Surface Of Tubules Research Articles

Formation of Surface Tubules and Fluorescent Foci in Arabidopsis thaliana Protoplasts Expressing a Fusion between the Green Fluorescent Protein and the Cauliflower Mosaic Virus Movement Protein

The movement protein (MP) of cauliflower mosaic virus (CaMV) is a multifunctional protein that potentiates the cell-to-cell and long distance movement of the virus. Functional domains in the CaMV MP were determined by analyzing deletions in green fluorescent protein (GFP)–MP fusions transfected into Arabidopsis thaliana leaf protoplasts. GFP–MP accumulated at fluorescent foci at the cell periphery and in tubular structures extending from the protoplast surface. A region located near the center of MP was required for targeting GFP–MP to foci, whereas a larger region extending nearly to the N-terminus was needed for tubule formation. Cytoskeletal assembly inhibitors did not disrupt tubule formation or the accumulation of GFP–MP at foci, but brefeldin A, which disrupts the plant cell endomembrane system, did interfere with the formation of tubules but not foci.

Renal Cryptosporidiosis (Cryptosporidium baileyi) in Specific-Pathogen-Free Chickens Experimentally Coinfected with Marek's Disease Virus

Renal Cryptosporidiosis was experimentally induced during a study to investigate the pathogenicity of Cryptosporidium baileyi in specific-pathogen-free (SPF) chickens coinfected with Marek's disease virus (MDV). Cryptosporidium baileyi was administered orally at 4 days of age to chickens previously infected at hatching (day 0) with the HPRS 16 strain of oncogenic MDV. Three control groups received MDV at hatching, C. baileyi on day 4, or placebo consisting of distilled water. Renal cryptosporidiosis lesions were induced in the group coinfected with MDV and C. baileyi. The kidneys were markedly swollen and pale, with visible urate crystals in the ureters and surface tubules. Oocysts of C. baileyi were demonstrated in six of seven cases tested by a scoring method with modified Sheather's sugar solution on renal tissue scrapings and were confirmed in three cases by histologic examination of paraffin-embedded kidney sections. Histologic study also revealed subacute interstitial nephritis, acute ureteritis, and attachment of cryptosporidia on the epithelial cell surface of the ureters and collecting ducts, collecting tubules, and distal convoluted tubules. Various developmental stages of the parasite were present in the kidney sections. To our knowledge, this is the first report of experimentally induced renal cryptosporidiosis in SPF chickens coinfected with MDV.

Glutamate transport and renal function.

Brush border gamma-glutamyltransferase-glutaminase activity and the high-affinity glutamate transporter EAAC1 function as a unit in generating and transporting extracellular glutamate into proximal tubules as a signal that modulates intracellular glutamine/glutamate metabolism, paracellular permeability, and urinary acidification. The reported presence of a second glutamate transporter, GLT1, on the antiluminal tubule surface points to specific functional roles for each subtype in physiological and pathophysiological processes.

Glutamate transport asymmetry and metabolism in the functioning kidney.

Renal glutamate extraction in vivo shows a preference for the uptake of D-glutamate on the antiluminal and L-glutamate on the luminal tubule surface. To characterize this functional asymmetry, we isolated rat kidneys and perfused them with an artificial plasma solution containing either D- or L-glutamate alone or in combination with the system X-AG specific transport inhibitor, D-aspartate. To confirm that removal of glutamate represented transport into tubule cells, we monitored products formed as the result of intracellular metabolism and related these to the uptake process. Perfusion with D-glutamate alone resulted in a removal rate that equaled or exceeded the L-glutamate removal rate, with uptake predominantly across the antiluminal surface; L-glutamate uptake occurred nearly equally across both luminal and antiluminal surfaces. Thus the preferential uptake of D-glutamate at the antiluminal and L-glutamate at the luminal surface confirms the transport asymmetry observed in vivo. Equimolar D-aspartate concentration blocked most of the antiluminal D-glutamate uptake and a significant portion of the luminal L-glutamate uptake, consistent with system X-AG activity at both sites. D-Glutamate uptake was associated with 5-oxo-D-proline production, whereas L-glutamate uptake supported both glutamine and 5-oxo-L-proline formation; D-aspartate reduced production of both 5-oxoproline and glutamine. The presence of system X-AG activity on both the luminal and antiluminal tubule surfaces, exhibiting different reactivity toward L- and D-glutamate suggests that functional asymmetry may reflect two different X-AG transporter subtypes.

