Mutations in cytochrome B gene effects female reproduction of Ghungroo pig.

Cytochrome B is an important polypeptide of the mitochondria helpful in energy metabolism through oxidative phosphorylation. Cytochrome B plays an immense role in the reproduction of animals and due to its mutation prone nature, it can affect the basic physiology of animals. Cytochrome B affects reproductive system in males and equally plays an important role in transferring and providing energy in the development of the embryo, zygote, and oocytes precisely in females. The present study was conducted on Ghungroo pig to study their molecular and reproductive traits and the effect of the cytochrome B gene in the female reproduction of the Ghungroo pig. Although studies are available for cytochrome B gene analysis for evolutionary studies through phylogenetic analysis. This is the first report for the study of Cytochrome B gene on reproduction in pigs. Cytochrome B gene was sequenced and seven SNPs were observed out of which three were non-synonymous. INDEL mutation was detected in Variant B which had lead to Frame Shift mutation resulting in a stop codon AGA. The effect in the reproductive traits of the sow was studied due to the occurrence of nucleotide substitution. Bioinformatics analysis (I-mutant, PROVEAN, and SIFT) had revealed that the mutations were deleterious for the mutant type. Mutation leading to alterations in post-translational modification sites as phosphorylation site, leucine-rich nuclear export signal, occurrence of transmembrane helices, arginine and lysine peptide cleavage site for the mutant variant had resulted in a reduced physiological response. 3 D protein structure, (predicted through bioinformatics analysis) for cytochrome B has revealed distinct structural differences in mutated form with truncated protein by RMSD analysis through TM-Align software. Associated studies of genotype variants with reproductive traits have revealed the significant effect of variants of cytochrome B gene on reproductive traits namely litter size at first, second and third furrowing, piglet mortality, age at first furrowing and furrowing interval. Mitochondrial gene as Cytochrome B variants might be used as a marker for studying female reproduction of Ghungroo sow in future.

Temperature dependence of the photodissociation of CO2 from high vibrational levels: 205-230 nm imaging studies of CO(X1Σ+) and O(3P, 1D) products.

The 205-230 nm photodissociation of vibrationally excited CO2 at temperatures up to 1800 K was studied using Resonance Enhanced Multiphoton Ionization (REMPI) and time-sliced Velocity Map Imaging (VMI). CO2 molecules seeded in He were heated in an SiC tube attached to a pulsed valve and supersonically expanded to create a molecular beam of rotationally cooled but vibrationally hot CO2. Photodissociation was observed from vibrationally excited CO2 with internal energies up to about 20 000 cm-1, and CO(X1Σ+), O(3P), and O(1D) products were detected by REMPI. The large enhancement in the absorption cross section with increasing CO2 vibrational excitation made this investigation feasible. The internal energies of heated CO2 molecules that absorbed 230 nm radiation were estimated from the kinetic energy release (KER) distributions of CO(X1Σ+) products in v″ = 0. At 230 nm, CO2 needs to have at least 4000 cm-1 of rovibrational energy to absorb the UV radiation and produce CO(X1Σ+) + O(3P). CO2 internal energies in excess of 16 000 cm-1 were confirmed by observing O(1D) products. It is likely that initial absorption from levels with high bending excitation accesses both the A1B2 and B1A2 states, explaining the nearly isotropic angular distributions of the products. CO(X1Σ+) product internal energies were estimated from REMPI spectroscopy, and the KER distributions of the CO(X1Σ+), O(3P), and O(1D) products were obtained by VMI. The CO product internal energy distributions change with increasing CO2 temperature, suggesting that more than one dynamical pathway is involved when the internal energy of CO2 (and the corresponding available energy) increases. The KER distributions of O(1D) and O(3P) show broad internal energy distributions in the CO(X1Σ+) cofragment, extending up to the maximum allowed by energy but peaking at low KER values. Although not all the observations can be explained at this time, with the aid of available theoretical studies of CO2 VUV photodissociation and O + CO recombination, it is proposed that following UV absorption, the two lowest lying triplet states, a3B2 and b3A2, and the ground electronic state are involved in the dynamical pathways that lead to product formation.

