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Trefoil family factor 2 (TFF2) is expressed in gastrointestinal epithelial cells where it serves to maintain mucosal integrity and promote epithelial repair. The peptide hormone, gastrin, stimulates acid secretion but also induces proliferation of the acid-secreting mucosa. Because the relationship between these peptides of overlapping function is not understood, we chose to investigate the regulatory effect of gastrin on TFF2 expression. The expression of mRNA and protein of TFF2 was determined by RT-PCR and immunohistochemical staining, respectively. A series of truncated and mutant murine TFF2 promoter constructs was generated. Promoter activity was assessed using dual luciferase reporter assays. Gastrin-responsive DNA-binding sites in the TFF2 promoter were evaluated by electrophoretic mobility shift assay. Gastrin significantly increased the level of endogenous mRNA of TFF2 in the gastrin receptor-expressing AGS-E gastric cancer cell line in a time- and dose-dependent manner. TFF2 protein expression in the gastric fundus was elevated in hypergastrinemic (INS-GAS) transgenic mice and reduced in gastrin-deficient mice. Gastrin treatment increased TFF2 promoter activity through cis-acting regions, containing CCAATA- and GC-rich enhancers. Pretreatment with Y-F476, a gastrin/CCKB receptor antagonist, abolished gastrin-dependent promoter activity. Inhibitors of protein kinase C (PKC), mitogen/extracellular signal-regulated kinase (MEK1), and phosphatidylinositol 3-kinase (PI 3-kinase) reduced gastrin-dependent TFF2 promoter activity, whereas an epithelial growth factor receptor (EGFR) inhibitor had no effect. We found that gastrin regulates TFF2 transcription through a GC-rich DNA-binding site and a PKC-, MEK1- and PI 3-kinase-dependent but EGFR-independent pathway. Regulation of TFF2 by gastrin may play a role in the maintenance and repair of the gastrointestinal mucosa.
- gastrin
- trefoil family factor 2
- gastrin-responsive element
- gene regulation
- signal transduction
the trefoil family factor (TFF) comprises a group of three small proteins bearing one or more trefoil motifs, triple loop structures maintained by disulfide bridges between highly conserved cysteine residues (36, 38, 40). Under normal conditions, TFF1, TFF2, and TFF3 are differentially expressed in surface (foveolar) mucus cells of the stomach, mucous neck cells of the stomach, and goblet cells of the intestinal epithelium in rodent, respectively (36). In humans, TFF2 is expressed in Brunner's glands in the duodenum, the basal portion of the antral/pyloric glands, and in the midzone of the gastric/body mucosa, where it is localized to mucous neck cells and rarely to a few deeper cells (15, 29). TFFs constitute an established class of regulatory peptides involved in mucosal protection and repair of the gastrointestinal tract (15, 29, 36, 38, 40). TFF2, also known as spasmolytic polypeptide, was the first member of the family to be identified. In addition to roles in gastric cytoprotection and repair (11, 15, 29, 39), TFF2 is also thought to participate in the mucosal immune response (9) and is regulated by both proinflammatory (27) and anti-inflammatory cytokine (5). Treatment with exogenous TFF peptides alleviates gastric damage (3, 30) and colon inflammation through upregulation of repairing mechanisms and modulation of the inflammatory response (13, 35).
Gastrin is a peptide hormone produced primarily by G cells, endocrine cells located in the gastric antrum that regulate acid secretion. However, gastrin also acts as a growth factor for normal gastric oxyntic mucosa, stimulating the proliferation and migration of acid-secreting cells (parietal cells) and histamine-producing cells (ECL cells; see Ref. 12). A number of studies have indicated that, in the setting of gastric ulceration or injury, gastrin can promote ulcer healing and repair (26, 37), and overexpression of gastrin in transgenic animals leads to mucosal hyperplasia (20). The physiological actions of gastrin are mediated by the gastrin-CCKB subtype receptors (CCKBR; see Ref. 12), which are expressed normally on ECL cells, parietal cells, and mucous neck cells (15). CCKBR has also been implicated in gastric mucosal wound healing, with increased CCKBR expression noted in epithelial cells at the regenerative mucosal ulcer margin (32). To date, a number of downstream target genes of gastrin have been reported, including matrix metalloproteinase 9 (44), plasminogen activator inhibitor 2 (41), tenascin-c, S100A6, myosin light chain kinase (25) and cyclooxygenase-2 (20), and TFF1 (23).
Hypergastrinemic (INS-GAS) transgenic mice, with or without Helicobacter infection, show increased mucosal proliferation and TFF2-expressing cell types that precedes the development of gastric cancer (42). In the rat stomach, the TFF2-expressing lineage, designated spasmolytic polypeptide-expressing metaplasia (SPEM), is typically found at the base of fundic glands, and the lineage is often positive for proliferating cell nuclear antigen (45). In Helicobacter-infected wild-type C57BL/6 and INS-GAS transgenic mice, the TFF2-expressing SPEM lineage is the precursor for invasive cancer (28, 42). One study has shown that H. pylori infection leads to a reduction in luminal TFF2 in the human stomach (18). However, other studies have suggested that H. pylori infection results in significantly increased expression of TFF2 in chronic fundic gastritis, particularly in association with dysplasia (14, 33). In one study, the SPEM lineage was present in 68% of fundic biopsies from patients with H. pylori-associated gastritis but was absent in biopsies of fundic mucosa from patients without H. pylori infection. The SPEM lineage was found in 91% of gastric cancer, typically located in mucosa adjacent to the carcinoma or areas of dysplasia (33). In another study, SPEM was noted within cancer cells in 62% of early gastric cancer, within dysplastic cells in 76% of resections where dysplasia was present, and in 82% of the biopsies obtained before the diagnosis of gastric cancer, compared with only 37% in the gastritis cohort (14). Because serum gastrin levels are usually elevated in the stomachs of H. pylori-infected patients and mice, this suggests a possible association between gastrin stimulation, the TFF2-expressing SPEM lineage, and gastric cancer.
Thus gastrin and TFF2 appear to share overlapping roles in gastric repair and neoplasia. Although the mechanism of action of trefoil factors remains undefined, the observation that H. pylori-induced hypergastrinemia is often associated with increased TFF2 expression in gastric tissues raises the possibility that gastrin may regulate TFF2 expression. Previous studies by our group and others have investigated the TFF2 promoter and shown it to be a complex promoter/enhancer responsive to several growth factors and basal transcription factors such as GATA-6 and hepatocyte nuclear factor (HNF)-3 (1, 4). In the current study, we show that TFF2 is in fact a gastrin-regulated gene and demonstrate for the first time that gastrin can regulate transcriptionally the expression of TFF2 through gastrin-responsive cis-acting elements.
Cell culture and animals.
The human cell line, AGS-E (ATCC, Manassas, VA), which stably expresses CCKBR, was used in this study (23). AGS-E cells were maintained under selection by supplementing medium with puromycin (2 μg/ml) every 4 wk. Cells were cultured in DMEM medium supplemented with 10% FBS and 100 IU/ml penicillin/streptomycin in a humidified incubator at 37°C under 5% CO2-95% O2 conditions. The transgenic animals used in this study have been described previously (22, 42). Gastrin knockout (GAS-KO) animals deficient in all forms of gastrin have a targeted deletion of the gastrin gene (22). INS-GAS transgenic mice express the gastrin gene in pancreatic β-cells and are hypergastrinemic (42). The background strains for GAS-KO and INS-GAS mice are C57BL/6 and FVB/N, respectively.
Cell transfection and luciferase assays.
AGS-E cells (1.0 × 105 cells/well) were seeded in 12-well plates 24 h before transfection. Cells were transfected with either 1.5 μg TFF2-luciferase construct plasmid or 1.5 μg empty reporter vector DNA using 4 μl Superfectin (Qiagen, Valencia, CA) for 2.5 h. To control for background luciferase activity, 0.05 μg/well of a Renilla luciferase reporter vector DNA, driven by a minimally active thymidine kinase promoter (pRL-TK; Promega), was cotransfected with all promoter constructs, and the ratio of firefly to Renilla luciferase activity was calculated. The total amount of DNA transfected was kept constant by addition of appropriate amounts of the pCMV vector. The cells were cultured in full medium for 24 h before luciferase measurements. Luciferase activity was determined using the dual-luciferase assay system (Promega) and a Monolight 3010 luminometer (Pharmingen). CCKBR antagonist YF-476 was a gift from Dr. Keiji Miyata and Dr. Hidenobu Yuki (Yamanouchi Pharmaceutical, Tsukuba, Japan).
Immunohistochemical staining.
Gastric expression of TFF2 was examined by immunohistochemical staining in 2-mo-old INS-GAS, GAS-KO, and control mice. Following death, midline strips removed from the lesser curvature of the stomach were fixed in 10% neutral buffered formalin overnight, processed routinely, and embedded in paraffin. Sections were cut at 5 μm followed by antigen retrieval, achieved by boiling sections for 15 min in 0.01 M citrate buffer, pH 6.0. The rabbit polyclonal TFF2 antibody was raised to the COOH-terminal 16 amino acids (EVPWCFFPQSVEDCHY) from mouse TFF2 but which are highly conserved (e.g., 15 of 16 residues) in the human COOH-terminal peptide. We have tested this antibody in immunohistochemical stains of human tissue and in Western blots against recombinant human TFF2. The antibody detects quite cleanly the 14-kDa human TFF2 peptide (unpublished data). Tissue sections were incubated with the TFF2 antibody at a dilution of 1:200 at 4°C overnight in a humidified chamber. Indirect labeling was performed with species-appropriate biotinylated secondary antibodies followed by avidin-biotin complex (ABC)-peroxidase (ABC kit; Vector Laboratories, Burlingame, CA) according to the manufacturer's protocol. 3-Amino-9-ethylcarbazole (Vector Laboratories) was used as chromogen, and slides were counterstained with Mayer's hematoxylin. Stained sections were examined by light microscopy (CX31; Olympus Optical, Cebu, Philippines). Double immunohistochemistry for the CCKBR and TFF2 was performed as described previously (23). Tissue sections were incubated with the anti-human gastrin/CCKBR antibody at 4°C overnight and visualized with fluorescence isothiocyanate-conjugated affiniPure goat anti-serum against rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA). Staining for TFF2 was visualized with a rhodamine-conjugated mouse antiserum against rabbit IgG (1:200; Jackson ImmunoResearch), and double-stained sections were examined with a fluorescence microscope (Olympus BX 51; Olympus Optical, Melville, MD).
