Use of hyaluronic acid associated with triamcinolone acetonide or ozone gas in the treatment of induced osteoarthritis in rabbits

Purpose: To evaluate the effects of the intra-articular application of hyaluronic acid associated with triamcinolone acetonide, and ozone gas in the treatment of induced osteoarthritis in rabbit’s stifles.Methods: Twenty-one Norfolk rabbits were submitted to cranial cruciate ligament transection of the left stifle. After six weeks of the surgery, the rabbits were randomized assigned into three groups: G1 (control) – saline solution (0.9%); G2 – hyaluronic acid associated with triamcinolone; G3 – ozone gas, submitted to three intra-articular applications every seven days. Results: Significant differences occurred: osteophytes at medial femoral condyle (G2 > G1, G2 > G3) on radiography exam; thickening of the medial condyle (G1 > G3, G2 > G3) on ultrasound exam; osteophytes at lateral tibial condyle (G2 > G1, G2 > G3), and medial femoral condyle (G1 > G2, G3 > G1) on computed tomography. Histologically, mean values of chondrocytes in the femur and tibia in G3 and G2 were statistically lower. Conclusions: The intra-articular injection of hyaluronic acid associated with triamcinolone accentuated degenerative joint disease by imaging and macroscopic evaluations, and by histological findings, this treatment and the ozone gas treatment showed similar effects and were inferior to the saline solution (0.9%).

Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes.

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

High-performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements.

Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.

Online preconcentration and determination of chondroitin sulfate, dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis.

Capillary electrophoresis with large-volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200 mM sodium dihydrogen phosphate, 200 mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and -16 kV. Exploiting large-volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1 mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.

Absorption of Orally Administered Hyaluronan.

Hyaluronan (HA) has been utilized as a supplement. However, the absorption of orally administrated HA remains controversial. The degradation and absorption of HA in the intestine were investigated in this study. HA excretion into the feces, degradation in the intestinal tract, absorption through the large intestine, and translocation to the blood and skin were examined. HA administered orally was not detected in rat feces. HA was degraded by cecal content, but not by artificial gastric juice and intestinal juice. Oligosaccharide HA passed through excised large intestine sacs. Furthermore, disaccharides, tetrasaccharides, and polysaccharides HA were distributed to the skin of rats following oral administration of high molecular weight HA (300 kDa). The results of the study suggest that orally administered HA is degraded to oligosaccharides by intestinal bacteria, and oligosaccharide HA is absorbed in the large intestine and is subsequently distributed throughout the tissues, including the skin.

[Effects of extracts of Dragon's blood on fibroblast proliferation and extracellular matrix hyaluronic acid].

OBJECTIVE: To investigate the effects of Dragon' s blood extract on proliferation and secret extracellular matrix function of fibroblasts in vitro. METHODS: Dragon' s blood was extracted by chloroform, acetoacetic ester, alcohol. Human fibroblast were cultured in vitro in media containing gradient dilutions of Dragon' s blood extracts (0.002, 0.02, 0.2, 2, 20 mg/ml) , which was followed by cell proliferation assessed with MTT assay on 0, 12, 24, 36, 48, 60, 72 h. Under the optimal concentration, the cell growth curves were drawn and the flow cytometry (FCM) was used to determine the changes of cell cycle. On 0, 12, 24, 36, 48, 60, 72 h, the concentration of hyaluronic acid in the supernatant of fibroblast culture was measured by radioimmunoassay. RESULTS: 0.2-2 mg/ml Dragon' s blood extracts enhanced the proliferation of fibroblasts in a dose-dependent manner. 2 mg/ml was the optimal dilution of Dragon's blood extract, and it increased the ratio of S cells in cell cycle [(25.80 ± 3.10)%] than control group [(7.50 ± 0.70)%, P < 0.01]. From 12 h to 72 h, in 2 mg/ml Dragon's blood group, concentration of Hyaluronic acid secreted by fibroblasts gradually increased, but were less than control (P < 0.01). CONCLUSIONS: Dragon's blood acetoacetic ester extract improved the proliferation of cultured human fibroblasts in vitro, might be beneficial to promote wound healing.

Michelia alba extract attenuates UVB-induced expression of matrix metalloproteinases via MAP kinase pathway in human dermal fibroblasts.