Effect of feeding chayote (Sechium edule) meal on growth performance and nutrient utilization in indigenous pig (Zovawk) of Mizoram.

AIM: This study was planned to investigate the effect of feeding different levels of chayote (Sechium edule) meal by replacing standard concentrate mixture (CM) on the growth parameters such as feed intake, body weight gain, average daily gain (ADG) and feed conversion ratio (FCR), and nutrient utilization in indigenous pig of Mizoram. MATERIALS AND METHODS: Twenty-four growing indigenous pigs (Zovawk) were used to study the effect of feeding chayote (Sechium edule) meal (fruits and leaves at the ratio 4:1) on growth performance and nutrient utilization. They were allocated randomly into 4 treatment groups (G1, G2, G3, and G4). Chayote meal was used to replace standard CM (pig grower ration) at 0% (G1), 20% (G2), 30% (G3), and 40% (G4). RESULTS: During the feeding trial of 90 days, it was found that the dry matter (DM) intake decreased as the level of chayote meal increased. For G1, G2, G3, and G4, the ADG (kg) was 0.24±0.04, 0.23±0.03, 0.18±0.02, and 0.18±0.02, respectively, and the feed conversion efficiency was 5.42±0.44, 4.93±0.17, 5.38±0.05, and 5.74±0.53, respectively. However, there was no significant difference (p>0.05) among the different treatment groups in respect to ADG and FCR. At the end of the feeding trial, digestibility trial was conducted to study the effect of feeding chayote meal in the digestibility of the different nutrients by the experimental animals. From the digestibility trial, it was revealed that the digestibility coefficient of DM, crude protein, and crude fiber were also similar (p>0.05), although the ether extract digestibility in G1 was significantly low (p<0.01) as compared to G2, G3, and G4. CONCLUSION: Chayote meal could safely replace the standard grower ration up to 40% in the diet of growing local pigs without causing any adverse effects on growth and nutrient utilization.

Prebiotic inulin: Useful dietary adjuncts to manipulate the livestock gut microflora.

In recent years, there has been a growing appreciation on the relevance of gastrointestinal microflora in both ruminants and non-ruminants owing to revelation of their role in several physiological functions including digestion, nutrient utilization, pathogen exclusion, gastrointestinal development, immunity system, gut gene expression and quality of animal products. The ban imposed on the use of antibiotics and hormones in feed has compelled animal researchers in finding an alternative which could overcome the issues of conventional feed additives. Though the concept of prebiotic was evolved keeping in mind the gastrointestinal flora of human beings, presently animal researchers are exploring the efficiency of prebiotic (inulin) for modulating the gut ecosystem of both ruminants and non-ruminants. It was revealed that prebiotic inulin is found to exhibit desirable changes in the gut of non-ruminants like poultry, swine, rabbit etc for augmenting gut health and improvement of product quality. Similarly, in ruminants the prebiotic reduces rumen ammonia nitrogen, methane production, increase microbial protein synthesis and live weight gains in calves. Unlike other feed additives, prebiotic exhibits its effect in multipronged ways for overall increase in the performances of the animals. In coming days, it is expected that prebiotics could be the part of diets in both ruminants and non-ruminants for enabling modulation of gut microflora vis a vis animals productivity in ecological ways.

Application of pigeon pea (Cajanus cajan) stalks as raw material for xylooligosaccharides production.

Pigeon pea (Cajanus cajan) is a perennial plant widely cultivated in tropical and subtropical regions of many countries. The present studies aimed to produce xylooligosaccharides (XOS) from pigeon pea stalks in order to do value addition. The chemical analysis of stalks revealed 18.33 ± 1.40 % hemicelluloses in addition to cellulose, protein, and lignin. Sodium hydroxide coupled with steam application enabled almost 96 % recovery of original xylan, present in the pigeon pea stalks. Enzymatic hydrolysis of xylan led to production of XOS namely, xylobiose and xylotriose. Response surface model indicated a maximum yield of xylobiose (0.502 mg/ml) under the hydrolysis conditions of pH 4.91, temperature at 48.11 °C, enzyme dose at 11.01 U, and incubation time at 15.65 h. The ideal conditions for higher xylotriose yield (0.204 mg/ml) were pH 5.44, temperature at 39.29 °C, enzyme dose at 3.23 U, and incubation time at 15.26 h. The present investigation was successful in assessing the prospect of using pigeon pea stalks as a raw material for xylan extraction vis-à-vis XOS production.

