Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two different culture media (TCM-199 and follicular fluid), two activation treatments (10 and 50 micromol calcium ionophore l(-1)) and three culture periods with cycloheximide were evaluated to find effective culture conditions for activation of cumulus-free equine oocytes. Oocytes were collected by scraping the follicle walls of ovaries obtained from an abattoir. Oocytes with expanded cumuli were matured at 38.2 degrees C in a humidified atmosphere of 5% CO(2) in air, in either TCM-199 with 10% fetal bovine serum (FBS) and 5 microU FSH ml(-1), or in 100% follicular fluid derived from a preovulatory follicle 24 h after injection of hCG. After 40--42 h of in vitro maturation, oocytes were denuded by gentle pipetting in TCM-199 plus 10% FBS with hyaluronidase. Oocytes with intact cytoplasmic membranes (n = 398; 94% presumed metaphase II) were treated in protein-free PBS with 10 or 50 micromol calcium ionophore l(-1) for 5 min. After washing, the oocytes were cultured in TCM-199 containing 10% FBS and 10 microg cycloheximide ml(-1) for 6 h, in cycloheximide for 6 h and then in cycloheximide-free medium for 18 h, or in cycloheximide for 24 h. The oocytes were fixed and evaluated by fluorescence microscopy. Oocytes with pronucleus I--II (dense to decondensing chromatin), pronucleus III--IV (decondensed chromatin) or progressing towards the first cleavage division were considered activated. The activation rate for oocytes matured in TCM-199 was significantly (P < 0.05) higher than for oocytes matured in follicular fluid (49% (99/204) versus 35% (60/171), respectively; P < 0.05). Culture with cycloheximide for 24 h resulted in a significantly higher rate of activation (67%, 74/111) than did the 6 h (33%, 44/136) or 6 h plus 18 h (32%, 41/128) treatments. The highest rate of activation (82%) was observed in oocytes matured in TCM-199, treated with 50 micromol calcium ionophore l(-1) and cultured with cycloheximide for 24 h.
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PMID:Activation of cumulus-free equine oocytes: effect of maturation medium, calcium ionophore concentration and duration of cycloheximide exposure. 1142 42

The objective was to compare culture media for in vitro maturation of equine oocytes and for in vitro culture of zygotes produced from IVF of partially zona-removed oocytes. Cumulus-oocyte complexes from slaughterhouse-derived ovaries were washed in m-Dulbecco's PBS and cultured in TCM-199, F10-DMEM or c-F10-DMEM (50% F10-DMEM + 50% F10-DMEM conditioned medium from culture of an equine trophoblast monolayer for 3 or 4 days). All media included FSH, LH, E2, and 10% FCS. After 28 to 30 h maturation, cumulus expansion was scored from 0 (no expansion) to 4 (fully expanded). Oocytes with a 1st polar body were selected for manipulation after removing cumulus cells using hyaluronidase. About one-third of the zona pellucida was cut using a fragment of a razor blade. For fertilization, fresh stallion semen was washed twice in BGM3 (a modified Tyrode's medium) and capacitated with 0.5 mM c-AMP for 3.5 h and 100 microM ionomycin for 15 min and added to oocytes in fert-TALP at 10(6) spermatozoa/mL. After 20 h, some presumptive zygotes were stained, and the rest were cultured in 100% TCM-DMEM conditioned medium. Cumulus expansion in F10-DMEM and c-F10-DMEM was higher (P<0.05) than the TCM-199 control (3.2, 3.5 vs 1.3, on a scale of 0 to 4). However, polar body formation rates were not different among treatments (47, 52 and 50%). The fertilization rates of equine oocytes matured in TCM-199, F10-DMEM and c-F10-DMEM determined by fixing and staining were 41, 35 and 29%, with no significant differences. There were no significant differences among treatments in cleavage rates (36 to 40%), development to morula (3 to 10%), or blastocyst stages (3 to 5%). On Day 14 of culture in c-F10-DMEM treatment, one blastocyst had more than 500 nuclei, but no capsule was formed. In a further study, cleavage rates (46 to 50%) and development to morula (5 to 10%) and blastocyst stages (3 to 8%) were not different (P>0.1) between TCM-DMEM and 100% conditioned TCM-DMEM for culturing embryos. Six embryos (2 morulae and 4 blastocysts) were nonsurgically transferred to 4 recipient mares, but no pregnancy continued.
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PMID:Developmental capacity of equine oocytes matured and cultured in equine trophoblast-conditioned media. 1148 Jun 24

