Expression of Kidney Injury Molecule-1 in Healthy and Diseased Feline Kidney Tissue.

Sensitive markers to detect acute kidney injury (AKI) in cats are lacking. Kidney injury molecule-1 (KIM-1) is a promising marker of acute tubular injury in humans, and sequence and structure of feline KIM-1 have been determined. KIM-1 is shed into urine of cats with natural AKI. The objectives of this study were to characterize temporal and cellular expression of KIM-1 in kidneys from cats without and with experimental and natural AKI using histopathology and immunohistochemistry. Tissue sections from 8 cats without kidney disease, 3 to 4 cats with experimentally induced AKI on each day 1, 3, 6, and 12 after unilateral ischemia/reperfusion, and 9 cats with natural AKI were assessed. In sections from cats without kidney disease, patterns of periodic acid-Schiff and aquaporin-1 staining allowed identification of 3 distinct segments of the proximal tubule. KIM-1 staining was absent in segments 1 (S1) and S2, and faint in S3. Injury of S3 in cats with experimental and natural AKI was characterized by cell loss and necrosis, and remaining intact cells had cytoplasmic blebs and reduced brush borders. In experimental AKI, intensity of KIM-1 expression increased in proportion to the severity of injury and was consistently present in S3 but only transiently in other segments. Vimentin was absent in proximal tubules of healthy cats but expressed in injured S3. These findings indicate that S3 is the proximal tubular segment most susceptible to ischemic injury and that KIM-1 is a sensitive tissue indicator of AKI in cats.

Localization and functional characterization of pulmonary bovine odorant-binding protein.

Bovine odorant-binding protein (OBP) may function in olfaction and defense against oxidative injury, but its role in inflammation and defense against bacterial infection has not been investigated. Expression of OBP was discovered in the bovine lung and found to undergo changes in abundance during glucocorticoid administration and stress. OBP was localized to nasal, tracheal, and bronchial mucosal glands with immunohistochemistry, with faint expression in airway surface epithelium and none in bronchioles or alveoli. Two isoforms of OBP were identified, appearing to be differentially regulated during lipopolysaccharide-induced pulmonary inflammation, but differences between these isoforms were not revealed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Functional studies showed no effect of OBP on in vitro growth of Escherichia coli or Mannheimia haemolytica under iron-replete or iron-depleted conditions, nor did OBP opsonize bacteria for an enhanced neutrophil oxidative burst. However, OBP did reduce the ability of supernatants from lipopolysaccharide-stimulated macrophages to induce neutrophil chemotaxis. These findings indicate that OBP may inhibit neutrophil recruitment by inflammatory mediators, and they suggest an ability to bind macrophage-derived inflammatory mediators within the airways.

Behavioral and spermatogenic hybrid male breakdown in Nasonia.

Several reproductive barriers exist within the Nasonia species complex, including allopatry, premating behavioral isolation, postzygotic inviability and Wolbachia-induced cytoplasmic incompatibility. Here we show that hybrid males suffer two additional reproductive disadvantages, an inability to properly court females and decreased sperm production. Hybrid behavioral sterility, characterized by a reduced ability of hybrids to perform necessary courtship behaviors, occurs in hybrids between two species of Nasonia. Hybrid males produced in crosses between N. vitripennis and N. giraulti courted females at a reduced frequency (23-69%), compared with wild-type N. vitripennis and N. giraulti males (>93%). Reduced courtship frequency was not a simple function of inactivity among hybrids. A strong effect of cytoplasmic (mitochondrial) background was also found in N. vitripennis and N. giraulti crosses; F2 hybrids with giraulti cytoplasm showing reduced ability at most stages of courtship. Hybrids produced between a younger species pair, N. giraulti and N. longicornis, were behaviorally fertile. All males possessed motile sperm, but sperm production is greatly reduced in hybrids between the older species pair, N. vitripennis and N. giraulti. This effect on hybrid males, lowered sperm counts rather than nonfunctional sperm, is different from most described cases of hybrid male sterility, and may represent an earlier stage of hybrid sperm breakdown. The results add to previous studies of F2 hybrid inviability and behavioral sterility, and indicate that Wolbachia-induced hybrid incompatibility has arisen early in species divergence, relative to behavioral sterility and spermatogenic infertility.

Living with water stress: evolution of osmolyte systems.