Synthesis and micro-structural study of one-dimensional nano-materials

Silicon nano-wires (SiNWs) and boron nitride nano-tubules (BN-NTs) were successfully synthesized by excimer laser ablation at high temperature. These one-dimensional nano-materials synthesized by this method have a very high yield, a uniform diameter distribution, and a high purity. Micro-structures of these nano-materials were investigated by transmission electron microscopy (TEM). The SiNWs have a high density of structural defects of microtwin, stacking faults, and low-angle boundary, which are closely related to the formation of SiNWs and the determination of morphology of the nano-wires. BN-NTs are mainly single atomic-layered and the outer surface of tubules is clean without any attachment. The formation of single atomic-layered tubule is attributed to the catalyst effect which makes the axial rate of BN-NTs much higher than the radial growth

Confocal microscopy of dentinal tubules in human tooth stained with alizarin red.

The present study was designed to analyze the structures of dentinal tubules by confocal microscopy. Undecalcified ground sections of human teeth were stained with alizarin red in 0.1% KOH aqueous solution, and examined by confocal microscopy. Alizarin red stained dentinal tubules, interglobular dentine, granular layer of Tomes, and the surface of dentine. Interglobular dentine was seen between the outer and middle layers of coronal dentine. At the outer layer of coronal dentine, the dentinal tubules were thin and showed numerous branches. At the middle layer of coronal dentine, dentinal tubules displayed two types. The type I tubules are the dentinal tubules that do not show any nodular structures and the type II tubules are the dentinal tubules that appear bamboo-like with many nodules. In the cross section through the type II tubules, the nodules appeared as fine circular tubules surrounding the dentinal tubules. The circular tubules of nodules adhered to one side of the dentinal tubules. When the fluorescence images were compared with the images taken by transmission light mode, the fluorescence of dentinal tubules was seen at the inner surface of dentinal tubules, and the fluorescence of nodules was seen at interface between peritubular and intertubular dentine. Most of the dentinal tubules were of the type II tubules in the teeth from older individuals, whereas the type II tubules were scarce in the teeth from younger individuals. At the inner layer of coronal dentine, the dentinal tubules have no nodules and branches were scarce. The dentinal tubules of radicular dentine were different from those of coronal dentine. Most of the dentinal tubules were the type I tubules. Numerous fine branches were seen at the outer and middle layers of radicular dentine. No interglobular dentine was seen in the root except at the cervical part, and the granular layer of Tomes was also positive with alizarin red. At the cervical part of the root, interglobular dentine was present and the dentinal tubules displayed types I and II.

Poxvirus virions: their surface ultrastructure and interaction with the surface membrane of host cells.

Virions of vaccinia and orf viruses were examined by ultrahigh-resolution scanning electron microscopy using a non-coating method. Intracellular mature particles of vaccinia virus appeared to be covered with a net and ultrastructurally their surface consists of many fine ridges and globules, while the surfaces of orf virus mature particles recovered from infected cells consist of spirally running protrusions. The ridge-like structures of vaccinia virus were presumed to correspond to surface tubules shown by negative staining of this virus, while the spiral protrusions of orf virus were presumed to correspond to spiral threads having a criss-cross appearance by the same staining. Using scanning electron microscopy in which the samples were prepared by the conventional method, we observed: (i) many virions, i.e. one or two hundreds, or occasionally more reaching about one thousand particles, of the IHD strain of vaccinia virus, (ii) many or a moderate number of virions, i.e. about one hundred or fewer particles, of the 58 strain of cowpox virus and (iii) rather few virions, i.e. several tens or fewer particles, of the Iwate strain of orf virus on the free surface of each cell infected with these viruses. It must be noted that the number of virions detected considerably differed in respective cells examined. Virus budding was frequently observed at the cell surface of monolayer cells infected with vaccinia virus but it was never detected with cowpox or orf virus, indicating a difference in the mechanism of virus release between vaccinia and the other two viruses. When whole cells infected with vaccinia virus were examined by a combination of high-voltage and scanning electron microscopies, virions on the cell surface and those inside the cells were clearly differentiated. All virions on the cell surface had an envelope, and some of the envelopes had a slack and/or one or more bulges.