Although the precise etiology of low back pain is disputed, degeneration of the intervertebral disc is believed to play an important role. Many animal models have been described which reproduce the changes found in degenerative disc disease, but none allow for efficient, large-scale testing of purported therapeutic agents. The purpose of this study was to develop a simple animal model resembling degenerative disc disease using the intervertebral discs found in the tails of rats. The proximal two intervertebral discs in the tails of 20 rats were injected with either chondroitinase ABC or control (phosphate buffered saline, PBS). The tails were harvested at 2 weeks, and measurements were made of intervertebral disc height (measured radiographically and histologically), biomechanics (stiffness, hysteresis, and residual deformation), and histologic appearance. Treatment with chondroitinase ABC resulted in a significant loss in intervertebral disc height (radiographic intervertebral disc height, p=0.001; histologic intervertebral disc height, p<0.001) and significant increases in all biomechanical parameters (stiffness, p<0.001; hysteresis, p=0.006; residual deformation, p=0.004) compared to PBS controls. Intervertebral discs treated with chondroitinase ABC had significantly lower histologic grades for each grading category (nucleus pulposus (NP), annulus fibrosus, and proteoglycan staining) compared to controls. The results of injury with chondroitinase ABC were similar to the findings in degenerative disc disease: reduced intervertebral disc height, diminished proteoglycan content, loss of NP cells, and increased stiffness of the disc. Thus, the model appears to be a reasonable tool for the preliminary in vivo evaluation of proposed treatments for degenerative disc disease.
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PMID:An in vivo model of degenerative disc disease. 1250 97

In this study, we measured the antiallergic activities of ginsenosides isolated from the root of Panax ginseng ( Araliaceae), and of their metabolites, as produced by human intestinal bacteria. Compound K, which was identified as a main metabolite, had the most potent inhibitory activity on beta-hexosaminidase release from RBL-2H3 cells and on the PCA reaction. The inhibitory activity of compound K was more potent than that of disodium cromoglycate, one of the commercial anti-allergic drugs. This compound demonstrated a membrane stabilizing action on differential scanning calorimetry. However, compound K did not inhibit the activation of hyaluronidase and did not scavenge active oxygen. These results suggest that the antiallergic action of compound K originates from its cell membrane stabilizing activity and that the ginsenosides of ginseng are prodrugs with extensive antiallergic properties. Abbreviations. compound K:20- O-beta- D-glucopyranosyl-20( S)-protopanaxadiol DNP:dinitrophenol DSCG:disodium cromoglycate DPPC:dipalmitoylphosphatidylcholine DPPH:1,1-diphenyl-2-picrylhydrazyl HSA:human serum albumin IC 50 :50% inhibitory concentration EC 50 :50% effective concentration XOD:xanthine oxidase ICR:Institute of Cancer Research PBS:phosphate buffered saline PCA:passive cutaneous anaphylaxis RAW264.7:mouse monocyte leukemiaRBL-2H3: rat basophil leukemia SD:Sprague-Dawley
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PMID:Antiallergic activity of ginseng and its ginsenosides. 1286 69

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.
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PMID:A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells. 1465 93

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 degrees C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.
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PMID:Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide. 1525 25

Efficient and controlled gene delivery from biodegradable materials can be employed to stimulate cellular processes that lead to tissue regeneration. In this report, a substrate-mediated approach was developed to deliver DNA from hyaluronic acid-collagen hydrogels. The hydrogels were formed by crosslinking HA with poly(ethylene glycol) diglycidyl ether. Poly(ethylene imine)(PEI)/DNA complexes were immobilized to the substrate using either biotin/neutravidin or non-specific adsorption. Complexes were formed in the presence or absence of salt to regulate complex size, and resulted in complexes with z-average diameters of 1221.7 +/- 152.3 and 139.4 +/- 1.3 nm, respectively. During 48-h incubation in PBS or hyaluronidase, DNA was released slowly from the hydrogel substrate (<30% of immobilized DNA), which was enhanced by incubation with conditioned media (approximately 50% of immobilized DNA). Transgene expression mediated by immobilized, large diameter complexes was 3 to 7-fold greater than for small diameter complexes. However, the percentage of cells expressing the transgene was greater for small diameter complexes (48.7%) than for large diameter complexes (22.3%). Spatially controlled gene transfer was achieved by topographically patterning the hydrogel to pattern cell adhesion. Biomaterial-based gene delivery can be applicable to numerous tissue engineering applications, or as a tool to examine tissue formation.
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PMID:DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach. 1552 59

