Effect of Rabbit Epididymal Antimicrobial Peptide, REHbβP, on LPS-Induced Proinflammatory Cytokine Responses in Human Vaginal Cells In Vitro

Antimicrobial peptides (AMP's) protect epithelial surfaces including epididymis against pathogens and play a key role in orchestrating various defensive responses. Recently, we have identified one such AMP, rabbit epididymal hemoglobin-β subuit (REHbβP) from the epididymal fluid of rabbit, Oryctologus cuniculus. The demonstration of a protective role of REHbβP in epididymal epithelial cells (EPEC's) led us to investigate: (1) the identification of LPS interactive domain in REHbβP, and (2) whether the REHbβP of rabbit origin mediates vaginal cellular immune responses of another species (human). HeLa-S3, human vaginal epithelial cells (hVECs) were exposed to LPS or the LPS-stimulated cells treated with REHbβP or neutral peptide, nREHbβP. Effect of LPS and cytokines (IL-6 and IL-1α) and chemokines (IL-8, MCP-1) levels was determined in the culture supernatants. In response to the LPS, hVECs synthesized these mediators and the levels were significantly higher than controls. This enhancing effect was ameliorated when the LPS-induced hVECs were treated with REHbβP. Similar results were obtained on NF-κB protein and hBD-1 mRNA expression. Confocal microscopy studies revealed that REHbβP attenuated the LPS-induced internalization of E. coli by macrophages. The chemotaxis studies performed using Boyden chamber Transwell assay, which showed elevated migration of U937 cells when the supernatants of LPS-induced hVECs were used, and the effect was inhibited by REHbβP. REHbβP was found to be localized on the acrosome of rabbit spermatozoa, suggesting its role in sperm protection beside sperm function. In conclusion, REHbβP may have the potential to develop as a therapeutic agent for reproductive tract infections (RTI's)

Effect of different shading intensities on growth and yield of cherry tomato

Cherry tomato (Solanum lycopersicon var cerasiforme) is small size fruits, with a bright red colour resembling to cherry and becoming popular in the retail chains which are marketed at a premium price. The field experiment was conducted to study the effect of different (35, 50 and 75 per cent) shading intensities on growth and yield (qha-1 ) of cherry tomato. Significantly maximum yield was recorded in 35 per cent shading intensity and genotype Kalash Seeds Product (KSP)-113 (579.44 and 503.88 q ha-1 , respectively). Among the different shading intensities and genotypes, maximum plant height was observed in 75 per cent shading intensity and genotype KSP -113 at 30 days interval (74.70 and 60.95 cm, respectively). The minimum days to 50% flowering of cherry tomato were observed in cherry tomatoes grown under 35 per cent shading intensity (45.00 days) as compared to other shading intensities while minimum days to 50% flowering were observed in genotype KSP-113 (44.00 days). The maximum length of the cluster (9.58 cm), the weight of cluster (27.67 g), number of fruits per cluster (9.42) and number of pickings (11.67) were observed in 35 per cent shading intensities and in genotype KSP-113. The cultivation of KSP-113 genotype under 35 per cent shading intensity was found to be most sustainable for improving growth and yield of cherry tomato during the summer season

Convective heat transfer in weak electrolytes under the action of electrolytic currents

Electrolytic currents are produced in a weak electrolyte giving rise to the generation of bubbles, the variation in the rate of heat transfer from a heated platinum wire to the surrounding electrolyte under the action of these currents has been studied. The enhancement of heat transfer with increase in the temperature difference (Δθ) between the heating surface and the surrounding electrolyte and with increasing bath temperatures has been graphically exhibited. It is found that whereas a single maximum is obtained in the (h/h0) versus log/graphs for different values of Δθ at lower electrolyte temperatures (h and ho are the heat-transfer coefficients in the presence and absence of electrolytic currents respectively and I is the strength of the electrolytic current), there appear two maxima for (h/h0) when the electrolyte temperature is raised sufficiently above the room temperature (>45°C in the present experiments). An attempt has been made to investigate the nature of these maxima and to offer a suitable explanation for them

Studies on the Effects of RDX Particle Size on the Burning Rate of Gun Propellants

The ballistic properties of RDX-based propellants are highly dependent on the particle size of RDX used. The effect of RDX particle size on the burning rate and pressure exponent of the gun propellant was studied. Propellant formulation containing RDX to extent of 60 per cent in the composition was processed with varying particle size of RDX. Finished propellants in heptatubular and cord geometry were evaluated for ballistic aspects by closed vessel firing in a 700 cc vessel at a loading density of 0.18 g/cc. The data obtained clearly indicate that increase in particle size of RDX increases the burning rate as well as the pressure exponent

Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

<div><p>Hemoglobin (Hb) is a major protein involved in transport of oxygen (O₂). It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish <i>in-vitro</i> culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-β proteins (4) to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-<i>κ</i>B to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11–7082 treatment (5μM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF-<i>κ</i>B to Hb-α promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-α and Hb-β and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-<i>κ</i>B regulatory sequences in Hb-α promoter, whose activation is associated with immune response of hPVECs, indicating Hb-α and Hb-β may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.</p></div

Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells.

<p>hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).</p

Hemoglobin Expression in Nonerythroid Cells: Novel or Ubiquitous?

Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). Red blood cells (RBCs) contain maximum amount of Hb and because of their unique structure and plasticity they transport O2 to various tissues of the body at an optimal concentration. Recently, it has been reported that, apart from RBCs, Hb is also expressed by nonerythroid cells such as epithelial cells of different origin. The cells expressing Hb are from the tissues where maintenance of O2 homeostasis is of paramount importance. Hb expression has been observed in the epithelial cells from human tissues including lungs, neurons, retina, and endometrium. Our group has recently demonstrated that Hb is expressed by the cervicovaginal epithelial cells. We further showed that, apart from maintaining O2 homeostasis, Hb and the peptides derived from it play an indispensable role in the protection of vaginal epithelium by exhibiting antimicrobial activity. In this review, we discuss the significance of Hb expression in vaginal epithelial cells and its role in the recognition of pathogens thereby reducing the risk and/or severity of inflammation and/or infections and the possible mechanism by which Hb exhibits antimicrobial and antioxidative functions

Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells.

<p>Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).</p