Functional activity of epidermal growth factor receptors in autosomal recessive polycystic kidney disease.

Evidence from a number of laboratories suggests a potential role for the epidermal growth factor (EGF)-transforming growth factor-alpha-epidermal growth factor receptor (EGF-R) axis in promoting epithelial hyperplasia and cyst formation in autosomal recessive polycystic kidney disease (ARPKD). As previously reported, in the C57BL-6Jcpk/cpk (CPK), BALB/c-bpk/bpk (BPK), and C3H-orpk/orpk (ORPK) murine models of ARPKD, as well as in human ARPKD and human ADPKD, the EGF-R is mislocated to the apical surface of cystic collecting tubule (CT) epithelial cells. The present studies demonstrate that cells from cystic and control CTs can be isolated and that these cells maintain their in vivo EGF-R phenotype in vitro. Domain-specific high-affinity ligand binding was assessed by standard Scatchard analysis, and selective ligand stimulation of apical vs. basolateral EGF-R in these cells was followed by measurement of receptor autophosphorylation and determination of cell proliferation. These studies demonstrate that in vitro apically expressed EGF-Rs exhibit high-affinity binding for EGF, autophosphorylate in response to EGF, and transmit a mitogenic signal when stimulated by the appropriate ligand.

Functional characterization of ORCTL2 - an organic cation transporter expressed in the renal proximal tubules

Chromosome 11p15.5 harbors a gene or genes involved in Beckwith-Wiedemann syndrome that confer(s) susceptibility to Wilms' tumor, rhabdomyosarcoma, and hepatoblastoma. We have previously identified a transcript at 11p15.5 which encodes a putative membrane transport protein, designated organic cation transporter-like 2 (ORCTL2), that shares homology with tetracycline resistance proteins and bacterial multidrug resistance proteins. In this report, we have investigated the transport properties of ORCTL2 and show that this protein can confer resistance to chloroquine and quinidine when overexpressed in bacteria. Immunohistochemistry analyses performed with anti-ORCTL2 polyc.onal antibodies on human renal sections indicate that ORCTL2 is localized on the apical membrane surface of the proximal tubules. These results suggest that ORCTL2 may play a role in the transport of chloroquine and quinidine related compounds in the kidney.

The axonemal microtubules of the Chlamydomonas flagellum differ in tubulin isoform content.

Little is known of the molecular basis for the diversity of microtubule structure and function found within the eukaryotic flagellum. Antibodies that discriminate between tyrosinated alpha tubulin and post-translationally detyrosinated alpha tubulin were used to localize these complementary tubulin isoforms in flagella of the single-celled green alga Chlamydomonas reinhardtii. Immunofluorescence analysis of intact axonemes detected both isoforms along most of the lengths of flagella; however, each had a short distal zone rich in tyrosinated tubulin. Localizations on splayed axonemes revealed that the microtubules of the central-pair apparatus were rich in tyrosinated tubulin, while outer doublets contained a mixture of both isoforms. Immunoelectron analysis of individual outer doublets revealed that while tyrosinated tubulin was present in both A and B tubules, detyrosinated tubulin was largely confined to the wall of the B hemi-tubules. The absence of detyrosinated tubulin from the A tubules of the outer doublets and the microtubules of the central pair, both of which extend past the B hemi-tubules of the outer doublets in the flagellar tip, explained the appearance of a tyrosinated tubulin-rich distal zone on intact axonemes. Localizations performed on cells regenerating flagella revealed that flagellar assembly used tyrosinated tubulin; detyrosination of the B tubule occurred during later stages of regeneration, well after microtubule polymerization. The developmental timing of detyrosination, which occurs over a period during which the regrowing flagella begin to beat more effectively, suggests that post-translational modification of the B tubule surface may enhance dynein/B tubule interactions that power flagellar beating.

