Apolipoprotein A-I in Labeo rohita: Cloning and functional characterisation reveal its broad spectrum antimicrobial property, and indicate significant role during ectoparasitic infection.

Apolipoprotein A-I (ApoA-I) is the most abundant and multifunctional high-density lipoprotein (HDL) having a major role in lipid transport and potent antimicrobial activity against a wide range of microbes. In this study, a complete CDS of 771 bp of Labeo rohita (rohu) ApoA-I (LrApoA-I) encoding a protein of 256 amino acids was amplified, cloned and sequenced. Tissue specific transcription analysis of LrApoA-I revealed its expression in a wide range of tissues, with a very high level of expression in liver and spleen. Ontogenic study of LrApoA-I expression showed presence of transcripts in milt and 3 h post-fertilization onwards in the larvae. The expression kinetics of LrApoA-I was studied upon infection with three different types of pathogens to elucidate its functional significance. Its expression was found to be up-regulated in the anterior kidney of L. rohita post-infection with Aeromonas hydrophila. Similarly following poly I:C (poly inosinic:cytidylic) stimulation, the transcript levels increased in both the anterior kidney and liver tissues. Significant up-regulation of LrApoA-I expression was observed in skin, mucous, liver and anterior kidney of the fish challenged with the ectoparasite Argulus siamensis. Immunomodulatory effect of recombinant LrApoA-I (rApoA-I) produced in Escherichia coli was demonstrated against A. hydrophila challenge in vivo. L. rohita administered with rApoA-I at a dose of 100 µg exhibited significantly higher protection (∼55%) upon challenge with A. hydrophila 12 h post-administration of the protein, in comparison to that observed in control group, along with higher level of expression of immune-related genes. The heightened expression of ApoA-I observed post-infection reflected its involvement in immune responses against a wide range of infections including bacterial, viral as well as parasitic pathogens. Our results also suggest the possibility of using rApoA-I as an immunostimulant, particularly rendering protection against A. hydrophila.

Four pro-inflammatory cytokines of rohu (Labeo rohita) during early developmental stages, their tissue distribution and expression by leucocytes upon in-vitro stimulation.

Cytokines are important components of both adaptive and innate immunity, and are required to initiate and regulate immune responses following infection. The ontogeny and tissue specific distribution of four pro-inflammatory cytokines, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), IL-8 and IL-1ß in rohu (Labeo rohita), and their responses by leucocytes from anterior-kidney/head-kidney (HKLs), spleen (SPLs) and peripheral blood (PBLs) following stimulation with concanavalin A (ConA), ConA with phorbol 12-myristate 13-acetate (ConA/PMA) and formalin-killed Aeromonas hydrophila cells (FAH) were studied. In ontogeny study, mRNA levels of IL-6 and IL-1ß were evident in unfertilized egg stages of L. rohita whereas IL-8 and TNF-α transcripts were found from 1 to 3 h post-fertilization (hpf) onwards till day 15 post-fertilization, respectively. Basal level of all four cytokines was observed in all twelve tissues (eye, brain, heart, gill, anterior kidney, posterior kidney, spleen, liver, skin, muscle, hindgut and foregut) of L. rohita juveniles. Expression levels of IL-6 and IL-8 were found to be the highest in liver and heart tissues, respectively, while TNF-α transcripts were high in anterior kidney and liver tissues. Transcripts of IL-1ß showed high expression in muscle, heart and spleen. Upon in vitro stimulation of leucocytes, there was variable up-regulation of all the four cytokines following different treatments throughout the experimental time period. Induction of cytokines was more pronounced in PBLs stimulated with FAH compared to other stimuli. However, an up-regulated IL-8 expression was evident in all the leucocytes following stimulation with FAH thus indicating IL-8 could be used as an indicator or indirect marker to monitor vaccine status or health status of L. rohita during bacterial infection.

Immune responses and protective efficacy of recombinant outer membrane protein R (rOmpR)-based vaccine of Aeromonas hydrophila with a modified adjuvant formulation in rohu (Labeo rohita).