Quantitative and semiquantitative PCR.
Total RNA was extracted from AGS-E cells and whole murine stomach tissues by TRIZOL (Invitrogen, Carlsbad, CA). RT was performed using SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's protocol. Semiquantitative PCR reactions were performed with a GeneAmp PCR System 9700 (Applied Biosystems) using PCR Core Kit. The quantity of cDNAs generated in PCR reactions were standardized against glyceraldehyde trisphosphate dehydrogenase amplification. The sense and the antisense mouse and human TFF2 primers were designed to cross exon-intron boundaries to avoid amplification from contaminating DNA. The sequences of human TFF2 primers were as follows: forward, 5′-atgggacggcgagacgccca-3′ and reverse, 5′-ttagtaatggc agtcttccacag-3′. The sequences of mouse TFF2 primer were as follows: forward, 5′-GCAGTGCTTT GATCTTGGATGC-3′ and reverse, 5′-TCAGGTTGGAAAAGCAGCAGTT-3′. The PCR products were analyzed by agarose gel electrophoresis.
Quantitative real-time PCR was performed with a three-step method using the Bio-Rad iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). Each reaction was carried out in a 50-μl mixture consisting of QuantiTect SYBR Green PCR (Qiagen). All primer pairs were optimized to amplify only a single band, with amplification curves in real-time PCR consistent with efficient amplification under the reaction conditions. The PCR conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s.
Western blot analysis.
Cells were lysed in buffer containing 50 mM Tris·HCl, pH 7.5, 250 mM NaCl, 0.1% Nonidet P-40, and 5 mM EGTA, 50 mM sodium fluoride, 60 mM β-glycerolphosphate, 0.5 mM sodium vanadate, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. Protein extracts of whole gastric fundus from 2-mo INS-GAS, GAS-KO, and control mice were prepared in lysis buffer. Protein samples were electrophoresed on 4–20% denaturing SDS gels (Invitrogen), and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Blots were fixed 0.2% glutaraldehyde for 25 min, incubated with rabbit polyclonal TFF2 antibody (6 μg/ml) overnight, and then incubated at room temperature for 1 h with a peroxidase-conjugated goat anti-rabbit secondary antibody (Amersham, Piscataway, NJ). Immunobands were visualized by enhanced chemiluminescence (Amersham).
Reporter constructs and PCR mutagenesis.
A 2.5-kb fragment of the TFF2 promoter was obtained through PCR amplification from mouse genomic DNA (Promega) using synthetic primers (Invitrogen) based on published sequences. Part of sequences of mouse promoter was shown in ⇓⇓Fig. 3A. The forward primer was located at 2583 nucleotides upstream of the published TFF2 transcriptional start site, and the reverse primer corresponded to nucleotides +20 to +34. The promoter fragment was cloned directly into pBluescript (Invitrogen) and subsequently subcloned into the Hind III-XhoI site of the luciferase reporter vector pGL2-basic to generate the TFF2-2583 plasmid. This construct was used as a reporter template to generate the other truncated promoter constructs, namely TFF2-1842, TFF2-965, TFF2-524, TFF2-305, TFF2-222, TFF2-79, and TFF2-46. To verify gastrin-responsive elements contained in the TFF2-305 (P-305) promoter construct, several additional constructs containing point mutations were generated derived from the P-305 construct using the Site-Directed Mutagenesis kit (Stratagene). All mutants were confirmed by sequencing.
Electrophoretic mobility shift assays.
Electrophoretic mobility shift assays (EMSAs) were performed as described previously (6) using nuclear extracts from AGS-E cells. Double-stranded oligonucleotides (5 pmol) were radiolabeled with [α-32P]dCTP with Klenow fragment of DNA polymerase. Probes (10 fmol) were incubated with nuclear extracts (6 μg). DNA-protein complexes were electrophoresed through 6% nondenaturing polyacrylamide gels, and the dried gels were exposed to phosphor storage screens. Images were revealed with a PhosphorImager (GE Healthcare Bio, Piscataway, NJ). The probes used were designed, and the sequences are shown in Table 1.
Statistical analyses.
Data are presented throughout as means ± SE. Statistically significant differences (P < 0.05) were decided by one-way ANOVA and unpaired t-test.
Gastrin upregulates TFF2 gene expression in vitro and in vivo.
We first investigated by RT-PCR the effect of gastrin on endogenous TFF2 mRNA expression in AGS-E cells that stably express CCKBR. The level of TFF2 mRNA was markedly increased in AGS-E cells in response to 10−9 to 10−7 M (1–100 nM) gastrin for 24 h (Fig. 1A). Real-time PCR confirmed that gastrin stimulated expression of TFF2 gene expression in a time- and dose-dependent manner (P < 0.05; Fig. 1B). Consistent with previous reports (6, 8), we failed to detect by Western blot TFF2 protein expression in human gastric cancer cell lines.
We next asked whether gastrin overexpression or deficiency influences the in vivo expression of TFF2 in the gastric mucosa. Because older INS-GAS and GAS-KO mice exhibit increased number of TFF2-expressing cells (22, 42), we examined younger (2-mo-old) INS-GAS and GAS-KO mice. Real-time PCR revealed that TFF2 mRNA expression was downregulated significantly in GAS-KO mice and upregulated significantly in INS-GAS mice compared with strain- and age-matched control animals (P < 0.05, Fig. 2A). Western blot indicated a similar downregulation in TFF2 protein levels in gastrin-deficient mice and upregulation in hypergastrinemic mice compared with strain- and age-matched control animals (Fig. 2B). Densitometric analysis of TFF2-specific bands noted on Western blots from cohorts of six mice per group showed a 2.3-fold decrease in the abundance of TFF2 in GAS-KO mice and a 1.89-fold increase in the abundance of TFF2 in INS-GAS mice compared with control mice (P < 0.05). In addition, immunohistochemical staining showed that TFF2 protein expression was reduced in the gastric fundus of GAS-KO mice compared with the C57BL/6 controls, whereas TFF2 protein expression was increased in INS-GAS mice (Fig. 2C). Expression of TFF2 protein was localized primarily to gastric mucous neck cells (Fig. 2C), whereas expression of the CCKBR was found in both neck cells and the deeper regions of the gastric glands (Fig. 2D). Nevertheless, some colocalization of TFF2 and CCKBR was observed in occasional glandular cells (Fig. 2E), raising the possibility that gastrin might directly stimulate TFF2 expression. These results strongly suggested that gastrin regulates the expression of TFF2 in vitro and in vivo.
Gastrin regulation of the TFF2 promoter is dependent on the CCKBR.
To determine whether gastrin-dependent upregulation of TFF2 gene expression could be attributed to stimulated transcription, a series of murine TFF2 promoter reporter constructs were generated containing the downstream 3′-untranslated sequence (+34) and various lengths of upstream mouse TFF2 5′-flanking sequences (Fig. 3, A and B). Basal promoter activity of the TFF2-2583 construct in AGS-E cells was >14-fold higher than that of the empty vector pGL-2. With deletion to −965 nucleotides (in the TFF2-965 construct), promoter activity was reduced significantly (P < 0.01), and, with further deletions, promoter activity continued to decline gradually (P < 0.05; Fig. 3B). The minimal basal promoter could be localized to the region between −46 and +34 bp (Fig. 3B).
Next, we determined the effect of gastrin on TFF2 promoter activity in AGS-E cells. Gastrin stimulation increased significantly the activity of all TFF2 promoter-reporter gene constructs (P < 0.01; Fig. 3C). The promoter activity of the TFF2-2583 construct showed minimal responses to lower concentrations of gastrin (10−11 to 10−9 M) but showed a 9- to 10-fold increase in response to higher concentrations of gastrin (10−8 to 10−7 M; P < 0.01; Fig. 4A). The promoter activity of the TFF2-2583 construct began to increase at 4 h after gastrin stimulation and peaked at 12 h (P < 0.01; Fig. 4B). The results showed that gastrin activates TFF2 transcription in a time- and dose-dependent manner.
To confirm that gastrin activation of TFF2 transcription is dependent on the CCKBR, AGS-E cells were pretreated with CCKBR antagonist, YF-476 (23), before gastrin stimulation. These results showed the YF-476 pretreatment significantly abolished gastrin-dependent TFF2 promoter activity in a dose-dependent manner (P < 0.05; Fig. 4C). The response of all TFF2 promoter constructs to 10−7 M ∼ 10−8 M of gastrin was completely inhibited in the presence of YF-476 at 10−7 M (P < 0.01; Figs. 3C and 4A). Thus gastrin-dependent TFF2 promoter activity is specifically dependent on signaling through the CCKBR.