Molecular cloning and functional expression of an LLC-PK1 cell taurine transporter that is adaptively regulated by taurine.

Studies have shown that the renal tubular epithelium adapts to alterations in the sulfur amino acid composition of the diet. The renal adaptive response has been described in man, mouse, rat, dog, and pig. The observed phenomenon involves increased or decreased initial rate activity of the NaCl-dependent taurine transporter at the brush border membrane surface of the proximal tubule following dietary manipulation of taurine. A cDNA encoding a taurine transporter has been isolated from LLC-PK1 cells, designated pTAUT, and its functional properties have been examined in Xenopus laevis oocytes. The nucleotide sequence of the clone predicts a 621-amino acid protein with about 90% homology to other cloned taurine transporter cDNAs. When expressed in oocytes the transporter displays a Km of 25 microM and is dependent on the presence of external sodium and chloride, characteristics similar to taurine uptake by LLC-PK1 cells. The abundance of pTAUT mRNA and protein were up-regulated in cells cultured in taurine-free medium as compared with cells cultured in medium containing 500 microM taurine. Activation of PKC by PMA had no effect on adaptive regulation of pTAUT mRNA and protein, indicating that down-regulation of LLC-PK1 cell taurine transport activity by PMA occurs at the post-translational level.

The role of complement in the pathogenesis of tubulointerstitial lesions in rat mesangial proliferative glomerulonephritis.

Persistent proteinuria and tubulointerstitial lesions are important signs of progressive renal disease. The purpose of this study was to assess the role of complement in the development of tubulointerstitial lesions in rats with proteinuria due to primary glomerulonephritis. Mesangial proliferative glomerulonephritis was induced in mononephrectomized rats by intravenous injection of monoclonal antibody (mAb) 1-22-3 (Clin Exp Immunol 102: 181-185, 1995). As early as 24 h after the injection, proteinuria became evident, persisted throughout the observation period, and was associated with mesangial cell proliferation and tubulointerstitial lesions when examined at 7 and 14 d after mAb administration. Deposition of rat C3 and C5b-9 was observed at the luminal surface of proximal tubules and in cellular debris present in the tubular lumen (group I). Rats injected with mAb 1-22-3 and depleted of complement by injections of cobra venom factor starting at day 3 developed glomerulonephritis and proteinuria comparable to rats of group I, but complement deposition in the tubules and the tubulointerstitial lesions were markedly reduced (group II). Rats in group III were injected with mAb and, from day 3, with soluble complement receptor type 1, which became detectable at the luminal surface of proximal tubules and in the urine. Deposition of C5b-9 in tubular cells was not detectable, and the severity of tubulointerstitial lesions was reduced compared with rats in group I. These results indicate that, in this model of primary mesangial proliferative glomerulonephritis with proteinuria, the development of tubulointerstitial lesions is associated with activation of serum complement at the level of tubular brush border, and tubulointerstitial lesions can be reduced by inhibition of complement activity.

Effect and distribution of intravenously injected 125I-guanylin in rat kidney examined by high-resolution light microscopic radioautography.

125I-guanylin was injected intravenously into rats, and their kidney and intestinal tract were processed for light microscopic radioautography using semithin sections to examine the binding sites. Various doses of unlabeled guanylin were also injected to examine the morphological effects of guanylin on the kidney. Dense labeling of silver grains due to 125I-guanylin were observed only in the kidney. In the cortex, silver grains were localized on the luminal side of the proximal tubules at 5-30 min. In the medulla, silver grains appeared at the basal side of the collecting ducts, capillaries and loops of Henle after 5 min. Silver grains then accumulated in the cytoplasm of the collecting ducts after 10 min, and disappeared after 30 min. The cell height of the inner medullary collecting ducts (IMCD) decreased and their luminal spaces increased dose-dependently 5 min after the injection of both labeled and unlabeled guanylin. These structural changes returned to control levels within 30 min. These results indicate a high density localization of guanylin receptors on the luminal surface of proximal tubules in the renal cortex and also rapid excretion of guanylin through the IMCD. The morphological changes of the IMCD suggest a diuretic effect of guanylin.