Solar ultraviolet (UV) radiation can cause skin photoaging by inducing secretion of matrix metalloproteinases (MMPs). It has been reported that MMPs, especially MMP-1, -3 and -9, reduce elasticity of the dermis by degrading collagen. Polyphenols are a group of compounds that exist mainly in glycosides in the plants and they may transform to aglycone after hydrolysis. Polyphenols can inhibit MMP expression and elastase activity. In this study, we investigated the effects of Michelia alba extract (MAE) on expression and activity of MMPs in human skin fibroblast cultures after UVB exposure. The results showed that MAE and its hydrolysates (MAH) inhibited collagenase and elastase activities. In addition, MAE exhibited antioxidant activity, elevated hyaluronic acid content and inhibited UVB-induced MMP-1, MMP-3 and MMP-9 expression. In addition, the zymography assay revealed that MAE also inhibited MMP-9 activity. We also found that MAE inhibited UVB-induced ERK and JNK kinase but not p38 kinase expression, suggesting that MAE may regulate the UVB-induced expression of MMP-1, MMP-3 and MMP-9 via the ERK and JNK kinase pathway. MAE could restore total collagen synthesis reduced by UVB. The results also suggest that MAE treatment may prevent UVB-induced extracellular matrix damage by inhibiting the expression of MMP-1, MMP-3 and MMP-9 through the MAP kinase pathway. Our findings imply that MAE is an effective agent against UVB-induced photodamage.

Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes.

With a view to search for optimal concentration of hyaluronan (HA) and plant protein (PP) in different culture systems for in vitro maturation of bovine oocytes, cumulus-oocyte complexes (COCs) were matured in vitro in 2 culture systems (first co-cultured with granulose cells and estrus calf serum (ECS) in 2 mL volume, second without co-culture where ECS was replaced by exogenous hormones and BSA or PP in 100 microL dose under mineral oil). Seven types of media were used; 3 in first system and 4 in second system. To evaluate HA and PP effect on oocytes after in vitro culture an estimation of meiosis stage and a level of DNA fragmentation was performed by TUNEL staining. The highest meiotic maturation (84%) was observed in oocytes cultured in medium enriched with ECS in co-culture with granulose cells (1st system). The lowest meiotic maturation was noted in medium with addition of BSA (43%). The addition of HA in the medium enriched with BSA significantly increased the rate of matured oocytes (67%) and also didn't affect the chromatin quality of individual oocytes. The addition of HA to the culture medium supplemented with a PP decreased the rate of matured oocytes to 54% but no statistical differences were noted. The results of the present study showed that HA supplementation didn't have a detrimental impact on oocyte chromatin integrity and improved bovine oocytes' meiotic maturation in medium supplemented only with BSA without co-culture of granulose cells.

1,2,3,4,6-Penta-O-galloyl-beta-D-glucose reduces renal crystallization and oxidative stress in a hyperoxaluric rat model.

Adhesion of calcium oxalate (CaOx) crystals to kidney cells may be a key event in the pathogenesis of kidney stones associated with marked hyperoxaluria. Previously, we found that 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), isolated from a traditional medicinal herb, reduced CaOx crystal adhesion to renal epithelial cells by acting on the cells as well as on the crystal surface. Here we used the ethylene glycol (EG)-mediated hyperoxaluric rat model and found evidence of oxidant stress as indicated by decreases in the activities of the renal antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, with increased kidney cell apoptosis and serum malondialdehyde levels, all evident by 21 days of EG treatment. These effects of hyperoxaluria were reversed by concurrent PGG treatment along with decreased urinary oxalate levels and CaOx supersaturation. Renal epithelial cell expression of the crystal binding molecule hyaluronan increased diffusely within 7 days of EG initiation, suggesting it is not a result of but precedes crystal deposition. Renal cell osteopontin (OPN) was also upregulated in EG-treated animals, and PGG significantly attenuated overexpression of both OPN and hyaluronan. Thus, our findings demonstrate that PGG reduces renal crystallization and oxidative renal cell injury, and may be a candidate chemopreventive agent for nephrolithiasis.

[Effect of moxibustion in treating knee joint osteoarthritis and its relation with contents of hyaluronic acid in serum and synovial fluid].