Despite the importance and success of developing a candidate vaccine against Aeromonas hydrophila infection in fish, little is known about the molecular mechanisms of the vaccine-induced immunoprotection in Indian major carp, Labeo rohita, primarily due to lack of information on most of the immune related genes of the species. In this study, a novel candidate antigen recombinant outer membrane protein R (rOmpR) of A. hydrophila was evaluated as a vaccine candidate along with a modified adjuvant formulation. Protective efficacy of the rOmpR immunization was assessed in terms of survival against A. hydrophila challenge as well as modulation of immune response in vaccinated fish after 1, 3, 6, 12, 24, 72 h and 10 days post-injection (using immune gene expression analysis) and 10, 28, 56 and 140 days post-injection (serum immune parameter analysis). The generated immune response was compared with a formalin-killed A. hydrophila antigen preparation using mineral oil only and modified adjuvant alone. We report a variable up-regulation of the immune-related genes viz., lysozyme G, complement factor 4, immunoglobulin M, ß2-microglobulin, major histocompatibility complex I and II, and interleukin-1ß in anterior kidney and spleen tissues at early time points post-immunization in all the groups, when compared to the control fish. The vaccinated fish also showed an increase in serum natural hemolysin titer, lysozyme and myeloperoxidase activities, and antibody titer irrespective of vaccine formulations as compared to control fish on days 10, 28 and 56. However, the increase in the serum parameters was more pronounced on day 140 in rOmpR-modified adjuvant injected group, indicating the modulatory role of this new vaccine formulation. Upon challenge with live A. hydrophila on days 56 and 140 post-immunization, significantly reduced percent mortality was noted in the group immunized with modified adjuvant based rOmpR vaccine formulation. Taken together, our results suggest that rOmpR along with modified adjuvant could potentially be used as a vaccine formulation to handle A. hydrophila infection on a long-term basis.

Ouabain-insensitive K-adenosine triphosphatase in distal nephron segments of the rabbit.

An electrogenic H-ATpase sensitive to inhibition by N-ethyl-maleimide has been reported to be present in renal distal tubules. In contrast to another H-ATPase (gastric H-K-ATPase), the renal enzyme is not stimulated by K+ and is not inhibited by vanadate. However, our preliminary observations indicated that a K-stimulated ATPase (K-ATPase) sensitive to inhibition by vanadate is present in renal medullary collecting duct (MCD). To localize and further characterize this renal tubular K-ATPase, we measured K-ATPase activity in eight specific segments of the rabbit nephron. K-ATPase activity was the difference in ATPase activity in the presence and absence of KCl but in the presence of ouabain (to inhibit Na-K-ATPase). ATPase activity was determined by a fluorometric microassay in which ATP hydrolysis is coupled to the oxidation of NADH. There was a significant K-ATPase activity (expressed as pmol.min-1.mm-1) in the connecting tubule (CNT, 17.0 +/- 3.3), cortical collecting duct (CCD, 6.6 +/- 0.7), and MCD (8.8 +/- 1.7), but not in the proximal segments and the thick ascending limbs. The renal tubular K-ATPase was not only inhibited by vanadate but also by omeprazole and SCH 28080 (relatively specific inhibitors of gastric H-K-ATPase). It is concluded that K-ATPase present in the CNT, CCD, and MCD has some properties in common with gastric H-K-ATPase. However, the physiological role of K-ATPase in the distal nephron segments remains to be elucidated.

Renal adaptation to potassium in the adrenalectomized rabbit. Role of distal tubular sodium-potassium adenosine triphosphatase.

Potassium secretion and sodium-potassium adenosine triphosphatase (Na-K-ATPase) activity in the distal nephron segments are known to be influenced by the dietary intake of K+. This has been attributed to a change in the plasma aldosterone level, which also influences K+ secretion and Na-K-ATPase activity in the distal nephron. To investigate whether or not dietary K+ can modulate Na-K-ATPase activity in the distal nephron independently of aldosterone, we determined Na-K-ATPase activity in four distinct nephron segments of adrenalectomized (adx) rabbits given four specific diets for 1 wk before experimentation. Na-K-ATPase activity was determined by a fluorometric microassay in which ATP hydrolysis is coupled to NADH oxidation. The nephron segments examined were the distal convoluted tubule (DCT), the connecting tubule (CNT), the cortical collecting duct (CCD), and the outer medullary collecting duct (MCD). All diets were similar in composition except for their K+ contents, which were 100, 300, 500, and 700 meq/kg in groups 1-4, respectively. In these adx animals, Na-K-ATPase activity increased greater than 200% in the CCD as the dietary intake of K+ increased. There was a linear relationship between K+ excretion and the enzyme activity in this segment. There was a 50% increase in Na-K-ATPase activity in the CNT as the dietary intake of K+ increased in adx animals. However, there were no significant differences in Na-K-ATPase activities in the DCT and MCD among the four treatment groups. It is concluded that dietary K+ intake can influence Na-K-ATPase activity in the CCD and CNT independently of plasma aldosterone levels.