Analysis of epithelial transport by measurement of K+, Cl- and pH gradients in extracellular unstirred layers: ion secretion and reabsorption by Malpighian tubules of Rhodnius prolixus

Summary The pH and concentrations of K+ and Cl- in the unstirred layer (USL) associated with the basolateral surfaces of the upper and lower Malpighian tubules of Rhodnius prolixus were measured using extracellular ion-selective microelectrodes. When stimulated with 5-hydroxytryptamine (5-HT) in vitro, the upper Malpighian tubule secretes Na+, K+, Cl- and water at high rates; the lower Malpighian tubule reabsorbs K+ and Cl- but not water. Concentrations of K+ and Cl- in the unstirred layer of the lower Malpighian tubule ([K+]USL, [Cl-]USL) were greater than those in the bathing saline, consistent with the accumulation of K+ and Cl- in the USL during 5-HT-stimulated KCl reabsorption. [K+]USL exceeded [K+]Bath by as much as 5.3-fold. Calculations of K+ flux based on measurements of [K+]USL at various distances from the tubule surface agreed well with flux calculated from the rate of fluid secretion and the change in K+ concentration of the secreted fluid during passage through the lower tubule. Concentrations of K+ in the unstirred layer of the upper Malpighian tubule were reduced relative to those in the bathing saline, consistent with depletion of K+ from the USL during 5-HT-stimulated secretion of K+ from bath to lumen. Changes in [K+]USL during 5-HT-stimulated K+ secretion from single upper Malpighian tubule cells could be resolved. Although differences between [K+]USL and [K+]Bath were apparent for upper and lower tubules in an in situ preparation, they were reduced relative to the differences measured using isolated tubules. We suggest that convective mixing of the fluids around the tubules by contractions of the midgut and hindgut reduces, but does not eliminate, differences between [K+]USL and [K+]Bath in situ. The USL was slightly acidic relative to the bath in 5-HT-stimulated upper and lower tubules; contributions to USL acidification are discussed. The results also show that the techniques described in this paper can resolve rapid and localized changes in ion transport across different regions of Malpighian tubules in response to stimulants or inhibitors of specific membrane transporters.

Expression of adhesion molecules in rat renal cortex during experimental hydronephrosis

Unilateral ureteral obstruction (UUO) is associated with an early and steadily increasing infiltration of macrophages into the renal cortical interstitium. As adhesion molecules may play an important role in macrophage recruitment following the mechanical disturbance after UUO, we delineated the time course of intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 mRNA and protein expression. A significant 6.6- (P < 0.001), 2.6- (P < 0.025), 2.6- (P < 0.01), and 2.0-fold (P < 0.005) increase in ICAM-1 mRNA expression was observed at 12, 24, 48, and 96 hours after obstruction, respectively, in comparison to the contralateral unobstructed kidney (CUK). Despite an apparent relief of obstruction, four weeks following reversal of obstruction mRNA levels of ICAM-1 remained equivalent to the 96-hour obstructed kidney group. No significant difference in VCAM-1 mRNA expression was observed between the obstructed kidneys and CUK specimens. Immunohistochemistry revealed focal labeling of ICAM-1 on the apical and basolateral surface of the renal tubules, peritubular interstitium, and vessels of the renal cortex by 12 hours after UUO. In contrast, only faint staining for ICAM-1 protein was observed in the cortex from CUK specimens. The obstructed and CUK specimens exhibited diffuse immunolocalization of VCAM-1 in the cortical tubules and Bowman's capsular epithelium. In situ hybridization showed mRNA transcription for ICAM-1 localized in the peritubular interstitium and cortical tubules from obstructed kidneys. To lend mechanistic insight into the response of ICAM-1 to the mechanical disturbance after UUO, the expression of ICAM-1 mRNA was examined when freshly isolated proximal tubules were exposed to angiotensin II (1 to 100 microM) immediately after preparation. Levels of ICAM-1 mRNA were elevated 1.4-, 7.1-, and 3.7-fold when exposed to 10 microM, 100 microM, and 1000 microM of angiotensin II for one hour, respectively, when compared to control cultures. The addition of losartan to proximal tubules for one hour prior to angiotensin II stimulation decreased ICAM-1 levels to control values. In summary, this investigation demonstrates that ICAM-1 is important in the initiation of macrophage recruitment into the renal cortex of the obstructed kidney. These findings provide evidence that angiotensin II, produced after ureteral ligation as a result of tubular injury and dysfunction, may play a central role in the release of ICAM-1 from the proximal tubule epithelial cells.