OBJECTIVE: To investigate the clinical effects of moxibustion (Mox) in treating knee joint osteoarthritis, and to detect the change of hyaluronic acid (HA) level in serum and synovial fluid (SF) for evaluating its significance. METHODS: Thirty OA patients were treated with Mox applied on inner and outer hsiyens and Ashi point for 10 min once a day, 5 times a week for 3 months and the therapeutic efficacy was evaluated. Serum and SF levels of HA were measured by radio-immunoassay before and after the 3-month treatment, and compared with those from 30 non-OA persons for normal control. RESULTS: After treatment, 19 patients (20 joints) out of the 30 patients were cured, the efficacy of Mox was evaluated as markedly effective in 8 patients on 10 joints, and as effective in 3 patients on 4 joints, the cure rate being 63.3%. Before treatment, HA level in serum (122.87 +/- 34.10 microg/L) was higher and in SF (0.98 +/- 0.17 g/L) was lower in OA patients than those in the normal controls (68.32 +/- 21.48 microg/L and 1.62 +/- 0.30 g/L, P<0.01), whereas after treatment, both the serum and SF levels of HA in patients changed toward normal range (70.29 +/- 27.30 microg/L and 1.58 +/- 0.26 g/L), showing insignificant difference as compared with those in the controls (P<0.01). CONCLUSION: Mox is an effective approach for treatment of OA, and the levels of HA in serum and SF can be taken as the quantitative indicators for evaluating the pathogenetic condition of OA patients.

Capillary electrophoresis for the quality control of chondroitin sulfates in raw materials and formulations.

Exogenous administration of chondroitin sulfate (CS) is widely practiced for the treatment of osteoarthritis, although the efficacy of this treatment has not been completely established by clinical studies. A reason for the inconsistency of the results may be the quality of the CS preparations, which are commercially available as dietary supplements. In this article, we describe the development of a new method of capillary electrophoresis (CE) for the quantification of CS concentrations, screening for other glycosaminoglycan or DNA impurities and determination of hyaluronan impurities in CS raw materials, tablets, hard capsules, and liquid formulations. Analysis is performed within 12 min in bare fused silica capillaries using reversed polarity and an operating phosphate buffer of low pH. The method has high sensitivity (lower limit of quantitation [LLOQ] values of 30.0 microg/ml for CS and 5.0 microg/ml for hyaluronan), high precision, and accuracy. Analysis of 11 commercially available products showed the presence of hyaluronan impurities in most of them (up to 1.5%). CE analysis of the samples after treatment with chondroitinase ABC and ACII, which depolymerize the chains to unsaturated disaccharides, with a previously described method (Karamanos et al., J. Chromatogr. A 696 (1995) 295-305) confirmed the results of hyaluronan determination and showed that the structural characteristics (i.e., disaccharide composition) of CS are very different, showing the different species or tissue origin and possibly affecting the therapeutic outcome.

Investigation of anti-hyaluronidase treatment on vocal fold wound healing.

SUMMARY: Phytochemical constituents of medicinal plants demonstrate inhibition of tissue and bacterial hyaluronidase. Echinacoside is a caffeoyl conjugate of Echinacea with known anti-hyaluronidase properties. The purpose of this study was to investigate the wound healing effects of Echinacea on vocal fold wound healing and functional voice outcomes. Pig animal model. METHODS: Vocal fold injury was induced in 18 pigs by unilateral vocal fold stripping. The uninjured vocal fold served as control. Three groups of six pigs randomly received a topical application of 300, 600, or 1,200 mg of standardized Echinacea on the injured side. Animals were euthanized after 3, 10, and 15 days of wound healing. Phonation threshold pressure and vocal economy measurements were obtained from excised larynges. Treatment outcomes were examined by comparing the animals receiving treatment with a set of 19 untreated and 5 historical controls. Treatment effects on wound healing were evaluated by histologic staining for hyaluronan and collagen. Treated larynges revealed improved vocal economy and phonation threshold pressure compared with untreated larynges. Histologically, treated vocal folds revealed stable hyaluronan content and no significant accumulation of collagen compared with control. Findings provide a favorable outcome of anti-hyaluronidase treatment on acute vocal fold wound healing and functional measures of voice.

Analysis of hyaluronan content in chondroitin sulfate preparations by using selective enzymatic digestion and electrospray ionization mass spectrometry.