We further sought to identify gastrin-responsive cis-acting elements in the TFF2 promoter. AGS-E cells were transfected with a series of truncated TFF2 promoter constructs and treated with and without gastrin stimulation before luciferase assays. These results demonstrated that gastrin treatment led to significant induction of all TFF2 promoter constructs (Fig. 3C). A significant reduction in stimulation occurred upon deleting from −305 to −222 and from −46 to pGL2, suggesting that gastrin-responsive elements may be located in both the −305 to −222 bp and the −46 to +34 bp regions, respectively (Fig. 3C).
The CCAATA enhancer element located at −35 to −25 bp of the TFF2 promoter is a gastrin-responsive site.
To further define the gastrin-responsive elements in the TFF2 promoter, we performed EMSA using AGS-E extracts and wild-type and mutant oligonucleotides derived from the sequences between −46 to −1 bp and −305 to −222 bp upstream of the TFF2 transcriptional start site. These EMSA experiments were performed to directly detect DNA-protein complex formation using 32P-labeled wild-type (WT) 1 to WT3 and mutant (M) 1 to M4 probes (Table 1). DNA-protein complexes could be detected using WT1 probe (−46 to −1), M1 to M3 probes (Fig. 5A), and WT2 (−305 to −264) and WT3 (−263 to −222) probes (Fig. 5B). Only the M4 (−46 to −1) probe with mutant sequences located at −34 to −25 bp significantly attenuated the formation of DNA-protein complexes (Fig. 5A). These results suggested that the sequences between −34 and −25 bp (M4), which contains the sequence 5′-GAGCCAATAA-3′, were responsible for the most prominent DNA-protein complexes observed in EMSA.
To further map the specific binding elements in this region, a series of additional oligonucleotides was generated for EMSA with 3 bp cluster mutations replacing the −34 to −25 bp region, and designated M5, M6, M7, and M8 (Table 1). EMSA results showed that M6 and M7 failed to form DNA-protein complexes, suggesting that the nucleotides contained in this region (−32 CCAATA −27) were directly responsible for DNA complex formation. EMSA probes M5 and M8, which contained mutations outside of the core CCAATA sequence, were still able to form DNA-protein complexes. These results demonstrate that the −32 to −27 bp sequence (−CCAATA−) is largely responsible for the DNA-protein complex formation.
A GC-rich element (CCCTGTGG) is also required for gastrin-dependent TFF2 promoter activity.
We next investigated the gastrin-responsive cis-acting elements in the −305 to −222 bp region of the TFF2 promoter using wild-type and mutant oligonucleotides in EMSA studies. The mutant oligonucleotides contained 10-bp mutations in the original −305 to −264 bp (WT2) and −263 to −222 bp (WT3) wild-type oligonucleotides and were designated M9A, M9B, M9C, and M9D, and M10A, M10B, M10C, and M10D, respectively (Table 1). EMSA studies employing these mutant oligos as competitors showed that the DNA-protein complexes formed with the WT2 probe were almost completely abolished by competition with unlabeled M9A, M9B, and M9D but not the M9C mutant oligo (Fig. 6A). Similarly, when used as competitors in EMSA, the M10A, M10B, M10C, and M10D oligos all reduced binding and complex formation with the WT3 wild-type probe (Fig. 6B). Thus the finding that the M9C mutant oligo failed to reduce the binding to the WT2 probe suggests that the sequences mutated in this oligo, ACCCTGTGGGTG between −285 to −274 bp regions, were critical for factor interaction with the TFF2 promoter.
To determine the functional contribution of the −285 to −274 bp region to gastrin responsiveness of the promoter, we generated a series of TFF2 promoter constructs in which point mutations in the −285 to −274 region were introduced on the backbone of TFF2-305 luciferase constructs. These constructs, which were designated TFF2-305-9CM1, TFF2-305-9CM2, TFF2-305-9CM3, and TFF2-305-9CM4 (Fig. 6C), were then tested for gastrin responsiveness. These studies revealed that the mutant promoter constructs TFF2-305-9CM1, -9CM2, and -9CM3, showed significantly decreased responses to gastrin (P < 0.05), whereas the activity of mutant TFF2-9CM4 promoter showed a normal gastrin response (Fig. 6D). These findings indicate that the sequence 5′-CCCTGTGG-3′ between −284 to −277 bp regions was required for full gastrin-dependent activation of the TFF2 promoter.
Gastrin activates the TFF2 promoter through a protein kinase C-, mitogen/extracellular signal-regulated kinase 1-, and phosphatidylinositol 3-kinase-dependent pathway.
Last, we used specific inhibitors to investigate the signal transduction pathways involved in gastrin-dependent regulation of TFF2 promoter activity. The protein kinase C (PKC)-specific inhibitor staurosporine (23) and the mitogen/extracellular signal-regulated kinase (MEK) 1 inhibitor PD-098059 (23) significantly reduced gastrin-dependent activity of the TFF2-305 promoter construct (P < 0.05; Fig. 7A). Similarly, the phosphatidylinositol 3-kinase (PI 3-kinas) inhibitor LY-294002 (23) almost completely reversed the effect of gastrin on the induction of TFF2 promoter activity (P < 0.01; Fig. 7A). Furthermore, the inhibitors staurosporine, PD-098059, and LY-294002 also suppressed the effects of gastrin on endogenous TFF2 mRNA expression in AGS-E cells (Fig. 7A). Intriguingly, these specific inhibitors had no effect on gastrin-dependent activity of the TFF2-46 promoter construct, suggesting that the upregulation by gastrin of this basal promoter may be mediated through other signaling pathways. Overall, these results indicate that gastrin-dependent regulation of the CCCTGTGG cis-acting element is largely dependent on the PKC, MEK1, and PI 3-kinase pathways.
We also investigated the effects of the epithelial growth factor (EGF) receptor (R)-specific inhibitor tyrphostin, AG-1478, on TFF2 promoter activity. Both EGF and gastrin stimulated the TFF2 promoter and upregulated the level of endogenous TFF2 mRNA (P < 0.01; Fig. 7, B and C). However, AG-1478 significantly reduced EGF-stimulated TFF2 gene expression and transcription (P < 0.01) but did not inhibit gastrin-stimulated TFF2 promoter activity and TFF2 gene expression in AGS-E cells (P > 0.05; Fig. 7, B and C). Thus gastrin-dependent activation of TFF2 transcription appears to be independent of EGFR signaling.
In this study, we found that gastrin regulates TFF2 gene expression both in vitro and in vivo. Expression of the TFF2 gene was reduced in the gastric fundus of gastrin-deficient mice and increased in the proximal stomach of our hypergastrinemic INS-GAS mice. Thus expression of the TFF2 gene appears to be highly responsive to the normal physiological range of circulating amidated gastrin. Gastrin treatment increased endogenous TFF2 mRNA expression in gastric cancer cells in a dose- and time-dependent manner. Using TFF2 promoter-reporter gene constructs, we showed that gastrin induced TFF2 transcription through two discrete gastrin-responsive promoter elements and through a PKC-, MEK1-, and PI 3-kinase-dependent but EGFR-independent pathway.
The TFF2 promoter contains a complex enhancer region responsive to a number of agents, including estrogens and phorbol esters (7, 21). In addition, basal transcription factors like GATA-6 and HNF-3 have been demonstrated to activate both the TFF1 and TFF2 promoters (1, 4). Studies from our group have shown that TFF2 is an immediate early gene capable of regulating its own expression through activation of the TFF2-promoter, and we have reported on a cis-acting element, designated spasmolytic polypeptide responsive element, located between −191 and −174 upstream of the TFF2 transcriptional initiation site (8), as well as a cell-specific enhancer element located in the TFF2 promoter 5′-flanking sequence at −32/−27, which contained a CCAAT/enhance binding proteins consensus-binding site. Mutation of this consensus site reduced the basal promoter activity by >50% in MCF-7 cells but had no effect on basal promoter activity in human gastric cancer cells (6). In the current study, we found that gastrin stimulated the transcription of TFF2 though both the proximal CCAATA elements located at −32 to −27 bp and the more distal CCCTGTGG element located at −284 to −277 bp upstream of the TFF2 transcription starter. Many studies have shown that the CCAAT sequences are consensus regulatory sites of a number of promoters and bind CCAAT/enhancer-binding proteins, a family of structurally related transcription factors critical for normal tissue development, proliferation, and differentiation (19). For the GC-rich element containing the CCCTGTGG sequence, we did not identify any homology to other transcriptional factors known by computer-based homology search, suggesting a novel gastrin-responsive cis-acting element. Mutation of both the CCAAT and CCCTGTGG sequences abolished gastrin-dependent TFF2 promoter activity, indicating that both elements are required for gastrin induction of TFF2 transcription. EMSA studies indicated binding by specific factor complexes, but further investigations will be required to define the precise nuclear factors involved in TFF2 transcriptional control through the CCAAT element.
Gastrin activation of TFF2 transcription is dependent on its receptor, the CCKBR. In this study, the CCKBR antagonist YF-476 pretreatment significantly abolished gastrin-dependent TFF2 promoter activity in a dose-dependent manner. YF-476 is an extremely potent selective human CCKBR antagonist with a reported inhibitory constant value of 0.068∼0.19 nM in different tissues. The response of all TFF2 promoter constructs to 10−7∼10−8 M of gastrin was completely inhibited in the presence of YF-476 at 10−7 M. Notably, YF-476 at 10−11 M inhibited 50% of the transactivating effects of 10−7 M gastrin on the TFF2 promoter in AGS-E cells. The greater apparent affinity of YF-476 for the CCKBR in this study, compared with previous studies, is not currently understood.