Prebiotic inulin: Useful dietary adjuncts to manipulate the livestock gut microflora

In recent years, there has been a growing appreciation on the relevance of gastrointestinal microflora in both ruminants and non-ruminants owing to revelation of their role in several physiological functions including digestion, nutrient utilization, pathogen exclusion, gastrointestinal development, immunity system, gut gene expression and quality of animal products. The ban imposed on the use of antibiotics and hormones in feed has compelled animal researchers in finding an alternative which could overcome the issues of conventional feed additives. Though the concept of prebiotic was evolved keeping in mind the gastrointestinal flora of human beings, presently animal researchers are exploring the efficiency of prebiotic (inulin) for modulating the gut ecosystem of both ruminants and non-ruminants. It was revealed that prebiotic inulin is found to exhibit desirable changes in the gut of non-ruminants like poultry, swine, rabbit etc for augmenting gut health and improvement of product quality. Similarly, in ruminants the prebiotic reduces rumen ammonia nitrogen, methane production, increase microbial protein synthesis and live weight gains in calves. Unlike other feed additives, prebiotic exhibits its effect in multipronged ways for overall increase in the performances of the animals. In coming days, it is expected that prebiotics could be the part of diets in both ruminants and non-ruminants for enabling modulation of gut microflora vis a vis animals productivity in ecological ways.

Enzymatic production of xylooligosaccharides from alkali solubilized xylan of natural grass (Sehima nervosum).

In this study, a process for producing XOS from Sehima nervosum grass was developed. The grass contains 28.1% hemicellulose. NaOH and steam application yielded 98% of original xylan in contrast to 85% by KOH application. Hydrolysis of xylan with commercial xylanase caused breakdown into XOS comprising of xylobiose, xylotriose along with xylose. Response surface model (RSM) revealed highest xylobiose yield (11 g/100g xylan) at pH 5.03, temperature 45.19°C, reaction time 10.11h with enzyme dose 17.41 U. Similarly for maximizing xylotriose yield, ideal hydrolysis conditions were pH 5.11, temperature 40.33°C, reaction time 16.55 h with enzyme dose 13.20 U. A two step process encompassing xylan fractionation and enzymatic hydrolysis enabled XOS production from the S. nervosum grass.

Cone milling of compacted flakes: process parameter selection by adopting the minimal fines approach.

Cone mill was well studied for milling of wet agglomerates. This study evaluated the effects of various process parameters of cone milling roller compacted flakes on the granules produced. Impeller sidearm shapes, screen surface profiles and impeller speeds were studied. Impeller speed was found to play a major role in determining the granule attributes. Besides this, median size, size distribution and percent fines of a milled granule population were mainly determined by the size reduction mechanisms of different impellers and screens. Pre-breaking followed by shearing and slicing of flakes inside the milling chamber was primarily responsible for determining the size, size distribution and percent fines of milled granules. The pre-breaking action could be achieved using teethed round sidearm impeller and lowered the need for screen-based size reduction, thus generating less fines. The shearing and slicing of flakes due to the raised impaction edges of the grater screen also helped to minimize the production of fines. Therefore, the lowest percentage of fines was observed when the teethed round sidearm impeller was used with a grater screen. The results indicated that fines can be reduced considerably with the judicious selection of a suitable impeller and screen combination in the cone mill.