Two important glycosaminoglycans (GAGs) with structural roles in the body's cartilage are hyaluronan (HA) and chondroitin sulfate (CS). A simple mass spectrometric method for determining the amount of HA that may be present in isolated CS samples is presented in this article. Samples are subjected to selective enzymatic digestion using a bacterial hyaluronidase (HA lyase, EC 4.2.2, from Streptococcus dysgalactiae) specific for HA. Undigested CS GAG is then removed by centrifugal filtration, and digested HA left in the filtrate is quantified by electrospray ionization mass spectrometry using an internal standard and selected ion monitoring. The described method was applied to the analysis of several CS samples prepared for use in nutritional supplements.

Dynamical influence of Cordyceps sinensis on the activity of hepatic insulinase of experimental liver cirrhosis.

BACKGROUND: Cordeceps sinensis (CS) is a herb which can inhibit the liver fibrosis. Hyperinsulinemia is common in liver cirrhosis patients. The activity of insulin degrading enzyme could reflect the metabolism of insulin. This study was to detect the dynamical effects and mechanisms of CS on the activity of hepatic insulinase in CCl4 induced liver cirrhosis in rats. METHODS: Rats were randomly allocated into three groups: normal group, model group and CS group. The rats in the normal group were sacrificed at the beginning of experiment, and the other two groups were sacrificed randomly at the end of the third, sixth and ninth weeks. Blood and tissue specimens were taken. Biochemical assays were used to determine the changes of alanine transaminase (ALT), albumin levels in serum. And radioimmunological assays were used to determine the changes of hyaluronic acid (HA), insulin levels in serum and the activity of hepatic insulinase. RESULTS: No significant differences were seen in the serum levels of ALT, albumin, HA between the CS group and the model group at the third and sixth weeks (P>0.05). The serum levels of ALT, HA in the CS group were lower than those in the model group at the ninth week (P<0.05), but the serum level of albumin in the CS group was higher than that in the model group at the ninth week (P<0.05). No significant differences were observed in the serum levels of insulin and the activity of hepatic insulinase between the CS and model groups at the third week and the normal group (P>0.05). The serum levels of insulin in the CS and model groups at the sixth and ninth weeks were higher than those in the normal group (P<0.05). But the activity of hepatic insulinase was lower than that in the normal group (P<0.05 or P<0.01). No significant differences were found in the serum levels of insulin and the activity of hepatic insulinase between the CS and model groups at the third, sixth and ninth weeks (P>0.05). CONCLUSIONS: CS may decrease the damage to hepatocyte by CCl4, and inhibit hepatic fibrogenesis. Six weeks after CCl4 administration, the activity of hepatic insulinase began decreasing. CS could not inhibit the decrease of the activity of hepatic insulinase.

Glucose-induced changes in the phenotype of human peritoneal mesothelial cells: effect of L-2-oxothiazolidine carboxylic acid.

BACKGROUND: During peritoneal dialysis, mesothelial cells are chronically exposed to high concentrations of glucose. Therefore, the cytotoxic effect of glucose may alter the function and reactivity of these cells. METHODS: For 4 weeks, human peritoneal mesothelial cells were cultured in vitroin medium supplemented with 45 mM glucose or 45 mM mannitol or with 45 mM glucose and 1 mM L-2-oxothiazolidine-4-carboxylic acid (OTZ), the latter being a precursor for glutathione synthesis. Peroxidation of the mesothelial cell lipids, synthetic activity and reaction of these cells to peritoneal dialysis fluids were studied. RESULTS: In contrast to mannitol, glucose enhanced the peroxidation of the cellular lipids (+65%, p < 0.01) an effect that was prevented by OTZ. Synthesis of hyaluronan and vascular endothelial growth factor was reduced in mesothelial cells treated with glucose by 36% (p < 0.01) and 44% (p < 0.05), respectively; both glucose effects were reversed when cells were incubated with glucose plus OTZ. Monocyte chemoattractant protein-1 synthesis by cells exposed to glucose was increased by 31% (p < 001), and again that effect was prevented by OTZ. Glucose and mannitol stimulated synthesis of fibronectin (+32%, p < 0.05). Mesothelial cells chronically exposed to glucose became activated after subsequent exposure to the dialysis fluid, as reflected by the increased release of interleukin (IL)-6, in contrast to control mesothelial cells, in which IL-6 synthesis was suppressed. CONCLUSIONS: Chronic exposure of mesothelial cells to glucose changes their synthetic activity and their reaction after exposure to dialysis fluids. Some of these effects are prevented by OTZ, which suggests that glucose-induced free radicals are responsible for a change in mesothelial cell phenotype under the conditions of peritoneal dialysis.