Sequential extraction was applied to investigate the proteoglycan (PG) organization in healthy laryngeal cartilage (HLC) and laryngeal cartilage squamous cell carcinoma (LCSCC). Highly stable aggrecan aggregates, extracted from both HLC and LCSCC with strong dissociative reagents, i.e., 4 M guanidine HCl (GdnHCl), represented 53% and 7%, respectively, of total extracted macromolecules. Less stable complexes/aggregates, extracted with mild dissociative reagents (1 and 2 M GdnHCl), represented 40% and 61% of total extracted PGs from healthy and cancerous cartilage, respectively. Interestingly, a relative high proportion (32%) of uronic acid (UA)-containing macromolecules were removed from the cancerous cartilage using associative extracting solutions (PBS and 0.5 M GdnHCl), which obviously represented molecules freely extractable from the tissue. In contrast, the corresponding proportion in HLC was impressively low (about 7%). The major proportion of these molecules was chondroitin sulfate-containing PGs (CSPGs), which identified mainly as aggrecan. Differential digestion of the sequential extracts with chondroitinase ABC and chondroitinase AC II demonstrated the presence of dermatan sulfate-containing PGs (DSPGs) in both HLC and LCSCC, being mainly present in the 1 M GdnHCl extract, and identified as decorin. All cancerous extracts were found to be rich in 4-sulfated disaccharides, mostly participating in DS structures. In conclusion, the applied procedure permitted the elucidation of the changes in the cartilage status, regarding the stability and identity of its proteoglycan aggregates/complexes, in both HLC and LCSCC.
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PMID:The extractability of extracellular matrix components as a marker of cartilage remodeling in laryngeal squamous cell carcinoma. 1565 82

The regional difference in the contribution of the mucous/glycocalyx layers in rat small intestine, as a diffusional or enzymatic barrier, to the absorption of insulin was investigated by in vitro studies. The mucous/glycocalyx layers from the duodenum, the jejunum, and the ileum in rat were successfully removed without damaging membrane integrity, by exposing them to a hyaluronidase solution in situ. In an in vitro transport experiment, the apparent permeability coefficient (P(app)) of insulin for the hyaluronidase-pretreated group was significantly increased compared to the PBS-pretreated (control) group in all small intestinal regions, and the P(app) of insulin in both PBS- and hyaluronidase-pretreated groups increased in the following order: duodenum < jejunum < ileum. On the other hand, irrespective of small intestinal regions, the P(app) of FD-4 and of antipyrine, respectively the passive para- and transcellular permeation marker, exhibited no significant differences between PBS- and hyaluronidase-pretreated group. In addition, a significant amount of insulin was degraded in the mucous/glycocalyx layers compartment removed by hyaluronidase pretreatment, and the degradation activity in the mucous/glycocalyx layers showed regional differences in the following order: duodenum > jejunum > ileum. These findings suggest that, irrespective of small intestinal regions, the mucous/glycocalyx layers contributed to insulin permeation predominantly as an enzymatic barrier, and not as a diffusional barrier. Furthermore, the variation of the enzymatic activities in the mucous/glycocalyx layers and in the brush-border membrane would be one factor that accounts for the regional differences in the transport of insulin.
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PMID:Region-dependent role of the mucous/glycocalyx layers in insulin permeation across rat small intestinal membrane. 1604 94

Chondroitin sulfate (CS) is a potential candidate for colon-specific drug carriers. However, the readily water-soluble nature limits its application as a solid-state drug-delivery vehicle. In this study, the CS formation of a polyelectrolyte complex (PEC) with Ca2+ (CS-Ca) was adapted to retain CS in a solid form for use in a drug-delivery system. Pre-treated CS with poly(ethylene glycol) diglycidyl ether (EX-810) followed by complexation with Ca2+ was also tested (CS-Ca-EX). Diclofenac sodium was used as a drug probe to evaluate the performance of the drug-release behavior of the complexes. The amount of diclofenac sodium released was higher in simulated intestinal fluid (SIF) than in simulated gastric fluid (SGF) due to the anionic groups on CS or the higher solubility of drug itself in PBS. The release profile of diclofenac sodium from CS-Ca-EX was most notably sustained when compared to other groups. Enzymatic degradation by chondroitinase ABC of CS, CS-Ca and CS-Ca-EX exhibited a similar degradation mechanism and GPC revealed the dissolution rate of CS from the three matrix types was, in decreasing order: CS, CS-Ca, CS-Ca-EX. The synergy of the anti-inflammatory activity of diclofenac sodium in CS-based complexes was evaluated using the carrageenan-induced edema rat test. The percentage of swelling was lower for all experimental groups as compared to the control, untreated group. The anti-inflammatory activity of diclofenac in the CS matrix gradually increased up to 9 h but CS-Ca or CS-Ca-EX matrices showed less potency than the CS matrix in reducing inflammation.
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PMID:Oral sustained delivery of diclofenac sodium using calcium chondroitin sulfate matrix. 1626 56


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