A simple and efficient diffusion technique for assay of endo b-1, 4-xylanase activity

Endo-β-1, 4-xylanases is thought to be of great significance for several industries namely paper, pharmaceuticals, food, feed etc. in addition to better utilization of lignocellulosic biomass. The present investigation was aimed to develop an easy, simple and efficient assay technique for endo-β-1, 4-xylanases secreted by the aerobic fungi. Under the proposed protocol, 9 g/L xylan containing agar was prepared in 100 mM phosphate buffer at different pH (4.5, 5.5 and 6.5). The sterilized xylan agar was dispensed in 90 mm petri dishes. 100 µl of culture supernatant of 12 fungal isolates was added to the wells and left overnight at 31±10C. The petri dishes were observed for zone of clearance by naked eye and diameter was measured. Congo red solution (1 g/L) was applied over the petri dishes as per the established protocol and thereafter plates were flooded with 1M Sodium chloride solution for the appearance of zone of clearance. The diameter for zone of clearance by the proposed method and the established protocol was almost identical and ranged from 21 to 42 mm at different pH depending upon the activity of endo-β-1, 4-xylanases. Change of pH towards alkaline side enabled similar or marginal decrease of diameter for the zone of clearance in most of the fungal isolates. The specific activities of these fungal isolates varied from 1.85 to 11.47 IU/mg protein. The present investigation revealed that the proposed simple diffusion technique gave similar results as compared to the established Congo red assay for endo-β-1, 4-xylanases. Moreover, the present technique avoided the cumbersome steps of staining by Congo red and de-staining by sodium chloride.

Instability range of microsolvated multiply charged negative ions: prediction from detachment energy of stable hydrated clusters.

We have presented a first-principle theory-based derivation of an exact expression for the solvent number-dependent electron-detachment energy of a solvated species in the thermodynamic limit. We also propose a generalized equation bridging the electron detachment energies for small and infinitely large clusters, thus providing a new route to calculate the ionization potential of a negatively charged ion from the electron-detachment energies of its stable hydrated clusters. Most importantly, it has the ability to predict the instability range of microhydrated anions. The calculated results for the ionization potential for a number of ions are found to be in good agreement with the available experimental results, and the predicted instability range for the doubly charged anions SO4²â» and C2O4²â» is also consistent with experimental and ab initio results.

A simple and efficient diffusion technique for assay of endo ß-1,4-xylanase activity.

Endo-ß-1, 4-xylanases is thought to be of great significance for several industries namely paper, pharmaceuticals, food, feed etc. in addition to better utilization of lignocellulosic biomass. The present investigation was aimed to develop an easy, simple and efficient assay technique for endo-ß-1, 4-xylanases secreted by the aerobic fungi. Under the proposed protocol, 9 g/L xylan containing agar was prepared in 100 mM phosphate buffer at different pH (4.5, 5.5 and 6.5). The sterilized xylan agar was dispensed in 90 mm petri dishes. 100 µl of culture supernatant of 12 fungal isolates was added to the wells and left overnight at 31±1(0)C. The petri dishes were observed for zone of clearance by naked eye and diameter was measured. Congo red solution (1 g/L) was applied over the petri dishes as per the established protocol and thereafter plates were flooded with 1M Sodium chloride solution for the appearance of zone of clearance. The diameter for zone of clearance by the proposed method and the established protocol was almost identical and ranged from 21 to 42 mm at different pH depending upon the activity of endo-ß-1, 4-xylanases. Change of pH towards alkaline side enabled similar or marginal decrease of diameter for the zone of clearance in most of the fungal isolates. The specific activities of these fungal isolates varied from 1.85 to 11.47 IU/mg protein. The present investigation revealed that the proposed simple diffusion technique gave similar results as compared to the established Congo red assay for endo-ß-1, 4-xylanases. Moreover, the present technique avoided the cumbersome steps of staining by Congo red and de-staining by sodium chloride.

Jak2 inhibition deactivates Lyn kinase through the SET-PP2A-SHP1 pathway, causing apoptosis in drug-resistant cells from chronic myelogenous leukemia patients.