Effects of recombinant human tissue factor pathway inhibitor on thrombus formation and its in vivo distribution in a rat DIC model.

Tissue factor pathway inhibitor (TFPI) plays a key role in modulating tissue factor-dependent blood coagulation. This study was done to determine not only the inhibitory effects of recombinant human TFPI (rTFPI) on thrombus formation in rat models with disseminated intravascular coagulation (DIC), but also to identify the distribution of exogenous TFPI in vivo. Disseminated intravascular coagulation was induced by administering a priming dose of carrageenan 10 mg/kg body weight and was followed 24 hours later by a provocative dose of lipopolysaccharide (LPS) 500 mg/kg body weight. The rTFPI was administered intravenously at a dose of either 1 or 4 mg/kg body weight immediately after LPS treatment. Exogenous rTFPI at a dose of 4 mg/kg significantly inhibited the consumption of fibrinogen, platelets and factor VIIa (P < .05) and also reduced the number of fibrin thrombi formed in the liver, lungs, kidneys, and spleen (P < .05), whereas rTFPI at a dose of 1 mg/kg had no significant inhibitory effect on these DIC parameters. Recombinant human rTFPI activity was rapidly cleared from the plasma; however, a significant amount of the inhibitor was still present in tissues even 3 to 6 hours after intravenous administration. Exogenous TFPI was mainly identified in Kupffer cells, macrophages, and on the microvascular endothelial lining of different organs. In the kidney, rTFPI was identified on both the abluminal surface of the renal tubules and the luminal surface of the proximal convoluted tubules. No rTFPI, however, was detected in the hepatocytes. Tissue factor was mainly expressed by monocytes/macrophages. These findings suggest that TFPI plays an important role in modulating TF-dependent thrombogenesis. The elucidation of the rTFPI distribution and interactions in vivo might thus provide valuable insight into its inhibitory mechanisms as well as its therapeutic implications in DIC.

Renal Na+-phosphate cotransporter gene expression in X-linked Hyp and Gy mice

The X-linked Hyp and Gy mutations are murine homologues of X-linked hypophosphatemia (XLH), a dominant disorder of phosphate (Pi) homeostasis characterized by growth retardation, rickets, hypophosphatemia and decreased renal tubular maximum for Pi reabsorption relative to glomerular filtration rate (Tmp/GFR). In Hyp and Gy mice, the decrease in Tmp/GFR is associated with a reduction in renal brush-border membrane (BBM) Na(+)-Pi cotransport that can be ascribed to a decrease in renal-specific, Na(+)-Pi cotransporter (NPT2) mRNA and protein abundance. Although renal NPT2 gene expression is reduced in Hyp and Gy mice, the NPT2 gene does not map to the X chromosome. These findings exclude NPT2 as a candidate gene for murine and human X-linked hypophosphatemias and suggest that genes at the Hyp, Gy and XLH (HYP) loci are involved in regulation of NPT2 gene expression. Both Hyp and Gy mice respond to low Pi diet with an increase in BBM Na(+)-Pi cotransport, NPT2 mRNA and protein. The increase in NPT2 protein in Pi-depleted mice far exceeds the increase in NPT2 mRNA, suggesting that translational or post-translational mechanisms are involved in the adaptive process. NPT2 protein is localized to the apical surface of the proximal tubule, where immunostaining in both normal and Hyp mice is increased in response to low Pi diet. Pi-deprived Hyp and Gy mice fail to show an increase in Tmp/GFR, indicating that adaptation at the BBM is not sufficient for the overall increase in Tmp/GFR in response to low Pi diet.