Gastrin-dependent TFF2 promoter activity was inhibited significantly by the MEK1 inhibitor (PD-98059) and the PKC inhibitor (staurosporine), suggesting activation of the CCKBR of a PKC- and MEK-dependent pathway. The CCKBR is a Gq-linked seven-transmembrane receptor, and gastrin has been shown to regulate numerous genes (e.g., TFF1, histidine decarboxylase and heparin-binding EGF) through a PKC-dependent HDC- and MEK1-dependent pathway (16, 17, 23, 46). Studies with recombinant CCKBR expressed in eukaryotic cells have indicated that the receptor showed near-maximal binding with gastrin concentrations between 10−9 and 10−10 M. In the original cloning of the CCKBR, the IC50 for gastrin was 0.26 nM (43); in the generation of CCKBR-expressing AGS cells, the IC50 for gastrin binding to AGS/CCKBR cells was 0.6 nM (17). With respect to transcriptional studies, CCKBR-expressing AGS cells have shown near-maximal transcriptional responses with gastrin concentrations ranging from 10−9 to 10−8 M or up to one log greater gastrin concentrations. Our previous publications have demonstrated that 5 × 10−9 M gastrin can induce 50% maximal promoter upregulation of the rat HDC promoter (17), the human HDC promoter (46), the mouse chromogranin A promoter (16), and the murine TFF1 promoter (23) when transfected in AGS cells. The reason for this slight discrepancy in curves for binding vs. transactivation is not clear but might relate to the need for a certain threshold occupancy level for signaling. In some cases, signaling from the CCKBR to the MEK/ERK pathway is direct, while in other cases it is indirect, involving transactivation of the EGFR by EGFR ligands. For example, previous studies have demonstrated that gastrin regulates the HB-EGF promoter via an EGFR-dependent mechanism (34). In addition, gastrin induction of TFF1 promoter activity is in part indirect through EGFR-dependent activation (23), and we have also shown previously that trefoil genes are capable of auto- and cross-induction through an EGFR-dependent pathway (7, 34). Nevertheless, in the current study, EGFR inhibitors did not attenuate gastrin-dependent TFF2 promoter activity, indicating that gastrin regulation of TFF2 expression was independent of the EGFR pathway.
TFF2 is upregulated in diverse pathological conditions of the gastrointestinal tract in human and mouse (38), such as gastric ulceration, duodenal ulceration, and Crohn's disease of the small intestine (36, 38, 40). It is in fact one of the earliest genes upregulated after injury to the gastrointestinal tract, preceding the activation of other trefoil genes and growth factors (31, 43). In experimentally induced gastric ulceration in rats, the increase in TFF2 mRNA expression occurs within minutes (43). Although the CCKBR is not so promptly induced, a similar pattern of altered expression of the CCKBR has been observed during the later stages of ulcer healing (32). During wound healing, CCKBR are specifically expressed and localized to the regenerative mucosal ulcer margin and correlated with proliferative activity in these tissues. Thus it seems likely that sustained elevations in TFF2 gene expression in the setting of wound healing or regeneration may be sustained in part through gastrin stimulation.
Gastrin is not only a trophic factor for normal gastric epithelial cells but also has been postulated to play a role in a number of malignancies (12). Expression of CCKBR has also been observed in mucous neck cells in the fundic mucosa of mouse, and, in H. pylori-infected patients, an increase in the number of TFF2-expressing mucosa neck cells has been observed in the mouse and human stomach (14, 42). Helicobacter-infected mice show increases in serum gastrin levels before the emergence of prenoplastic and neoplastic TFF2-expressing gastric metaplasia and dysplasia (42). Both gastrin and TFF2 have been linked to cellular migration, and TFF2 has been shown to be expressed by some human gastric cancers where it is associated with a significantly shorter disease-free survival (10). Thus induction by gastrin of TFF2 expression could potentially contribute to cancer cell migration, invasion, and spread. Further studies will be required to determine the importance of the gastrin-TFF2 link in neoplastic disease.
This work was supported by a National Institute of Diabetes and Digestive and Kidney Diseases Grant R01.DK-58889-01 to T. C. Wang.
We thank Dr. S. Lei for technical assistance in EMSA experiments.
↵* S. Tu and A. L. Chi contributed equally in this study.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society


text:
Why is it I have trouble spelling words like very and cousin but I can spell combative Radiology department with ease even when I was little like 5 I couldn't spell blue or yellow but I could spell casino microraptor and Brachiosaurus whats wrong with me?
Most Helpful Girl
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I have always been this way it doesn't affect my grades so I never found it nesasary to get it checked out. i get my D's Bs Ms Ws and PS Q's mixed up but I look over my work to make sure it's right why?
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subset: commoncrawl


text:
Herbal Baby Massage Oil
Passion Moon Potions
$ 8.00
This oil is made with calendula infused oil and blended with roman chamomile and french lavender essential oil.
For a soothing and relaxing massage. This oil can also be added to the bath water.
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This oil is made to order in small batches with lots of aloha.
Comes in a 2oz bottle.


text:
Smart the New Cool Car with IPod Integration Device - The Latest Gadgets in Store
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Dec 21, 1999
|Filing date
|Dec 7, 1998
|Priority date
|Sep 27, 1995
|Fee status
|Publication number
|Thomas P. Walker, Tristan E. Juergens
|Original Assignee
|Bolder Technologies Corporation
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (56), Referenced by (59), Classifications (29), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 08/784,853, filed Jan. 15, 1997, now U.S. Pat. No. 5,895,728 which is a continuation-in-part of application Ser. No. 08/534,790, filed Sep. 27, 1995, now U.S. Pat. No. 5,677,078. These two applications are incorporated herein by reference.
The present invention relates to the field of electrochemical battery cells and cell cases, and more particularly to a battery that contains a plurality of lead-acid cells in separated gas spaces and that may interchangeably mate with a variety of electrical connectors such as those integrated within power tools.
The use of portable, rechargeable electrochemical cells has been rapidly increasing in recent years. With developments in rechargeable battery technology, rechargeable batteries are used for power not only in traditional areas such as flashlights and small electronic devices but also in heavy duty power tools, temporary power sources, and even vehicles. Of course, rechargeable batteries are also widely used in the starting systems of internal combustion vehicles and tools. Many of the rechargeable batteries in use today are nickel-cadmium, nickel-metal hydride or lithium batteries. Recent advances in lead-acid batteries, such as those disclosed in U.S. Pat. No. 5,047,300 to Juergens and assigned to the assignee of the present invention, have increased the utility and the popularity of portable, rechargeable lead-acid battery systems.
Of course, some case or housing must be used to contain the cells that comprise a battery so that the cells' electrical energy may be safely and conveniently used and transported. Since many uses require the battery to be frequently recharged, the battery should be easily manipulable between a battery-powered device and a recharger. It is also desirable that a battery be compatible, perhaps with adaptations, with a variety of the connectors that are often found on battery-powered devices, so that the same battery may potentially be used with many different devices. A case that contains several cells is often termed a "battery pack" or a "power pack." In the present application, it should be understood that what is referred to as a "battery case" may contain several electrochemical cells, wherein each cell may comprise a plurality of electrodes or, for example, two long spirally-wound electrodes.
Many rechargeable battery cases and rechargers and tools useful therewith have been developed, and these devices address a variety of uses. U.S. Pat. Nos. 5,144,217 and 5,391,972 to Gardner et al. disclose a combination battery back/recharger wherein the charger may accommodate several battery configurations. U.S. Pat. No. 5,200,280 to Karasa discloses a device that surrounds a battery to prevent accidental discharge. U.S. Pat. No. 4,339,201 discloses a battery case to prevent terminal short-circuiting. U.S. Pat. Nos. 4,871,629 to Bunyea; 3,883,789 to Achenbach et al.; 3,999,110 to Ramstrom et al.; 5,368,954 to Bruns; 5,213,913 to Anthony et al., 5,208,525 to Lopic et al.; 4,084,123 to Lineback et al.; 5,089,738 to Bergquist et al.; 5,336,953 to Alleman; 4,616,169 to Proffitt; 4,447,749 to Reeb, Jr. et al.; 4,339,201 to Nagahara; and 3,757,194 to Weber et al. disclose latching arrangements and other mechanical and electrical connections between battery cases and electric devices.
U.S. Pat. No. 4,751,452 to Kilmer et al. discloses a battery case that precludes the battery from attempted recharge when the battery is of a non-rechargeable type. U.S. Pat. No. 5,296,314 to Millauer provides a battery case keyed to provide the appropriate voltage to selected electric devices.
None of these prior art devices, however, address the difficulties associated with lead-acid cells, such as filling, venting, and attaching end connectors. Some of these devices are meant to function in several electric tools; however, none of these devices are designed to allow the same unit of cells to be adapted to a variety of tools by substituting external connectors.
The present invention includes a cell case (sometimes called a "monoblock") that houses a plurality of electrochemical cells, preferably of the lead-acid type. The individual cells are located in hermetically-sealed compartments, so that there is no atmospheric communication between the several cells. The cells are electrically connected to each other, such as in a series configuration, so that they may be attached to a tool or other electrically-powered device and function as one electrical unit. The monoblock includes interchangeable adapters including electrical connectors, so that the same cells may be used with a variety of tools or other devices. The adapters may also include electrical circuitry, such as recharging circuitry. If included, the recharging circuitry may incorporate a variety of features, such as a heat sensor or a voltage sensor that modulates the battery charging parameters, such as time of charge and charge current, to the charging condition of the battery. Other circuity may be included in addition to or instead of a charger, such as a safety cut-off that electrically disconnects the battery if the battery temperature exceeds a preselected value.
The individual electrochemical cells are preferably spirally-wound ultra-thin plate lead acid cells, which allow for about twice the useable energy of competing cells, such as nickel-cadmium cells, at high discharge rates. The increase in usable energy primarily results from the lead-acid cells' ability to supply a relatively constant voltage as the cells discharge. Nickel-cadmium cells, in contrast, lose a significant amount of voltage during discharge, so that much of the energy theoretically available may not be supplied at a power level sufficient for many applications. For instance, nickel-cadmium batteries may not be able to supply the voltage required to provide enough torque to a drill to allow the drill to penetrate many materials or to spin the drill bit fast enough to provide satisfactory drilling speeds after the cells have become partially discharged.