Striking convergent evolution is found in the properties of the organic osmotic solute (osmolyte) systems observed in bacteria, plants, and animals. Polyhydric alcohols, free amino acids and their derivatives, and combinations of urea and methylamines are the three types of osmolyte systems found in all water-stressed organisms except the halobacteria. The selective advantages of the organic osmolyte systems are, first, a compatibility with macromolecular structure and function at high or variable (or both) osmolyte concentrations, and, second, greatly reduced needs for modifying proteins to function in concentrated intracellular solutions. Osmolyte compatibility is proposed to result from the absence of osmolyte interactions with substrates and cofactors, and the nonperturbing or favorable effects of osmolytes on macromolecular-solvent interactions.

Clara cell secretory protein is reduced in equine recurrent airway obstruction.

Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horses. Recombinant equine CCSP and specific antiserum were produced, and percutaneous lung biopsies were obtained from 3 healthy horses and from 3 RAO-affected horses before and after induction of RAO. CCSP relative gene expression in tissue, as well as protein concentration in lung lavage fluid, was determined. Immunocytochemical analysis, using both light and immunogold ultrastructural methods, demonstrated reduced CCSP staining in lung tissue of animals with RAO. Immunogold label in Clara cell granules was less in animals with chronic RAO than in normal animals, and absent in animals that had active disease. Median lung lavage CCSP concentration was 132 and 129 ng/ml in healthy horses, and 62 and 24 ng/ml in RAO horses before and after challenge, respectively. CCSP lung gene expression was significantly higher in healthy animals than in animals with chronic RAO. Together, these preliminary findings suggest that reduced production of CCSP and subcellular changes in Clara cells are features of chronic environmentally induced lung inflammation in horses.

Wolbachia plays no role in the one-way reproductive incompatibility between the hybridizing field crickets Gryllus firmus and G. pennsylvanicus.

Wolbachia are cytoplasmically inherited alpha-proteobacteria that can cause cytoplasmic incompatibility (CI) in insects. This incompatibility between sperm and egg is evident when uninfected females mate with infected males. Wolbachia-driven reproductive incompatibilities are of special interest because they may play a role in speciation. However, the presence of Wolbachia does not always imply incompatibility. The field crickets Gryllus firmus and G. pennsylvanicus exhibit a very clear unidirectional incompatibility and have been cited as a possible example of Wolbachia-induced CI. Here, we conduct curing experiments, intra- and interspecific crosses, cytological examination of Wolbachia in testes and Wolbachia quantifications through real-time PCR. All of our data strongly suggest that Wolbachia are not involved in the reproductive incompatibility between G. firmus and G. pennsylvanicus.

Wolbachia modification of sperm does not always require residence within developing sperm.

Wolbachia are maternally inherited intracellular bacteria known to manipulate the reproduction of their arthropod hosts. Wolbachia commonly affect the sperm of infected arthropods. Wolbachia-modified sperm cannot successfully fertilize unless the female is infected with the same Wolbachia type. A study of spermatogenesis in the parasitic wasp Nasonia vitripennis reveals that Wolbachia are not required in individual spermatocytes or spermatids to modify sperm. In N. vitripennis, Wolbachia modify nearly all sperm, but are found only in approximately 28% of developing sperm, and are also found in surrounding cyst and sheath cells. In the beetle Chelymorpha alternans, Wolbachia can modify up to 90% of sperm, but were never observed within the developing sperm or within the surrounding cyst cells; they were abundant within the outer testis sheath. We conclude that the residence within a developing sperm is not a prerequisite for Wolbachia-induced sperm modification, suggesting that Wolbachia modification of sperm may occur across multiple tissue membranes or act upstream of spermiogenesis.

The POU homeodomain transcription factor Oct-1 is essential for activity of the gonadotropin-releasing hormone neuron-specific enhancer.

The mechanisms of specification of gene expression in a complex tissue such as the brain remain poorly understood. To provide a model system for the study of gene regulation in a specific subpopulation of differentiated neurons, we have derived cell lines from tumors created in transgenic mice by targeting simian virus 40 T antigen expression by using the regulatory regions of the gene for gonadotropin-releasing hormone (GnRH), a decapeptide released from specialized neurons in the hypothalamus. Transfections into the cultured GnRH-secreting hypothalamic neuronal cell line GT1 have identified a neuron-specific enhancer, 1.5 kb upstream of the GnRH gene, which binds multiple GT1 nuclear proteins. In particular, one AT-rich protein-binding region, AT-a, is critical for enhancer activity. In this study, we used electrophoretic mobility shift assays to detect a GT1 nuclear protein complex that binds the AT-a region. Close inspection of the AT-a bottom-strand sequence revealed homology to the octamer motif, a sequence known to bind members of the POU homeodomain transcription factor family. Although we demonstrate expression of a number of POU homeodomain genes in GT1 cells, a supershift assay with Oct-1 antibody demonstrates that Oct-1 is the protein binding the enhancer. Finally, specific mutations in the AT-a region that affected Oct-1 binding were correlated with decreased transcription. Thus, Oct-1 binds to the GnRH enhancer in vitro, and this binding is critical to the transcriptional activity of this neuron-specific enhancer in GT1 cells.