Autocrine inhibition of Na+/K(+)-ATPase by nitric oxide in mouse proximal tubule epithelial cells.

An inducible nitric oxide synthase has recently been described in proximal tubule epithelium. To investigate the effects of proximal tubule NO on Na+/K(+)-ATPase, we induced NO production in mouse proximal tubule epithelial cells by treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) followed by determinations of ouabain-sensitive ATPase activity. Na+/K(+)-ATPase activity decreased after 4 h of LPS/IFN gamma treatment, reaching maximal inhibition after 24 h (34% reduction in activity). The inhibition of Na+/K(+)-ATPase activity by LPS/IFN gamma was prevented by simultaneous incubation with N omega-nitro L-arginine and markedly blunted by removal of L-arginine from the medium. The NO donors sodium nitroprusside and SIN-1 also inhibited Na+/K(+)-ATPase activity to a similar extent than LPS/IFN gamma. However, treatment with 8-pCPT-cGMP only modestly reduced Na+/K(+)-ATPase activity. Interestingly, superoxide dismutase prevented the inhibitory effects of NO on Na+/K(+)-ATPase activity, suggesting a role for peroxynitrite in this inhibition. We conclude that NO generated by mouse proximal tubule epithelial cell iNOS inhibits Na/K ATPase activity in an autocrine fashion and that this inhibition is accompanied by a reduction in Na-dependent solute transport.

Stimulation of phosphoinositide hydrolysis in renal medulla by vasopressin.

Arginine vasopressin (AVP) interacts with V1 and V2 receptors to stimulate hydrolysis of phosphoinositides (PI) and formation of cAMP, respectively. The effects of AVP on V2 receptors in the kidney are well characterized. In order to determine whether V1 receptors, coupled to phospholipase C for hydrolysis of PI, are also present in the kidney, we investigated the effects of AVP on PI hydrolysis in tissue slices from the cortex, outer medulla, and inner medulla of the rabbit kidney. We found that 10(-6) M AVP produced a significant increase in PI hydrolysis in the inner and outer medulla but not in the cortex. In the inner medulla, AVP (10(-10) M) produced a greater than 50% increase in PI hydrolysis; the effect was much greater at higher concentrations. AVP-stimulated PI hydrolysis was blocked by a V1 antagonist but not by a V2 antagonist. Increasing the osmolality of the incubation to 600 mosmol/kg water also abolished the effect of AVP on PI hydrolysis in the inner medulla. Furthermore, AVP did not stimulate PI hydrolysis (even in isoosmotic media) in isolated inner medullary collecting duct cells which make a major portion of the inner medulla. Our results indicate: 1) V1 receptors linked to PI system are not present in the inner medullary collecting duct cells but are probably present in blood vessels and/or interstitial cells of the renal medulla; and 2) AVP-stimulated PI hydrolysis in the inner medulla is modulated by the osmolality of the extracellular fluid.

Production of the beta-subunit of human chorionic gonadotropin in Escherichia coli and its export mediated by the heat-labile enterotoxin chain-B signal sequence.

The PCR-amplified beta-subunit of the human chorionic gonadotropin structural gene (betahCG) was cloned under the control of the tac promoter and the heat-labile enterotoxin chain B (LTB) signal sequence (LTBss). BetahCG was successfully produced, processed and exported to the periplasmic space in Escherichia coli. Expression of betahCG was confirmed by immunoblot analysis using an anti-betahCG polyclonal antibody. The processing of the protein was very efficient, as only the processed band could be detected at all time points during the course of induction. Expression was evident soon after the addition of the lactose analogue, IPTG. These results demonstrate that E. coli cells can synthesize, process and export betahCG using the LTBss.

High-level production and one-step purification of biologically active human growth hormone in Escherichia coli.

A plasmid has been constructed to direct the synthesis of recombinant human growth hormone (re-hGH) in Escherichia coli as a fusion protein containing a His6 tag at the N-terminus under the control of the T5 promoter. The re-hGH was synthesized in large amounts and accumulated in the form of inclusion bodies upon induction with IPTG. Inclusion bodies were solubilized in 6 M guanidine.HCl and the re-hGH was purified by single-step affinity chromatography on Ni(2+)-nitrilotriacetic acid (NTA) agarose. At the shake flask level, the purified re-hGH was obtained with a yield of 30 mg/l of culture. The re-hGH was biologically active in a node rat lymphoma (Nb2) cell bioassay.