Chronic myelogenous leukemia (CML) patients treated with imatinib mesylate (IM) become drug resistant by mutations within the kinase domain of Bcr-Abl, and by other changes that cause progression to advanced stage (blast crisis) and increased expression of the Lyn tyrosine kinase, the regulation of which is not understood yet. In Bcr-Abl+ cells inhibition of Jak2, a downstream target of Bcr-Abl, by either Jak2 inhibitors or Jak2-specific short interfering RNA (siRNA) reduced the level of the SET protein, and increased PP2A Ser/Thr phosphatase and Shp1 tyrosine phosphatase activities, which led to decreased levels of activated Lyn. Activation of PP2A combined with Jak2 inhibition enhanced the reduction of activated Lyn kinase compared with Jak2 inhibition alone. In contrast, inhibition of either PP2A or Shp1 combined with Jak2 inhibition interfered with the loss of Lyn kinase activation more so than Jak2 inhibition alone, indicating the involvement of PP2A and Shp1 in the inactivation of the Lyn kinase caused by Jak2 inhibition. Inhibition of Jak2 induced apoptosis and reduced colony formation in IM-sensitive and -resistant Bcr-Abl mutant cell lines. Jak2 inhibition also induced apoptosis in CML cells from blast crisis patients but not in normal hematopoietic cells. These results indicate that Lyn is downstream of Jak2, and Jak2 maintains activated Lyn kinase in CML through the SET-PP2A-Shp1 pathway.

BCR-ABL oncogenic transformation of NIH 3T3 fibroblasts requires the IL-3 receptor.

Oncogenic transformation of hematopoietic cells by the Bcr-Abl oncoprotein directly involves the activation Jak2 tyrosine kinase and the Stat5 transcription factor. Both proteins are normally linked to the interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor receptors for growth and survival. Since fibroblastic cells are not targets of BCR-ABL-induced oncogenesis, we determined whether forced expression of the IL-3 receptor would allow oncogenic transformation of NIH 3T3 fibroblasts known to be resistant to transformation by BCR-ABL. NIH 3T3 cells transduced with the human IL-3 receptor alpha and beta chains were highly susceptible to oncogenic transformation by expression of BCR-ABL. Forced expression of both receptor chains but not either one alone allowed efficient foci formation of NIH 3T3 cells expressing BCR-ABL (triple positive cells), and these cells formed colonies in soft agar, whereas BCR-ABL+ NIH 3T3 cells lacking IL-3 receptor expression did not. Signaling studies indicate that the BCR-ABL/IL-3 receptor+ NIH 3T3 cells utilize the Gab2/PI-3 kinase pathway activated by Jak2, and the Stat5 pathway activated separately by Bcr-Abl, whereas BCR-ABL+ NIH 3T3 cells lacking the IL-3 receptor do not utilize the Jak2 pathway, but still maintain activation of Stat5. The Bcr-Abl kinase inhibitor imatinib mesylate (1 microM) and two Jak2 kinase inhibitors strongly inhibited agar colony formation and the activation of Gab2 caused by Jak2. All of these findings indicate that Bcr-Abl oncoprotein requires the IL-3 receptor/Jak2/Stat5 pathways for oncogenic transformation of NIH 3T3 fibroblasts.

The ischemic preconditioning paradox in deceased donor liver transplantation-evidence from a prospective randomized single blind clinical trial.

While animal studies show that ischemic preconditioning (IPC) is beneficial in liver transplantation (LT), evidence from few smaller clinical trials is conflicting. From October 2003 to July 2006, 101 deceased donors (DD) were randomized to 10 min IPC (n = 50) or No IPC (n = 51). Primary objective was efficacy of IPC to decrease reperfusion (RP) injury. Both groups had similar donor risk index (DRI) (1.54 vs. 1.57). Aminotransferases on days 1 and 2 were significantly greater (p < 0.05) in IPC recipients. In multivariate analyses, IPC had an independent effect only on day 2 aspartate transferase. Prothrombin time, bilirubin and histological injury were similar in both groups. IPC had no significant effect on plasma TNF-alpha, IL-6 and IL-10 in the donor and TNF-alpha and IL-6 in the recipient. In contrast, IPC recipients had a significant rise in systemic IL-10 levels after RP (p < 0.05) and had fewer moderate/severe rejections within 30 days (p = 0.09). Hospital stay was similar in both groups. One-year patient and graft survival in IPC versus No IPC were 88% versus 78% (p = 0.1) and 86 versus 76% (p = 0.25), respectively. IPC increases RP injury after DDLT, an 'IPC paradox'. Other potential benefits of IPC are limited. IPC may be more effective in combination with other preconditioning regimens.