The monoblock is preferably constructed of several components that allow for the lead-acid cells to be essentially permanently grouped together, and for interchangeable adapters to be connected thereto. The monoblock also allows for the cells to be filled with electrolyte and to vent gas that may form within the cells, and to contain any electrolyte that may otherwise leak from the cell. The central unit consists of connected plastic containers, where each container is shaped to hold an individual spirally wound cell. The containers are preferably arranged in an array of rows and columns, so that the monoblock will have a rectangular block-like shape. The containers are preferably cylindrical, so that interstices are formed between the rows and columns. These interstices allow a sensor, such as a temperature sensor, to be inserted into the core of the monoblock.
The containers are manufactured with open-ended bottoms, so that the spirally wound cells may be inserted through these bottoms into the containers. After the cells are inserted into the container, a bottom cover plate is attached to the bottom of the containers, so that cells are sealed within. The bottom cover plate contains ventilation ports, such as Bunsen valves, beneath each container, so that excess gas may be released. Additionally, the cover plate contains a hole beneath each container, so that an end connector may be attached to each cell. Preferably, two rivet-type end connectors are used within each cell, one each for the positive and negative connectors. A bottom cap is attached beneath the cover plate to form the bottom surface of the monoblock. The bottom cap encloses the electrical connections between the cells, as well as any electrolyte that may happen to leak from the battery. Absorbent, foraminous material may be placed between the bottom cap and the cover plate to absorb any leaked electrolyte. The bottom cap preferably also includes ventilation ports, to release any gas that is vented from the containers into the area between the cover plate and the bottom cap. The cover plate may be attached to the containers by any of several suitable methods, such as ultrasonic or thermal welding or by adhesives. The bottom cap may be similarly attached to the cover plate.
The main body contains an integral top that forms the top of each container. Above each container, the integral top has two holes. One hole receives a rivet that serves as the electrical connector of the opposite polarity from the connector that attaches to the container bottom. The second hole is a fill port that allows electrolyte to be introduced into each container. After the electrolyte has been introduced, this fill port may be sealed, such as by a plug.
A top unit removably attaches to the main body, and serves several functions. The top unit has two electrical connectors that mate with the electrical connectors of the main body. As described above, the electrical connectors of each cell are connected to one another, such as in a series configuration. The positive and negative connectors of the series assembly mate with the positive and negative connectors, respectively, of the top unit. The top unit is physically configured to mate with a specific electric tool or family of tools. It may also contain additional electrical features such as a recharger or a temperature sensor, or both. The top unit may be removed from the main body, allowing a user to power different battery operated devices. Another top unit that physically connects with another electrical device may be mounted onto the main body, and the monoblock may again be used.
It should therefore be appreciated that the present invention allows a group of batteries, specifically lead-acid batteries, to power a variety of battery powered devices. The invention provides a safe, durable, and convenient method of combining a number of lead-acid batteries together, and may include supplementary electronics such as a recharger.
FIG. 1 is a side elevation view of an embodiment of the present invention.
FIG. 2 is bottom plan view of the main body of the present invention.
FIG. 3 is a top plan view of the main body of the present invention.
FIG. 4 is a top plan view of the bottom cover of the present invention.
FIG. 4A is a detailed side profile view of a Bunsen valve of the bottom cover.
FIG. 5 is top plan view of a top cap according on one embodiment of the present invention.
FIG. 6 is a top plan view of the bottom cap of the present invention.
FIG. 7 is a diagrammatic horizontal cross section of the electrochemically active components of a spirally wound cell of the type preferably contained within the main body of FIG. 1.
FIG. 8 is voltage discharge graph comparing of the performance of a battery according to present invention with a competing battery.
FIG. 9 is a perspective view of the present invention.
Referring to FIG. 9, an embodiment of the present invention 11 has a main body 10 that includes a plurality of connected cell compartments. As can be seen in FIGS. 1 and 2, the main body 10 includes six compartments, 20, 30, 40, 50, 60, and 70, arranged in two rows and three columns. Each compartment may enclose a separate electrochemical cell, such as representative cell 22 contained within compartment 20. The compartments are contiguous, and each compartment has a sidewall that is partially shared with a portion of a neighboring, contiguous compartment. For instance, compartment 20 shares a portion of its sidewall with compartment 30 and also shares a portion of its sidewall with compartment 50.
The main body 10 includes an integral top 80 that covers each of the compartments (see FIG. 3). The top 80 contains two openings associated with each compartment. One opening, such as opening 84 associated with compartment 20, is a fill port that allows electrolyte to be added to the cell 22. The second opening, such as opening 82, also associated with compartment 20, allows the insertion of a cell terminal so that the electricity produced within each compartment may be utilized. Preferably, the terminal is a rivet connector of the type disclosed in U.S. Pat. No. 5,677,078.
The top 80 also contains two additional openings 86, 88 that extend between compartment groupings. The compartments are preferably cylindrical, so that a separation exists at the compartment interfaces. The openings 86, 88 allow ventilation through the top 80 along the entire depth of the compartments. Optionally, a temperature sensor (not shown) may be inserted into either the opening 86 or the opening 88. The cells may be disconnected from their load if the sensor indicates that the battery is operating at an unsafe or otherwise undesirable temperature.
With reference to FIG. 2, the bottom of the main body 10 is essentially open, allowing the cells (such as cell 22) to be inserted into the compartments (such as compartment 20). The cells cannot be inserted from above, because the integral top 80 substantially blocks access to the compartments. After the cells are inserted into the compartments, a bottom cover plate 90 is attached to the bottom of the main body 10 in order to contain the cells within the compartments (see FIG. 4). The bottom cover plate 90 has the same cross sectional dimensions as the main body 10, and may be attached thereto by ultrasonic or thermal welding, adhesives, or other suitable means to form an hermetic seal.
The bottom cover plate 90 has two openings associated with each compartment. The first opening, such as opening 92 associated with compartment 20, allows the insertion of a cell terminal into the compartment. This corresponds to the opening 82 in the top sheet 80. However, the cell terminal inserted through the opening 92 will be connected to plates of opposite polarity than is the separate terminal inserted through the opening 82.
The second opening, such as opening 94 associated with compartment 20, serves as a valve that vents the compartment 20 of excess gas that may develop therein during the chemical reactions. Preferably, the opening 94 is through a valve stem 96 that projects downwards from the bottom cover plate 90 as shown in the detail of FIG. 4A. While many one-way valves may be used, a Bunsen valve is preferred because of its simplicity and reliability. In a Bunsen valve, an elastomeric sheath 95 is fitted over the valve stem 96. When the gas pressure within the compartment 20 exceeds the external gas pressure, the sheath 95 is displaced from the valve stem 96, so that gas may be vented from the compartment 20. When the pressure within the compartment 20 is reduced to closer to the ambient pressure, the elastomeric quality of the sheath 95 causes it re-seat onto the valve stem 96.
Referring to FIG. 6, a bottom cap 100 attaches beneath the cover plate 90. The bottom cap 100 has substantially the same cross sectional dimensions as the bottom cover plate 90. A raised ridge 102 surrounds a central platform 104 of the bottom cap, so that the ridge 102 contacts the bottom cover plate 90 and the central platform 104 is spaced away from the cover plate 90. This space between the bottom cover plate 90 and the bottom cap 100 provides space for routing terminal connector wire to connect the bottom terminals of the cells contained in the compartments, so that the electronics are shielded from exterior contact. Additionally, the space maintained between the cover plate and the bottom cap serves as a reservoir to collect any electrolyte that may leak from the cells. Foraminous, absorbent material may be included in the space 106 (see FIG. 1) between the bottom cover plate 90 and the bottom cap 100 to absorb any leaked electrolyte. However, the type of lead-acid cells preferably contained within the compartments maintain electrolyte absorbed within separator material of the cell, so that electrolyte leakage is uncommon. The bottom cap is preferably perforated, such as with micro-perforations 108, to allow any pressure caused by gas transport through the valve stem openings to escape.
A variety of top caps, such as top cap 110, may fit over the top 80 of the main body (see FIG. 5). The top cap 110 provides the electrical connectors that join the battery terminals of the cells contained within the main body 10 to an electric tool or other device. Two terminal cutouts, 112, 114, are formed in the top cap 110. The terminals may take a variety of forms depending on the specific application. For instance, if the battery is to be used as a starter power source for a vehicle, such as a motorcycle, simple lugs may protrude from or be accessible through the cutouts 112, 114.
More specialized connectors may be used to connect with specific connectors for other applications. Preferably, the top cap is removable from the top 80. A variety of top caps may then be used with the same main body 10, allowing the same battery to be used in a variety of applications. The top caps will vary in size, shape and type of electrical connectors, depending on the application. The attachment between top caps and the top 80 may be any suitable means, such as by adhesive ultrasonic welding or screw.
A sensor cutout 116 may also be formed in the top cap, directly above the opening 86 that extends between the compartments 20, 30, 50, and 60. The sensor cut out 116 provides an opening through which a sensing probe may be inserted into the core of the main body 10. The operation of the battery may be controlled depending upon parameters such as temperature that may be sensed by the sensor probe in the opening 86 (see FIG. 3).