Efficient processing and export of human growth hormone by heat labile enterotoxin chain B signal sequence.

The heat-labile enterotoxin chain B (LTB) signal sequence was used for the processing and export of human growth hormone (hGH). The protein was completely processed and exported across the cell membrane to accumulate in the periplasmic space in Escherichia coli. The human growth hormone cDNA was cloned as a PCR amplified fragment under the control of tac promoter and translationally fused to the LTB signal sequence. The rate of processing of hGH under the control of the LTB signal sequence was equal to or more than the rate of induction of expression, indicating efficient processing. The receptor binding activity of the processed periplasmic protein was established in a radio receptor assay.

Efficient expression, processing and secretion of a biologically active mammalian protein by Vibrio cholerae.

The use of Vibrio cholerae as a secretory expression system for the expression of a mammalian protein, namely human growth hormone, under the control of the heat labile enterotoxin chain B signal sequence is reported. The protein is efficiently expressed and processed. The mature protein is exported to the periplasm after which it is secreted to the extracellular milieu. The expressed and secreted hGH actively binds to its receptor as established by its receptor binding activity. The biological activity of the protein is demonstrated in vitro in a Nb2 proliferation assay.

N-terminus of mature heat-labile enterotoxin chain B is critical for its extracellular secretion in Vibrio cholerae.

The effects of addition of a few amino acids to the amino- and carboxy-terminal regions of the mature portion of the heat-labile enterotoxin chain B (LTB) of Escherichia coli on protein export, secretion and assembly were investigated. In E. coli, LTB (secretory protein) with or without the extension at the N- or C-terminus accumulated in the periplasmic fraction. For Vibrio cholerae, LTB with the extension at the C-terminus was exported to the periplasm followed by secretion to the extracellular milieu. However, LTB with the N-terminus extension was exported to the periplasm only. Our findings suggest that in the case of V. cholerae, the N-terminus of the mature LTB plays an important role in its secretion to the extracellular milieu.

Translational fusion of heat labile enterotoxin chain B and beta-subunit of human chorionic gonadotropin: periplasmic expression in Escherichia coli and its immunogenicity.

A fusion gene was constructed consisting of heat labile enterotoxin chain B (LTB) of E. coli genetically linked at its C-terminus to the beta-subunit of human chorionic gonadotropin in translational fusion, under the control of tac promoter and LTB signal sequence. Expression of the fusion gene (about 5 microgram/ml) in E. coli was confirmed by immunoblot analysis using both anti-LTB and anti-betahCG polyclonal antibodies. The fusion protein was efficiently processed and exported to the periplasmic space. LTB in the fusion protein retained its ability to bind to GM1 ganglioside receptor. Mice immunized with the fusion protein produce antibodies that recognize recombinant betahCG and the native hCG suggesting its potential use as a contraceptive vaccine.

Evidence of a high-activity C type of carbonic anhydrase in human ciliary processes.

Carbonic anhydrase activity was found in the ciliary process of fresh human donor eyes, originating from an enzyme antigenically similar to the erythrocyte high-activity isoenzyme HCA C. It was sensitive to inhibition by acetazolamide and resistant to inhibition by halides like HCA C. The enzyme is probably identical with HCA C. Its tissue concentration was one fifth to one tenth of that in the human kidney. The erythrocyte low-activity isoenzyme HCA B was also found in the processes as a contaminant.

Induction of differentiation in murine erythroleukemia cells by 1 alpha,25-dihydroxy vitamin D3.

The Friend murine erythroleukemia (MEL) cells can be stimulated to differentiate in response to a variety of chemical inducing agents. In the present study, the effect of 1 alpha,25-dihydroxyvitamin D3 on differentiation of MEL cells was investigated. Vitamin D3 induced differentiation of MEL cells in culture as determined by elevated hemoglobin content, a rise in the number of benzidine-positive cells and increase in acetylcholine esterase activity. The optimum concentration of the vitamin required to induce differentiation of MEL cells was found to be 750 nM. The pattern of induction of differentiation was similar to that observed with DMSO and the induction of differentiation by vitamin D3 was inhibited by dexamethasone.