Regulation of chemokine receptor CCR5 and production of RANTES and MIP-1alpha by interferon-beta.

Trafficking of inflammatory T cells into the brain is associated with interactions of certain chemokines with their receptors, which plays an important role in the pathogenesis of multiple sclerosis (MS). We examined whether interferon-beta (IFN-beta) had the ability to regulate the production of chemokines and the expression of their receptors in T cells derived from patients with MS. It was demonstrated for the first time that in vitro exposure of T cells to IFN-beta-1a selectively inhibited mRNA expression for RANTES and MIP-1alpha and their receptor CCR5. T cell surface expression of CCR5 was significantly reduced in MS patients treated with IFN-beta, correlating with decreased T cell transmigration toward RANTES and MIP-1alpha. The study provides new evidence suggesting that IFN-beta treatment impairs chemokine-induced T cell trafficking by reducing the production of RANTES and MIP-1alpha and the expression of their receptors CCR5.

Aberrant T cell migration toward RANTES and MIP-1 alpha in patients with multiple sclerosis. Overexpression of chemokine receptor CCR5.

Trafficking of inflammatory T cells into the central nervous system (CNS) plays an important role in the pathogenesis of multiple sclerosis. The directional migratory ability of peripheral T cells is associated with interactions of chemokines with their receptors expressed on T cells. In this study, transmigration of peripheral T cells toward a panel of chemokines was examined in patients with multiple sclerosis and healthy individuals using Boyden chemotactic transwells. A significantly increased migratory rate preferentially toward RANTES and MIP-1alpha, but not other chemokines, was found in T cells obtained from multiple sclerosis patients as opposed to healthy individuals (P: < 0.001). The migratory T-cell populations represented predominantly Th1/Th0 cells while non-migratory T cells were enriched for Th2-like cells. The study demonstrated further that aberrant migration of multiple sclerosis-derived T cells toward RANTES and MIP-1 alpha resulted from overexpression of their receptors (CCR5) and could be blocked by anti-CCR5 antibodies. These findings have important implications for our understanding of the mechanism underlying aberrant T cell trafficking in multiple sclerosis.

Hamycin inhibits IL-8-induced biologic response by modulating its receptor in human polymorphonuclear neutrophils.

IL-8, a neutrophil chemotactic agent, is involved in a large number of neutrophil-driven acute and chronic inflammatory diseases. We have found that hamycin, an antifungal agent, reduces IL-8-induced migration and binding of 125I-labeled IL-8 to neutrophils by 66 and 75%, respectively. Other IL-8-induced biologic functions, such as superoxide generation, intracellular Ca2+ mobilization, and enzyme release were also reduced in hamycin-treated cells by 50 to 75%. Anti-IL-8R Ab (C-X-CR1) and IL-8 itself failed to protect the cells from the effect of hamycin. Scatchard analysis of IL-8 binding data demonstrated that while the normal cells expressed 23,000 +/- 1,704 receptors/cell (Kd = 3.5 nM), the number was reduced to 8,000 +/- 592 receptors/cell (Kd = 3.43 nM) in hamycin-treated cells. Chemical cross-linking of 125I-labeled IL-8 to its receptor followed by 10% SDS-PAGE analysis and autoradiography showed that the signals in hamycin-treated cells were considerably reduced compared with those in controls. In the immunoblot, however, the signals in control and hamycin-treated cells were almost identical. The intensity of the fluorescence emission of diphenyl hexatriene at 430 nm and membrane microviscosity measured by diphenyl hexatriene were considerably reduced in hamycin-treated cells, resulting in a reduced number of functional IL-8R, presumably by conformational change in the receptor. The study suggests that hamycin may be a potent immunomodulator of the IL-8R for alleviation of inflammatory distress.