While the present battery has application with a variety of electrochemical cells, it is particularly suited for use with an embodiment of the lead-acid cells of the type described in U.S. Pat. No. 5,677,078 and other patents in the chain of this application, the contents of which are hereby incorporated. With reference to FIG. 7, such batteries are generally spirally wound, and include a negative plate 180 and a positive plate 190 that are separated from each other by separator material 200. Acceptable active positive plate materials include, among others, sulfated lead pastes, PbO, Pb3 O4, or leady oxide. Acceptable active negative plate material includes, among others, sulfated lead paste, PbO, or leady oxide. An example of suitable separator material is a glass mat composed of microfiber material consisting of 90% fibers 1 to 4 microns in diameter and 10% fibers that are somewhat larger.
As described in U.S. Pat. No. 5,677,078, the cells preferably use cast-on end connectors to connect the positive and negative plates of each cell to their respective terminals. For example, a rivet extends through opening 82 (see FIG. 3) into the compartment 20 (see FIG. 1), and contacts the cast on connector. Expanding the rivet mandrel causes the body of the rivet to form a strong interference fit with the cast-on connector. The fit between the terminal and the connector is further strengthened by an elastomeric sleeve disposed between the connector and compartment 20. The expansion of the rivet compresses the elastomeric sleeve, thereby forming a fluid seal around the compartment opening 82 as well as a mechanically and electrically strong connection.
The cell contained within compartment 20 is filled by introducing electrolyte through fill port 84. Perforations in the cast-on connector allow the introduced electrolyte to disperse within the region of compartment 20 containing the plates and separator. After the compartment has been filled, the fill port 84 is permanently sealed, such as by welding or otherwise attaching a connector plug into the fill port 84.
Venting of the compartment 20 is preferably accomplished by the venting opening 94 located in the valve stem 96 of the bottom cover plate 90.
While a preferred embodiment has been described, it should be apparent that many variations could be made to the monoblock that would not essentially alter its functionality. As one example, some of the components that have been described as being placed on the top of the monoblock could be placed on the bottom, and vice versa.
A monoblock according to the present invention was constructed using six one-inch diameter cells with a depth of 2.350 inches. The lead-acid cells were of the type described in the above referenced patents and applications of which this application is a continuation-in-part. The monoblock was used to power a drill to repeatedly drill holes through a wooden test board. Referring to FIG. 8, curve A shows the voltage across the board as a function of time. Curve B shows the voltage versus time results of a nickel-cadmium battery pack of the same approximate size, subject to the same test conditions. The results indicate a much more rapid decline in voltage for the nickel-cadmium battery. Energy within the nickel-cadmium battery may not be available at sufficient voltages to satisfy certain applications. The monoblock of the present invention, in contrast, supplied most of its energy at useable voltages. The voltage was maintained within 10 per cent of a nominal rating for about 350 seconds; the voltage began to rapidly decline at about 370 seconds.
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|Feb 22, 2010
|Apr 19, 2016
|Andreas Stihl Ag & Co. Kg
|Battery pack for an electric power tool
|Apr 1, 2014
|Apr 4, 2017
|Makita Corporation
|Battery pack for electric power tool
|Apr 29, 2014
|Jul 4, 2017
|Robert Bosch Gmbh
|Battery having a cover that has predetermined breaking points
|Mar 25, 2002
|Feb 20, 2003
|Zguris George C.
|Lead acid battery
|Sep 30, 2002
|Mar 6, 2003
|Squannacook Technologies Llc, A Delaware Corporation
|Battery paste
|Feb 26, 2002
|Aug 28, 2003
|Sabongi Gebran J.
|Self-monitoring radio network
|Mar 25, 2002
|Sep 25, 2003
|Zguris George C.
|Glass fibers
|Mar 25, 2002
|Oct 2, 2003
|Reher Ronald D.
|Methods of modifying fibers
|Jan 9, 2004
|Sep 2, 2004
|Johnson Controls Technology Company
|Battery and battery container
|Aug 31, 2004
|Mar 24, 2005
|New Bright Industrial Co., Ltd.
|High performance rechargeable battery pack for toy vehicles
|May 25, 2005
|Nov 24, 2005
|Yoshinori Shibata
|Battery powered table saws
|Aug 29, 2005
|Feb 16, 2006
|Tsang Francis Y
|Nuclear voltaic cell
|Jun 25, 2004
|Jun 29, 2006
|Rainer Glauning
|Battery pack comprising heat-diffusing means
|Feb 18, 2005
|Aug 24, 2006
|Shimano, Inc.
|Water resisting apparatus for a bicycle electrical component
|Jun 5, 2006
|Oct 12, 2006
|Reher Ronald D
|Methods of modifying fibers
|Aug 29, 2005
|Nov 9, 2006
|Tsang Francis Y
|Nuclear voltaic cell
|Jul 27, 2006
|Nov 23, 2006
|New Bright Industrial Co., Ltd.
|High performance rechargeable battery pack for toy vehicles
|Sep 14, 2006
|Mar 20, 2008
|Joseph Barrella
|Foil cell fill port
|Jan 14, 2008
|May 15, 2008
|Johnson Controls Technology Company
|Battery and battery container
|May 7, 2007
|Nov 13, 2008
|Ajith Kuttannair Kumar
|System and Method for Cooling a Battery
|May 7, 2007
|Nov 13, 2008
|Ajith Kuttannair Kumar
|System and Method for Cooling a Battery
|May 7, 2007
|Nov 13, 2008
|Ajith Kuttannair Kumar
|Battery mechanical packaging
|May 23, 2007
|Nov 27, 2008
|Ajith Kuttannair Kumar
|System and method for connecting a battery to a mounting system
|May 31, 2006
|Jan 29, 2009
|Florin Vantu
|Pressure relief valve for a battery charger
|Apr 11, 2006
|Jun 4, 2009
|Xi Shen
|Lithium Ion Battery
|Feb 22, 2010
|Sep 2, 2010
|Andreas Stihl Ag & Co. Kg
|Battery Pack for an Electric Power Tool
|Apr 1, 2014
|Oct 9, 2014
|Makita Corporation
|Battery pack for electric power tool
|Apr 29, 2014
|Nov 6, 2014
|Robert Bosch Gmbh
|Battery having a cover that has predetermined breaking points
|Apr 11, 2006
|Aug 6, 2008
|Byd Company Limited
|A lithium ion battery
|Apr 11, 2006
|Jan 7, 2009
|Byd Co Ltd
|A lithium ion battery
|Feb 3, 2010
|Jun 27, 2012
|Andreas Stihl AG & Co. KG
|Battery pack for an electrical tool
|U.S. Classification
|429/53, 429/177, 429/178, 429/159, 429/163
|International Classification
|Cooperative Classification
|European Classification
|Jan 28, 2002
Owner name: GP BATTERIES INTERNATIONAL, LTD., CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BOLDER TECHNOLOGIES CORPORATION;REEL/FRAME:012569/0800
Effective date: 20011214
|Jun 19, 2003
|Fee payment
Year of fee payment: 4
|Jul 9, 2003
|Maintenance fee reminder mailed
|Jun 15, 2007
|Fee payment
Year of fee payment: 8
|Jul 25, 2011
|Maintenance fee reminder mailed
|Dec 21, 2011
|Lapse for failure to pay maintenance fees
|Feb 7, 2012
|Expired due to failure to pay maintenance fee
Effective date: 20111221


text:
It was so timely that I got a new Asus ZenPower Pro power bank as I was shopping around for … More
Tag: Reviews
Starmobile Play Plus: What a Php 2,290 Smartphone Brings
While most people would drool over the latest high-end smartphones in the market, there is still a segment for budget … More
Field Test | Runtastic GPS Watch and Heart Rate Monitor
Runners these days have it easy. With a multitude of wearables lately, gone are the two-fingers-on-wrist pulse count for the heart rate method as these new wearables can instantly show information such as heart rate, distance, elevation, pace and calories burned at ease. Runtastic GPS Watch and Heart Rate Monitor is the offering from the famed Runtastic Apps brand. I reviewed their other wearable Runtastic Orbit which is a cheaper fitness tracker. The Runtastic GPS Watch has more features for people seriously starting or are already into running.
Express Review | The Nikon D750 Field Test in Tacloban
I have this misconception that Full-frame cameras are humongous DSLR monsters where one have to take an extra effort to bring it to trips. I was recently asked to test out the latest of the Nikon DSLR FX-format, the Nikon D750. I was pretty surprised by how light it was basing it on the body itself. I've been using the DX-format Nikon D7100 for a couple of months already and I actually found the D750 slightly lighter and much solid in construction. That is until I fitted the camera with the Nikon 24–140mm f4 lens which is the standard kit. I had a scheduled trip to Tacloban and I thought it was the perfect time to try the camera.
Review | Lenovo Yoga Tablet 8: Innovative Multimode Tablet
It was just timely this little wonder of a tablet came in when my 2-year old Apple iPad 2 is conking out. The Lenovo Yoga Tablet 8 is one of the many Android-based tablets currently saturating the market and this definitely gets curious looks for its unusual design that clearly sets it apart from the common and boring flat-rectangular style tablets. As a disclaimer, Lenovo Philippines provided this Lenovo Yoga Tablet 8 when I was invited to join their 18-hour challenge testing the tablet's battery life and for review. After our challenge, I took the tablet for a trip to really see if it could work with how I travel. Don't expect this review to be technical but as a practical user experience review.
Field Review | The Osprey Shuttle 22: A Rugged Wheeler Pack
I'm more of a backpack type of guy when I travel and seldom uses trolley-type bags unless I needed to. The wheeler I had before was a convertible Wenger, the other is a huge World Traveler luggage when I moved to a new location temporarily. Recently I went on a week-long trip up north and brought with me the Osprey Shuttle 22″/40L bag. Now here's one bag I wouldn't mind hauling around. Read on why.


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A colleague just let me know that I'm mentioned in the "Best of the Web" section of Genetic Engineering & Biotechnology News. Yay! It's a positive review!
