electron microscopy. ISEL-positive nuclei (apoptotic particles) were counted in blinded fashion using image analysis with NIH Image. Cell proliferation was assessed by detection of mRNA for histone by in situ hybridization, followed by counting of positive cells using NIH Image. RESULTS: Animals injected with saline showed very low levels of apoptosis on image analysis. BSA-injected rats had heavy proteinuria and showed both cortical and medullary apoptosis on ISEL. This was predominantly seen in the tubules and, to a lesser extent, in the interstitial compartment. Overall, the animals injected with BSA showed a significant 30-fold increase in the number of cortical apoptotic particles. Electron microscopy of tubular cells in a BSA-injected animal showed a progression of ultrastructural changes consistent with tubular cell apoptosis. The BSA-injected animals also displayed a significant increase in proximal tubular cell proliferation. This increased proliferation was less marked than the degree of apoptosis. CONCLUSION: Protein-overload proteinuria in rats induces tubular cell apoptosis. This effect is only partially balanced by proliferation and potentially provides a direct mechanism whereby heavy proteinuria can induce tubular atrophy and progressive renal failure. (+info)
Treponema brennaborense sp. nov., a novel spirochaete isolated from a dairy cow suffering from digital dermatitis.
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A novel Treponema species was isolated from an ulcerative lesion of a cow suffering from digital dermatitis (DD), a disease which causes painful ulcerations along the coronary band. Among other anaerobic bacteria, high numbers of spirochaetes have been regularly found in DD lesions. Here data are presented of a spirochaete isolated from a DD ulcer. By chemotaxonomy, protein analysis and comparative 16S rDNA sequence analysis this isolate was classified as a treponeme that differed from all Treponema species described previously. The only isolate, DD5/3T, for which the name Treponema brennaborense is proposed, is designated the type strain of the novel species. The strain is a small, highly motile spirochaete that has two periplasmic flagella, one flagellum being attached at each cell pole. Strain DD5/3T exhibits alpha-glucosidase and N-acetyl-beta-glucosaminidase activity and growth is inhibited by rabbit serum. T. brennaborense was phylogenetically most closely related (89.5% 16S rRNA similarity) to Treponema maltophilum, an oral spirochaete isolated from a periodontitis patient. (+info)
Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen.
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Strain 130ZT was isolated from the bovine rumen. It is a facultatively anaerobic, pleomorphic, Gram-negative rod. It exhibits a 'Morse code' form of morphology, which is characteristic of the genus Actinobacillus. Strain 130ZT is a capnophilic, osmotolerant succinogen that utilizes a broad range of sugars. It accumulates high concentrations of succinic acid (> 70 g l-1). Strain 130ZT is positive for catalase, oxidase, alkaline phosphatase and beta-galactosidase, but does not produce indole or urease. Acid but no gas is produced from D-glucose and D-fructose. 16S rRNA sequence analysis places strain 130ZT within the family Pasteurellaceae; the most closely related members of the family Pasteurellaceae have 16S rRNA similarities of 95.5% or less with strain 130ZT. Strain 130ZT was compared with Actinobacillus lignieresii and the related Bisgaard Taxa 6 and 10. Based upon morphological and biochemical properties, strain 130ZT is most similar to members of the genus Actinobacillus within the family Pasteurellaceae. It is proposed that strain 130ZT be classified as a new species, Actinobacillus succinogenes. The type strain of Actinobacillus succinogenes sp. nov. is ATCC 55618T. (+info)
Isolation of novel GRO genes and a phylogenetic analysis of the CXC chemokine subfamily in mammals.