Characterization and modification of the carboxy-terminal sequences of bluetongue virus type 10 NS1 protein in relation to tubule formation and location of an antigenic epitope in the vicinity of the carboxy terminus of the protein

Bluetongue virus produces large numbers of tubules during infection. The tubules are formed from a 552-amino-acid, 64-kDa NS1 protein encoded by the viral double-stranded RNA segment M6. A series of deletion and extension mutants of bluetongue virus serotype 10 NS1 has been generated and expressed in insect cells in order to identify the carboxy-terminal components of the protein which are important for tubule formation. The mutants AcCT5 and AcCT10, lacking 5 and 10 of the carboxy-terminal residues, respectively, were prepared. By analyzing their abilities to form tubules, it was shown that AcCT5 was capable of this function whereas AcCT10 was not, indicating that the last five amino acids are not strongly involved in NS1 tubule formation. Extension mutants including foreign antigenic sequences involving up to 16 amino acids added to the C terminus of NS1 were shown to form tubules, although an extension of 19 amino acids inhibited tubule formation. Analysis of a panel of monoclonal antibodies has established that an NS1 antigenic site is located near the carboxy terminus of the protein. It appears to be exposed on the surface of tubules. The opportunities to develop new vaccines using recombinant NS1 to deliver foreign epitopes are discussed.

Effect of barium on potassium diffusion across the proximal convoluted tubule of the anesthetized rat.

The role of diffusion in transepithelial potassium flux and the importance of potassium channels in the luminal cell membrane to this process were examined by applying a luminal microperfusion technique to surface tubules in kidneys of anesthetized rats. Potassium concentration gradients were applied by altering the concentration of KCl in perfusates. To some perfusates, 2 mmol/l BaCl2 was added to block potassium channels in the luminal cell membrane. The mean applied potassium concentration gradient was highly predictive of net potassium transport in the absence of any change in fluid reabsorption, with an apparent potassium permeability of 22 x 10(-5) cm/s. Thus potassium transport in the proximal tubule may have an important diffusive component. Luminal barium significantly reduced the concentration of potassium in collected fluid under conditions of net potassium secretion, although a substantial barium-insensitive potassium permeability was also observed. However, the site of action of luminally applied barium is uncertain in proximal tubule, since barium was reabsorbed by the tubule at a rate of 13.6 pmol.mm-1.min-1. We conclude that diffusion is a significant driving force for potassium reabsorption in proximal tubule and that most diffusive potassium transport occurs via a barium-insensitive route, possibly the paracellular pathway.

Characterization and localization of oxytocin receptors in the rat testis

In this study oxytocin (OT) receptors have been characterized and localized in the testis of the rat using the radioiodinated OT receptor antagonist 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr9-NH2]-vasotocin (OTA). Receptor density and localization have been compared with the rat testis arginine vasopressin (AVP) receptor using the radioiodinated AVP V1a receptor antagonist 125I-labelled d(CH2)5Sar7-AVP and the radioiodinated linear AVP V1a antagonist 125I-labelled [(C6H5-CH2CO)-O-methyl-D-Tyr-Phe-Gln-Asn-Arg-Pro- Arg-Pro-Arg-Tyr-NH2]. 125I-labelled OTA bound with high affinity to membrane fractions of the rat testis (Ka = 13.8 +/- 1.25 litres/nmol), mammary tissue (Ka = 20.3 +/- 4.36 litres/nmol) and uterus (Ka = 27.8 +/- 0.74 litres/nmol). Competition studies with various OT and AVP receptor agonists and antagonists confirmed that the binding was to OT receptors. AVP receptors in the testis were found to be identical to AVP V1a receptors in the liver. The AVP receptor density in the testis was much higher than the OT receptor density (109 +/- 12.3 vs 5.2 +/- 0.79 (mean +/- S.E.M.) fmol/mg protein). Autoradiographical localization showed that both OT and AVP receptors were present in the interstitial spaces in the testis consistent with binding to Leydig cells. AVP receptors were also localized on the epithelial surfaces of the seminiferous tubules and on testicular blood vessels. This study has, for the first time, found OT receptors in the testis of the rat which have similar ligand-binding characteristics to mammary and uterine OT receptors. The receptor localizations are consistent with binding to Leydig cells.