Binding of Escherichia coli heat-stable enterotoxin and rise of cyclic GMP in COLO 205 human colonic carcinoma cells.

Escherichia coli heat-stable enterotoxin (STa) was found to bind on the surface of human colonic (COLO 205) cells. The binding of [125I]STa to cell membranes was found to be specific, reversible and saturable. Scatchard analysis of the equilibrium binding demonstrated a single class of binding sites with a Kd of 0.5 x 10(-10) M. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed the specific incorporation of [125I]STa into a single STa binding protein with a molecular mass of 95 kDa. Following incubation of COLO 205 cells with STa, a rise of intracellular cGMP was also evident.

Receptor-mediated endocytosis of IL-8: a fluorescent microscopic evidence and implication of the process in ligand-induced biological response in human neutrophils.

Interleukin 8 (IL-8), a neurophil-activating and chemotactic cytokine, is known to play a key role in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. Although the cytokine is rapidly internalized at 37 degrees C with its receptors, there was no direct evidence for the ligand-induced endocytosis of the receptor or that of the interaction of receptor ligand complex at 37 degrees C. As a result, our understanding about the regulation of Il-8 induced biological response is very limited. In the present study, using FITC-IL-8 conjugate as a probe, we have demonstrated the time- and temperature-dependent endocytosis of IL-8 under fluorescent microscope. We have also shown that the bright fluorescent light on the surface of neutrophils gradually disappears and it becomes almost dark after 120 min of incubation. Monodansyl cadaverine (MDC, 900 microM), however, was found to retain the fluorescent light of FITC coupled with Il-8 on the cells. MDC and ouabain (2.5 mM) can inhibit the ligand induced endocytosis by 76% and 96%, respectively, compared to control. With respect to control, IL-8 induced biological responses e.g. IL-8 directed migration, intracellular Ca2+ release and superoxide release are significantly reduced by 77%, 94% and 76%, respectively, in presence of MDC. The study presents a direct visual evidence of the time and temperature-dependent receptor-mediated endocytosis of IL-8 which is inhibited by MDC and ouabain. This information is useful for understanding the ligand receptor interaction at 37 degrees C and may be useful for developing anti-inflammatory agents against IL-8.

A Ca2+-dependent autoregulation of lipopolysaccharide-induced IL-8 receptor expression in human polymorphonuclear neutrophils.

IL-8, a potent neutrophil chemotactic agent, is known to be a key mediator in several inflammatory diseases. We found that 10 ng/ml of serum-activated LPS (Escherichia coli) efficiently up-regulated IL-8R on the surface of neutrophils within 30 min of LPS stimulation by 115 to 120% through de novo protein synthesis. After 30 min of LPS stimulation, reduction of IL-8R level was initiated and the normal level was restored within 2 h of LPS interaction. EDTA or EGTA and bestatin separately inhibited the receptor down-regulation by 98%, indicating the involvement of metalloprotease(s), more specifically an aminopeptidase in the process. Induction and subsequent reduction of IL-8 binding in serum-activated LPS-stimulated cells have been demonstrated in autoradiography. Intracellular Ca2+ level in these stimulated neutrophils was increased and decreased with alteration of IL-8R level. Although IL-8 binding was drastically reduced, the total IL-8R level, as detected by anti-IL-8R Ab measured by 125I-labeled anti-rabbit IgG, remained almost unaltered, indicating that minimal proteolysis occurred in IL-8R. Anti-IL-8R Ab and IL-8 itself could prevent this down-regulation significantly, suggesting that the susceptible epitope(s) might be in the IL-8 binding domain of the receptor. Under Ca2+-depleted conditions, the proteolysis was inhibited, which was accelerated upon addition of 1 mM of CaCl2. The study demonstrates that LPS-induced up-regulation of IL-8R leads to amplified IL-8-mediated biologic responses of neutrophils that are restored to normal level by activation of a Ca2+-dependent aminopeptidase. This may be useful for understanding the regulation of LPS-mediated inflammatory responses of neutrophils during bacterial infection.