It's in the April 15 issue for all you subscribers.
A colleague just let me know that I'm mentioned in the "Best of the Web" section of Genetic Engineering & Biotechnology News. Yay! It's a positive review!
It's in the April 15 issue for all you subscribers.
They even spelled your name correctly. How much did that cost you?
But they didn't call you attractive and cephalopod-friendly.
'The author appears to be P. Z. Myers…'
Hmmm, I wonder who else the author appears to be? Martha Stewart?
Yes- who's this 'Morris' and why is he appearing to be P. Z. Myers? I want some answers.
Heh, heh.
GEN started showing up in my mailbox about two years ago, and now I can look forward to an approximately monthly call asking if I want to continue my free personal subscription, to which I say, "No" as I know I'll still get the magazine and the phone calls but not have to answer their stupid questionnaire.
Bad Press for the Army…
It's ok to descriminate against atheists… they have no religion, so it's not religious bigotry.
That rather reminds me of restaurant reviews in newspapers. Good going, your restaurant should be getting some more grazers.
"Evolution, development, and random ejaculations from a godless liberal"
HA! that makes it sound disgusting….
What does the "nothing significant" refer to?
According to their key, the red X icon means "weak points". That is, PZ's "weak point" is that he writes about "nothing significant" — which is (a) bollocks and (b) not really consistent with the review text itself. Perhaps the reviewer's estimation of the harm "antiscience folks" are causing is not concordant with mine.
Also, the blue ribbon means "excellent". How can you be "excellent" while writing "nothing significant" I don't know. Maybe I'm reading this completely backwards, and they really mean that PZ has no significant weak points?
Blake- I think you'll find that it means there are no significant weak points. At least, that's my interpretation.
uh… I think it means that there are no significant weak points…
Pros: "Well written, topical."
Cons: "Nothing significant."
I think it's interesting to see which entry was chosen for the screen shot: "We Need A New Gender," which was, as I recall, a spirited exchanged.
GEN had a nice article 15 Mar 2006, by Alan McHughen, of UC Riverside, "Implications of Faith-Based Science" starting
"Pressure is mounting to install faith-based beliefs into science classrooms. This movement subverts the very foundation of science–a search for the truthand turns science curricula into religious education. In schools and courts, religious crusaders equate faith-based beliefs with scientific theories, arguing, for example, that because the theory of evolution is unproved, alternative faith-based theories are equally valid and should be taught in science classes. ."
My attempts to embed links don't work, but http://www.genengnews.com/articles/chitem.aspx?aid=1410
Several creationists complained, and McHughen had a great last word, in May 2006, but I can't find letters to the editor in the GEN archives. GEN does have more than its share of right-wing editorials, though
Aaaaaaah I seeee … University of Minnesota *COMMA* Morris and his…
I feel (slightly) less dumb now..
Pharyngula was featured blog in Focus, the BBC's science and technology mag, too. They particularly liked the idea of a belly-dancing commenter.
New comments have been temporarily disabled. Please check back soon.


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The Northwood Farmers Market is a Sunday destination in Palm Beach County's hippest hood: Northwood Village. Stop by every Sunday to enjoy a family fun day at 400 block of Northwood Rd.
This new Farmers Market will be offering arts and crafts, fresh plants/flowers, fresh produce, and other types of wonderful food and entertainment every Sunday morning all year round, as the neighborhood has become more popular with year round residents. Bring the kids, bring the pets, and stroll the shops and galleries after market shopping. There will be local entertainers and friendly neighbors and vendors.
More About Northwood Farmers Market
Address: 400 Northwood Rd., West Palm Beach, 33407
Dates: Every Sunday | Events Hours: From 9:00am until 1:00pm
Northwood just keeps getting better and the addition of this Farmers Market is a sure sign of community involvement and commitment. As more great vendors in the area like Olivia's Special Teas, Moonlight Radiance Soaps, ArtHaus Gelateria, and various art and antique businesses open up, the more special this hood becomes. The annual Mango Festival and the monthly Art Walks all contribute to the vibe. Parking is Free!
Northwood Farmers Market in Pictures
Coming Soon!
Directions for Northwood Farmers Market
Don't Miss a Single event of the Northwood Farmers Market in West Palm Beach. Bookmark this page to find more information about hours, parking, what to buy, calendar of art events, participating food vendors, music performances and much more.


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Mazda MX-5 Coupe Likely to Arrive in 2017
An all-new Mazda MX-5 will be revealed in just a few short days, and it looks like a coupe model is in the works.
According to Automotive News, the new Miata will offer a hardtop version roughly one year after it launches, complete with fastback styling. That means we should see the car sometime in 2017.
WHAT WE KNOW: 2016 Mazda MX-5
The car is expected to weigh about the same as the original Miata, tipping the scales at 2,100 pounds. Under the hood, a 2.0-liter four-cylinder engine will be fitted, connected to a six-speed manual transmission. Mazda also says the new Miata will be stiffer and slightly smaller than the previous car.
Check back for live coverage on September 3 when Mazda pulls the wraps off the new MX-5 Miata.
[Source: Automotive News]
Discuss this story at our Mazda Miata Forum


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Enjoy the cruise of a lifetime on this wonderful cruise ship. Blue Cruise offers this wonderful Bodrum - Gukova - Bodrum itinerary for holidaymakers to enjoy. This wonderful cruise offers a great option for couples, singles and families who want to explore the area of a breathtaking cruise. You can make the most of the 74-foot long boat, which comes with spacious and comfortable rooms, and a wide range of facilities and features on offer. Guest can enjoy the en suite cabins as well as the excellent meals served on board in the charming on boa dining room. You will find plenty of opportunities to socialise with other guests as well as time to get off the boat and enjoy the many stops along the way. A trip on a Turkish Blue Cruise is a great option for many offering a different holiday experience with visits to the many secluded coves and ancient ruins along the way. Take a look at the ruins at Cleopatra Island and be sure to explore the marine life with a snorkel at the Gokova Gulf. You will have an unforgettable holiday with plenty of variation and interest to offer first time and experienced cruise ship passengers.
Day 1: Bodrum: Check in, dinner & overnight on board -
Day 2: Morning sail: Bodrum to Alakisla Bay, breakfast on board. At Kraada Island you can swim and enjoy the bay. Dinner & overnight at Cokertme.
Day 3: Morning sail: breakfast on board, Cokertme to Cati Lagoon for lunchtime. Dinner & overnight in Cati.
Day 4: Cruise to Yeduadalar for lunchtime. Anchor in English Harbour to enjoy the area. Dinner & overnight stay here.
Day 5: Cruising to Sedir Island. Visit to Cleopatra Beach. Sail to Karacasogut village for dinner and overnight. Optional tours Marmaris on request.
Day 6: Sail to Tuzla, free afternoon then sail to Ballisu for dinner & overnight in Longoz.
Day 7: Sailing to Pbuc after breakfast. Heremten bay after lunch and swimming break. After tea sail to Pabucburnu and swimming break. Sail back to Bodrum for last dinner and night over.
Day 8: Breakfast and Check Out.
**To make sure that the excursions mentioned are included in your holiday, please book the room type stating that excursions are included**
Double cabins come fully equipped on board with: en suite bathrooms, comfortable beds, spacious accommodation and shower.
Please note: This cruise departs only on Fridays, Saturdays and Mondays. The cruise can be booked for seven night only, and cannot accommodate children.
Eating and Drinking:
Guests can make the most of on board breakfast, lunch and dinner all cooked by a talented on board chef. Food is inspired by the region as well as by international cuisines. You are also welcome to eat off the boat with many wonderful restaurants and cafes along the route.
Right choice:
The Blue Cruise Bodrum - Gokova - Bodrum route is a great choice for first time cruise passengers as well as those who want to get the most out of a trip to the area. There is so much to see and do along the route as well as make the most of the wonderful surroundings and excellent service and facilities. The cruise is a great choice for couples who want to enjoy the beauty and culture of the area as well as a relaxing and idyllic cruise.
Suitable for:
A great holiday for couples


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text: My Style Mara Colson 2 things that Represent Me (Related to the Class) My bedroom Book shelf found on Pinterest. 5 Things that Represent Me(Unrelated to the class) I play soccer. Decoration from my room. My favorite color - Crimson/Red One of my favorite things is IU basketball. My dog, Dixie.


text:
latex distortion
Used to deejay some years ago: Preferably experimental techno but later moved to deep house. Now ears wide open for all kinds of music but with an addiction to indie, rock, folk, soul, singer/ songwriter music. However, electronic music is still in the heart and will always be. Therefore mixes will also be a mix of various styles.What else? Co-founded the netlabel one bit wonder (www.1bit-wonder.com) in 2005 and buried it in 2008.Loves his daughter, movies, music, beer.
Press play to try again.
Please update your browser or install another such as Google Chrome.Mixcloud requires HTML5 audio with MP3 and MP4 support.


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|"Stephen Colebourne" <scolebou...@btopenworld.com>
|[collections] Serializable decorators
|Tue, 06 Apr 2004 19:56:59 GMT
I had a look at making the decorators serializable, and it will take some time (tedious mostly). However I did find a design decision. The collection that a decorator decorates may or may not be serializable. But does the decorator need to represent this? This leads to two options. 1) Make all decorators implement serializable, but some will fail at runtime if you decorate a non-serializable instance. 2) Implement serializable subclasses of each decorator, and use the factory method to create either the serializable or non-serializable subclass as required. #1 is easy, #2 will take much longer. Any views? #1 is the default choice. Stephen --------------------------------------------------------------------- To unsubscribe, e-mail: firstname.lastname@example.com For additional commands, e-mail: firstname.lastname@example.com
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text:
Question: If the probability density of X is given by Find the
If the probability density of X is given by
View Solution:
Find the probability density of Y = X3. Also, plot the graphs of the probability densities of X and Y and indicate the respective areas under the curves that represent P(1/2 < X < 1) and P(1/8 < Y < 1).