Ocular hypotensive effects of timolol in cat eyes.

The effects of timolol on the aqueous humor (AH) formation and AH outflow in cat eyes were simultaneously studied with a continuous infusion method under constant intraocular pressure. The rate of AH formation was reduced 28%, 56%, and 71%, respectively, by 0.005%, 0.025%, and 0.15% of timolol solution infused intracamerally. There was no significant change of AH outflow after the administration of timolol. It was noted that the pupil was dilated by 0.025% and 0.15% timolol solutions. To determine the action mechanism of timolol to inhibit AH formation, experiments of carbonate dehydratase inhibition were performed with various concentrations of timolol. No appreciable enzyme inhibition was noted with timolol at concentrations up to 0.5%. It is thus concluded that the action mechanism of timolol to inhibit AH formation differs from that of carbonate dehydratase inhibitors.

Hyperoxia reduces plasma membrane fluidity: a mechanism for endothelial cell dysfunction.

To evaluate the relative contributions of three possible mechanisms that can be advanced to explain the observation that hyperoxia decreases serotonin uptake by endothelial cells, we examined the effect of high O2 tensions on Na+-K+-ATPase activity, ATP content, and plasma membrane fluidity in cultured endothelial cells. Confluent monolayers of pulmonary artery and aortic endothelial cells were exposed to 95% O2 (hyperoxia) or 20% O2 (controls) in 5% CO2 at 1 ATA for 4-42 h. Exposure to high O2 tensions had no effect on Na+-K+-ATPase activity or ATP content in pulmonary artery or aortic endothelial cells in culture. However, hyperoxia decreased the fluidity of the plasma membrane of pulmonary artery and aortic endothelial cells in culture, and the time course for the decrease in fluidity parallels that of the hyperoxic inhibition of serotonin transport. These results indicate that hyperoxia decreases fluidity in the hydrophobic core of the plasma membranes of cultured endothelial cells. Such decreases in plasma membrane fluidity may be responsible for hyperoxia-induced alterations in membrane function including decreases in transmembrane transport of amines.

Synthesis and release of acetylcholine in the rabbit kidney cortex.

Several cholinergic processes were demonstrated and partially characterized in rabbit kidney cortical minces: choline uptake, acetylcholine synthesis and calcium-dependent release. Minces took up labelled choline, acetylated it, and stored it in a pool that was not readily accessible to physostigmine-sensitive cholinesterase activity. [3H]Acetylcholine synthesis but not [3H]choline uptake was inhibited by the removal of sodium ions or incubation at 0 degrees C. The release of newly synthesized [3H]acetylcholine was increased by 300 mOsmol urea in a calcium-dependent manner, but not by potassium depolarization (300 mOsmol), vasopressin (10 microM), or bradykinin (10 microM). These results suggest that acetylcholine may be synthesized by non-neuronal rabbit kidney cortical cells and that this transmitter may be released in response to physiological levels of urea.

Correlative evidence of hypertension and altered cochlear microhomeostasis: electrophysiological changes in the spontaneously hypertensive rat.

The spontaneously hypertensive rat model has been used to show that hypertension is an important pathophysiological risk factor in age-related hearing loss. In the present study, compound action potential (CAP), electrochemical potential (ECP), and potassium concentration (CK+) measurements were taken from the cochlea of genetically predisposed, spontaneously hypertensive rats (SHR) and from normotensive Wistar-Kyoto (WKY) rats. In the SHR model, as the duration of hypertension increased with the animal's age (from 3 to 8 months), CAP thresholds increased, ECP increased in marginal cells only, and CK+ increased in both endolymph and marginal cells. Collectively, the data suggest that ionic alternations of cellular potentials are involved in hearing changes in the hypertensive state. Ultimately, such data may assist in understanding hearing loss in individuals who are diagnosed with hypertension.

Molecular cloning and sequence analysis of bubaline lactoferrin promoter.

The proximal promoter for bubaline lactoferin-encoding gene has been isolated, cloned and sequenced. A 468 bp fragment of the 5' flanking region of the lactoferrin gene was PCR amplified and cloned into pUC18 vector. Sequence analysis of the amplified fragment revealed the presence of one TATA box, one TATA like element, two GC boxes and one motif resembling cAMP response element (CRE) in this region. Bubaline lactoferrin promoter shares 93%, 53%, 52% and 48% homology with cattle, pig, mouse and human lactoferrin 5' flanking region, respectively.