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Approximately 15 different alpha, or CXC, chemokines have thus far been isolated from 11 species of mammals. Among the best studied chemokines are the 12 human proteins that are encoded by 11 paralogous genes. In order to better understand the evolution and function of this group of genes, we isolated and characterized six novel GRO and GRO-related cDNA sequences from the cow (Bos taurus), the sheep (Ovis aries), the rabbit (Oryctolagus cuniculus), and the guinea pig (Cavia porcellus). The amino acid sequence of the diverged guinea pig GRO or KC gene is only 50%-60% similar to presumed orthologs from other species, while the sheep and cow GRO proteins are 90%-99% similar to each other. The presence of multiple GRO genes in the cow, the rabbit, and the sheep is consistent with what has been observed for humans. Phylogenetic analyses of amino acid sequences from 44 proteins indicate that genes orthologous to many of the 11 known from humans exist in other species. One such gene, interleukin 8, or IL8, has been isolated from nine species, including the rodent guinea pig; however, this gene is absent in the rat and the mouse, indicating a unique gene loss event in the rat/mouse (muroid rodent) lineage. The KC (or MIP2) gene of rodents appears to be orthologous to the GRO gene found in other taxonomic orders. Combined evidence from different sources suggests that IP10 and MIG share sister taxon relationships on the evolutionary tree, while the remaining paralogous genes represent independent lineages, with limited evidence for kinship between them. This observation indicates that these genes originated nearly contemporaneously via a series of gene duplication events. Relative-rate tests for synonymous and nonsynonymous nucleotide substitutions in the KC and IL8 genes did not detect rate heterogeneity; however, there are several notable features regarding the IL8 genes. For example, the IL8 proteins from two Old World monkeys are as similar to one another as they are to the IL8 protein from humans, and all observed nucleotide differences between the IL8 genes of the two monkeys cause amino acid changes; in other words, there are no synonymous differences between them. (+info)
Ovine MHC class II DRB1 alleles associated with resistance or susceptibility to development of bovine leukemia virus-induced ovine lymphoma.
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For the further characterization of bovine leukemia virus (BLV)-induced leukemogenesis, we investigated the association between polymorphism of ovine leukocyte antigen (OLA)-DRB1 gene and tumor development after infection of sheep with BLV. We infected 28 sheep with BLV and cloned exon 2 of the OLA-DRB1 gene from asymptomatic animals and from animals with lymphoma Sequence analysis revealed that, among 12 healthy sheep without any evidence of tumor, ten (83.3%) carried DRB1 alleles encoding Arg-Lys (RK) at positions beta70/71 as compared with only 6 (37.5%) of the 16 sheep with lymphoma, which suggested that alleles encoding the RK motif might protect against development of tumors after infection by BLV. By contrast, alleles encoding Ser-Arg (SR) at positions beta70/71 were present at a significantly elevated frequency in sheep with lymphoma as compared with the healthy carriers, which indicated that OLA-DRB1 alleles encoding the SR motif might be positively related to susceptibility to tumor development. The two amino acids in these motifs line a pocket that accommodates the side chain of a bound peptide according to a model of the crystal structure of human leukocyte antigen (HLA)-DR1. To analyze immunoreactions of sheep with alleles that encoded RK or SR at beta70/71, we selected sheep with either the RK/SR genotypes or the SR/SR genotypes and immunized them with a mixture of multiple synthetic antigenic peptides that corresponded to T-helper, T-cytotoxic, and B-cell epitopes of the BLV envelope glycoprotein gp51. Two weeks after the last immunization, all of the sheep were challenged with BLV. Sheep with the RK/SR genotype produced neutralizing antibodies against BLV; they eliminated BLV completely within 28 weeks of the BLV challenge, and they gave strong lymphocyte-proliferative responses to the peptides used for immunization. Moreover, such animals did not develop lymphoma. By contrast, sheep with the SR/SR genotype continued to produce BLV throughout the experimental period and developed terminal disease. Our results indicate that the differences in immunoresponse were due to differences in major histocompatibility complex class II alleles and reflected the risk of BLV-induced leukemogenesis. In addition, it appears that susceptibility to tumor development may be determined to some extent by polymorphic residues binding to antigenic peptides directly within the binding cleft of the OLA-DR molecule. (+info)
The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium.
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The recent identification of nonvisual opsins has revealed an expanding family of vertebrate opsin genes. The retinal pigment epithelium (RPE) and Muller cells contain a blue and UV light-absorbing opsin, the RPE retinal G protein-coupled receptor (RGR, or RGR opsin). The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore through a covalent Schiff base bond. In this study, the isomeric structure of the endogenous chromophore of RGR was identified by the hydroxylamine derivatization method. The retinaloximes derived from RGR in the dark consisted predominantly of the all-trans isomer. Irradiation of RGR with 470-nm monochromatic or near-UV light resulted in stereospecific isomerization of the bound all-trans-retinal to an 11-cis configuration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitro conditions, the photosensitivity of RGR is at least 34% that of bovine rhodopsin. These results provide evidence that RGR is bound in vivo primarily to all-trans-retinal and is capable of operating as a stereospecific photoisomerase that generates 11-cis-retinal in the pigment epithelium. (+info)
Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear.
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Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress. (+info)
Identification of a domain in guanylyl cyclase-activating protein 1 that interacts with a complex of guanylyl cyclase and tubulin in photoreceptors.
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The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments. (+info)