View Solution:
Answer to relevant QuestionsIf X has a uniform density with α = 0 and β = 1, show that the random variable Y = –2 ∙ lnX has a gamma distribution. What are its parameters? If the joint probability distribution of X1 and X2 is given by f(x1, x2) = x1x2 / 36 For x1 = 1, 2, 3 and x2 = 1, 2, 3, find (a) The probability distribution of X1X2; (b) The probability distribution of X1/ X2. If X has the uniform density with the parameters α = 0 and β = 1, use the distribution function technique to find the probability density of the random variable Y = √X. Let X and Y be two independent random variables having identical gamma distributions. (a) Find the joint probability density of the random variables U = X / X + Y and V = X + Y. (b) Find and identify the marginal density ...Use the result of Exercise 7.45 to show that, if n independent random variables Xi have normal distributions with the means µi and the standard deviations σi, then Y = α1X1 + α2X2 + · · · + anXn has a normal ...
Post your question


text:
- Details
DetailsFeatures: Weight Capacity: 600 lbs. Designed to accommodate Bariatric patients. 600 lb. weight capacity. 10 ft. long gray laminate platform with anti-slip surface treads and tapered ends for wheelchair use. (6) steel uprights. Width adjusts from 22" to 35". 38" standard clearance between uprights. 1 1/2" diameter one-piece stainless steel handrails. Heavy gauge steel base plates, uprights and fittings. Black powder coated protective finish. Each upright telescopes in 1 1/2" increments and locks into (10) height positions with fail-safe locking pin. Width adjusts with ergonomic control knobs on each upright. 1386
- Specifications
Additional Information
Old Manufacturer Part Number (MPN) 4002-078 GREY/GRAY Manufacturer Hausmann Unit of Measure (UOM) Each Shipping Method Tailgate Delivery Model 4002 Country of Origin United States Of America
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Sur Niragas Ho - Katyar Kaljat Ghusli | Shankar Mahadevan & Anandi Joshi | Shankar - Ehsaan - Loy MP3
Catch the official video of Sur Niragas Ho from Katyar Kaljat Ghusli starring Shankar Mahadevan Song: Sur Niragas Ho Singer: Shankar Mahadevan & Anandi ...
Diwali Special : Shankar Mahadevan & Rahul Deshpande Best Performance (Jugalbandhi) at Shivaji Park MP3
Mumbai: Shankar Mahadevan & Rahul Deshpande Best Performance.
Shankar Mahadevan & Zakir Hussain jugalbandi Full Episode in Pahava Vitthal Programme MP3
Amazing Flute player with Shankar Mahadevan Notes & Sargam MP3
Best Of Shankar Mahadevan - Jukebox - Full Songs MP3
Shankar Mahadevan is an Indian music composer and singer. He is a part of the Shankar--Ehsaan--Loy trio team that composes for Indian films and a playback ...
Hariharan and Shankar Mahadevan Rocking Birmingham!! MP3
This was when these legends jammed at the Vijay TV star night, Birmingham,october 2011.
Ekdantaya Vakratundaya Gauri Tanaya by Shankar Mahadevan in Mumbai Youth Concert MP3
Ekdantaya Vakratundaya Gauri Tanaya : Shankar Mahadevan performing Ekdantaya Vakratundaya Gauri Tanaya in Art of Living Youth concert in Mumbai ...
Shankar Mahadevan performs at GiMA Awards 2015 MP3
Shankar Mahadevan's performance at GiMA Awards 2015.
Shankar Mahadevan performs "Breathless" LIVE at the 7th Mirchi Music Awards MP3
Watch the very talented Shankar Mahadevan perform to his songs on the stage as a tribute to Javed Akhtar the 7th Royal Stag Mirchi Music Awards. Drop in your ...
Shankar Mahadevan sung ek pyaar ka nagma hai at GiMA Awards. memorable song!!!!!!!!!!!!!!!!!!! MP3
Indian Idol 5 Sreeram and Shankar Mahadevan MP3
Shankar Mahadevan & Rahul Deshpande Jugalbandi MP3
Shankar Mahadevan & Rahul Deshpande Jugalbandi.
Ekadantaya Vakratundaya Song By Shankar Mahadevan- YouTube.flv MP3
This Is The Most Famous Karnatic Fusion Song Of Lord Ganapathi Ji Composed By The Energetic Singer Shankar Mahadevan Sir.......Watch,Listen & Enjoy.
FULL PERFORMANCE - Shankar and Shafqat's Musical Medley at the 7th Royal Stag Mirchi Music Awards! MP3
Watch Shankar Mahadevan and Shafqat Amanat Ali's unedited Musical medley on some of the Bollywood hits at the 7th Royal Stag Mirchi Music Awards!
Allah Hi Rehem,Shankar Mahadevan,Coke Studio @ MTV,S01,E02 MP3
A sufi composition by Shankar Ehsaan Loy for 'My Name Is Khan'. Music conceived and created by Lesle Lewis. www.leslelewis.com Singer - Shankar ...
shankar Mahadevan & Rahul Deshpande - Ghei Chand Makrand. MP3
The 2 Versions Of Masterpiece 'Ghei Chand Makrand..Classic.
Breathless by Shankar mahadevan lyrics. MP3
Both breathless and breathless the reprise in one song. Breathless lyrics:- Koi jo mila to mujhe aisa lagta tha jaise meri saari duniya main geeton ki rut aur ...
Sivamani, Shankar Mahadevan And Stephen Devassy Performace At Arimanambi Audio Launch MP3
Log-on to http://www.cineulagam.com Subscribe: http://www.youtube.com/subscription_center?add_user=cineulagam Facebook: ...
Aao Na Rasika N Shankar Mahadevan @ Singapore 19th Oct 13 MP3
A mesmerizing flautist Rasika and Shankar ,just fabulous !!
Shankar Mahadevan Breathless at AURA 2015 GIT MP3
KLS Gogte Institute Of Technology Belgaum.
Zakir Hussain & Shankar Mahadevan Jugalbandi : Pahava Vitthal MP3
Song Sajda Shankar Mahadevan & Rasika Shekar at AURA 2015 GIT MP3
KLS Gogte Institute Of Technology Belgaum.
Govind Bolo by Shankar Mahadevan MP3
This exuberant chant to Lord Krishna is a national favourite. Express your devotion to the Lord with abandon. Hare Krishna. Singer -- Shankar Mahadevan ...
Shankar Mahadevan Telugu Hit Songs || Jukebox (VOL-5) MP3
Subscribe to our Youtube Channel - http://goo.gl/tVbmAU Enjoy and stay connected with us!! Like us: http://www.facebook.com/adityamusic Follow us ...
Man Mandira - Shivam Mahadevan | Katyar Kaljat Ghusli | Shankar Mahadevan & Sachin Pilgaonkar MP3
Catch the official video of Man Mandira from Katyar Kaljat Ghusli starring Sachin Pilgaonkar & Shankar Mahadevan Song: Man Mandira Singer: Shivam ...
Musical journey, experiences and more | Shankar Mahadevan || converSAtions MP3
In this exclusive converSAtion, one of India's best known singers and music composer, Shankar Mahadevan shares a lot that is not know about him. Shankar not ...
Breathless | Famous Bollywood Video Song | Shankar Mahadevan MP3
Song: Breathless Singer: Shankar Mahadevan This is the song that shot Shakar Mahadevan to fame and made him a household name. For more updates ...
Shankar Mahadevan : Secret Of Breathless Song | LIVE performance @WWI MP3
Click here to Subscribe to Our 5th Veda Channel https://www.youtube.com/channel/UCQ_C6_PpqmKKWc7k-4KcGtw?sub_confirmation=1 Follow us on Twitter: ...
Katyar Kaljat Ghusali - Shankar Mahadevan's Acting Debut - Part 1 - Subodh Bhave, Amruta Khanvilkar! MP3
Katyar Kaljat Ghusali - Shankar Mahadevan's Acting Debut - Subodh Bhave, Sachin Pilgaonkar, Amruta Khanvilkar! Check out the special interaction with ...


text:
Screenshots on Virtual Skipper Wiki.
How to contribute Edit
- To upload content, visit Special:Upload or Special:MultipleUpload or Special:ImportFreeImages
- When uploading a file, please use the Summary input box to provide:
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- License: see Virtual Skipper Wiki:Licensing guide for help
- a brief description of the image/audio/video/ZIP archive/Open office document/etc.
- After a successful upload all content will have been added to the Uploads category but users may specify a sub-category from among:
- Images
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- of course you may create any suitable sub-category to suit your needs
This will prove helpful when you go to search for something later on.
ZIP archives
Please upload a ZIP archive for only the following purposes:
- Boat models
- Boat skins
- Collections of custom race tracks
- Race track .GBX files are tiny (<1kB) so bundle as many as you can into one ZIP archive and paste a comma-separated list of files into the Summary: input box.
- For step by step instructions please see Project:List contents of an archive.
Media in category "Screenshots"
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- Overstand layline for ...
- Radar toggle laylines.png


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Why editing of form is not saving?Asked by eatsops on April 12, 2016 at 12:30 PM
Hello JotForm Team;
I make modifications but the preview doesn't take count of them ( even if I save my JotForm).
Could you help me?
Best Regards
JotForm Support
Can you tell us which exactly modifications you did?
Also, can you try to reset your browser's cache and see if that helps.
We'll wait for your response.