A transcriptionally active form of TFIIIC is modified in poliovirus-infected HeLa cells.

In HeLa cells, RNA polymerase III (pol III)-mediated transcription is severely inhibited by poliovirus infection. This inhibition is due primarily to the reduction in transcriptional activity of the pol III transcription factor TFIIIC in poliovirus-infected cells. However, the specific binding of TFIIIC to the VAI gene B-box sequence, as assayed by DNase I footprinting, is not altered by poliovirus infection. We have used gel retardation analysis to analyze TFIIIC-DNA complexes formed in nuclear extracts prepared from mock- and poliovirus-infected cells. In mock-infected cell extracts, two closely migrating TFIIIC-containing complexes, complexes I and II, were detected in the gel retardation assay. The slower migrating complex, complex I, was absent in poliovirus-infected cell extracts, and an increase occurred in the intensity of the faster-migrating complex (complex II). Also, in poliovirus-infected cell extracts, a new, rapidly migrating complex, complex III, was formed. Complex III may have been the result of limited proteolysis of complex I or II. These changes in TFIIIC-containing complexes in poliovirus-infected cell extracts correlated kinetically with the decrease in TFIIIC transcriptional activity. Complexes I, II, and III were chromatographically separated; only complex I was transcriptionally active and specifically restored pol III transcription when added to poliovirus-infected cell extracts. Acid phosphatase treatment partially converted complex I to complex II but did not affect the binding of complex II or III. Dephosphorylation and limited proteolysis of TFIIIC are discussed as possible mechanisms for the inhibition of pol III-mediated transcription by poliovirus.

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro.

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant (p<0.01), and these effects were significantly (p<0.02, p<0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant (p<0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.

Physiological effects on demography: a long-term experimental study of testosterone's effects on fitness.

Understanding physiological and behavioral mechanisms underlying the diversity of observed life-history strategies is challenging because of difficulties in obtaining long-term measures of fitness and in relating fitness to these mechanisms. We evaluated effects of experimentally elevated testosterone on male fitness in a population of dark-eyed juncos studied over nine breeding seasons using a demographic modeling approach. Elevated levels of testosterone decreased survival rates but increased success of producing extra-pair offspring. Higher overall fitness for testosterone-treated males was unexpected and led us to consider indirect effects of testosterone on offspring and females. Nest success was similar for testosterone-treated and control males, but testosterone-treated males produced smaller offspring, and smaller offspring had lower postfledging survival. Older, more experienced females preferred to mate with older males and realized higher reproductive success when they did so. Treatment of young males increased their ability to attract older females yet resulted in poor reproductive performance. The higher fitness of testosterone-treated males in the absence of a comparable natural phenotype suggests that the natural phenotype may be constrained. If this phenotype were to arise, the negative social effects on offspring and mates suggest that these effects might prevent high-testosterone phenotypes from spreading in the population.

The Otx2 homeoprotein regulates expression from the gonadotropin-releasing hormone proximal promoter.

The GnRH gene is expressed exclusively in a highly restricted population of approximately 800 neurons in the mediobasal hypothalamus in the mouse. The Otx2 homeoprotein has been shown to colocalize with GnRH in embryonic mouse brain. We have identified a highly conserved bicoid-related Otx target sequence within the proximal promoter region of the GnRH gene from several species. This element from the rat GnRH promoter binds baculovirus-expressed Otx2 protein and Otx2 protein in nuclear extracts of a hypothalamic GnRH-expressing neuronal cell line, GT1-7. Transient transfection assays indicate that the GnRH promoter Otx/bicoid site is required for specific expression of the GnRH gene in GT1-7 cells and that it can confer specificity to a neutral Rous sarcoma virus (RSV) promoter in GT1-7 cells but not in NIH3T3 cells. Overexpression of mouse Otx2 in GT1-7 cells induces expression of a GnRH promoter plasmid, an effect that is dependent upon the Otx binding site. Thus, the GnRH proximal promoter is regulated by the Otx2 homeoprotein. Finally, we have now demonstrated the presence of Otx2 protein in the GnRH neurons of the adult mouse hypothalamus. These data suggest that Otx2 is important in the development of the GnRH neuron and/or in the maintenance of GnRH expression in the adult mouse hypothalamus.

Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos.

The formation of transgenic chimeric chickens for use in developmental studies and as intermediates in the production of transgenic chickens requires the incorporation of stably transfected blastodermal cells into a chimera. To obtain blastodermal cells, area pellucidae of stage X (Eyal-Giladi and Kochav, Dev. Biol. 49:321-337, 1976:E.-G.&K.) embryos were collected from unincubated, freshly oviposited Barred Plymouth Rock eggs and dissociated in 0.25% trypsin/0.04% EDTA (w/v) and 2% (v/v) chicken serum in phosphate-buffered saline (Ca2+ and Mg2+ free) at 4 degrees C for 10 min. The blastodermal cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM) and transfected by lipofection with superhelical pmiwZ, a plasmid containing a hybrid lacZ gene encoding bacterial beta-galactosidase (beta-gal) under the control of a chicken beta-actin/Rous sarcoma virus promoter. A mixture of 2.5 micrograms Lipofectin and 1.56 micrograms pmiwZ in 250 microliters DMEM was incubated for 30 min at 37 degrees C and added to 500 microliters of 20-40,000 cells in suspension. Cells incubated with the transfection reagents in the presence or absence of pmiwZ were either plated and cultured for 48 h at 37 degrees C in 5% CO2/95% air, or injected through a shell window into the subgerminal cavity of White Leghorn stage X (E.-G.&K.) embryos previously exposed to 500-600 rads from a 60Co source, after which the window was sealed and the egg incubated at 38 +/- 1 degrees C for 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of envelope glycoproteins of human T cell lymphotropic virus type I (HTLV-I) by affinity chromatography.

The external envelope glycoprotein (gp46) and transmembrane glycoprotein (gp21) of human T-cell lymphotropic virus type I (HTLV-I) were isolated from lysates of HTLV-I-infected HUT-102 cells by affinity chromatography. Fifty ml aliquots of packed HUT-102 cells were extracted with 1% Triton X-100, and lysates were treated sequentially with an affinity column containing IgG from an HTLV-I+ human subject followed by chromatography of the bound fraction over a lentil lectin column. The identity of the purified envelope proteins was confirmed with a human monoclonal antibody (0.5 alpha) to gp46 and with rabbit antisera raised to a synthetic peptide from the C-terminus of gp21. Affinity-purified envelope glycoproteins were bound to microtiter wells and used in radioimmunoassay to detect murine and human anti-envelope antibodies to gp46 and gp21 molecules.

Natural and surgically imposed anastomoses of the circle of Willis.

The efficacy of the circle of Willis as a flow equalizer is well known. Most cerebral macrovasculatures also contain other natural anastomoses which are activated in times of stenotic stress. For the past several decades, neurosurgeons have surgically augmented the cerebral network with additional vessels which further increase the flow of blood to a defrauded region of the brain. It is desirable to know quantitatively what role these anastomoses play in the delivery of blood. Apart from computer simulation, such knowledge remains out of reach to the medical community but with modern simulation techniques, a wealth of information can be made available. This paper presents both time-dependent and period-averaged results of a detailed study of cerebral anastomoses. Four different models of the macrovasculature in the circle of Willis vicinity have been developed, two of which contain an extracranial-intracranial (EC-IC) anastomosis. Five cases were developed to show how the amount of blood flow is related to the sizes of the anastomoses. Since the EC-IC bypass is only marginally beneficial in those patients whose cerebral circulations are well-equipped with naturally occurring anastomotic vessels, procedures should be developed to screen for their presence or absence. The fluid mechanics associated with the EC-IC bypass operation dictate a beneficial result. Since the surgical procedures fail to consistently show reduction in risk even when good grafts have been made, there is an enigma in the study group results.

On the rupture of an aneurysm.

The intracranial aneurysm, with an estimated occurrence of up to 4% in the general population, belongs among the most dangerous of cerebrovascular diseases. Although less than one-fifth of these cases results in a subarachnoid haemorrhage, the resulting disability and mortality rate is too high. More understanding is needed regarding aneurysm rupture and its deleterious effects. This study presents a physiologically-feasible explanation for the development of an enormous haemodynamic stress in the vicinity of the aneurysm in the cerebrovascular bed. Using a computer circulation model, this build-up of pressure was developed by sequentially imposing a series of interventions on the normal system.