Effects of estradiol and progesterone on vertebral collagen, glycosaminoglycans and phosphatases in ovariectomized adult rats.

Vertebral collagen, glycosaminoglycans (GAGs), tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were measured in ovariectomized (ovx) adult Wistar rats treated with estradiol (E 2 ) (10 micro g/kg BW for 35 days on alternate days, and progesterone (P 4 ) (140 micro g/kg BW for 35 days on alternate days) in E 2 + P 4 treated rats. P 4 given alone or in combination with E 2 significantly increased the levels of collagen in the vertebral bone. Neither ovx nor E 2 treatment altered the concentration of collagen in these rats. Administration of E 2 or P 4 significantly decreased the concentration of hyaluronic acid (HA), but remaining unaffected when a combination of these steroids was given. In contrast to their effect on HA, E 2 and P 4 each significantly increased the levels of chondroitin sulfate (CS) in the vertebral bone. The specific activity of ALP was decreased after ovx. E 2 and P 4 alone or in combination also registered a significant decrease in the activities of ALP and TRAP. The results suggest that E 2 and P 4 each exert definite effects on vertebral bone turnover in ovariectomized rats.

Endogenous nitric oxide and exogenous nitric oxide supplementation in hepatic ischemia-reperfusion injury in the rat.

BACKGROUND: Although nitric oxide (NO) is thought to be beneficial in hepatic ischemia-reperfusion (I/R), the mechanisms for this effect are not well established. METHODS: To investigate the effects of endogenous NO and exogenous NO supplementation on hepatic I/R injury and their pathogenic mechanisms, serum ALT and hyaluronic acid (endothelial cell damage), and hepatic malondialdehyde and H2O2 (oxidative stress), myeloperoxidase activity (leukocyte accumulation), and endothelin (vasoconstrictor peptide opposite to NO) were determined at different reperfusion periods in untreated rats and rats receiving L-NAME, L-NAME+L-arginine, and spermine NONOate (exogenous NO donor). RESULTS: After reperfusion every parameter increased in untreated animals. Endogenous NO synthesis inhibition by L-NAME increased hepatocyte and endothelial damage as compared to untreated rats, which was reverted and even improved by the addition of L-arginine. Spermine NONOate also improved this damage. However, different mechanisms account for the beneficial effect of endogenous and exogenous NO. Oxidative stress decreased by both L-NAME and L-NAME+L-arginine, but remained unmodified by spermine NONOate. Myeloperoxidase increased by L-NAME and this effect was reverted by the addition of L-arginine, whereas no change was observed with spermine NONOate. Endothelin levels were not modified by L-NAME and L-NAME+L-arginine, but decreased with spermine NONOate. CONCLUSIONS: These results suggest that, although both endogenous and exogenous NO exert a protective role in experimental hepatic I/R injury, the mechanisms of the beneficial effect of the two sources of NO are different.

Induction of dog sperm capacitation by glycosaminoglycans and glycosaminoglycan amounts of oviductal and uterine fluids in bitches.

Ejaculated sperm collected from 12 beagle dogs were incubated in canine capacitation medium (CCM), supplemented with 5 microg/ml chondroitin sulfate A (CS), 5 microg/ml hyaluronic acid (HA), or 5 microg/ml heparin (HP) for 7 hr at 38 degrees C in a 5% CO2 in air atmosphere to investigate the effects of glycosaminoglycans (GAGs) on dog sperm capacitation. The percentages of motile sperm, hyperactivated sperm (%HY), and acrosome-reacted sperm (%AR) in all media were examined after 4 hr and 7 hr of incubation. The oviducts and uteri of 9 anestrous and 18 estrous beagle bitches were removed under halothane inhalation anesthesia to measure the total GAG amounts in oviductal and uterine fluids. The lumens of the ampulla of the oviducts, isthmus of the oviducts, and the uterine horns were each flushed with 1 ml HEPES-EDTA fluid. Total GAG amounts in the flush fluids obtained were measured with a spectrophotometer. Sperm motility (51-59%), %HY (79-86%), and %AR (31-36%) in CCM supplemented with CS, HA, or HP were significantly higher after 7 hr of incubation than when incubated in CCM without GAGs (P<0.01 or 0.05). The mean total GAG amounts in the fluids from the ampulla and isthmus of the oviducts and the uterine horns in the estrous bitches were higher than in the anestrous bitches. These results indicate that GAGs in the oviductal and uterine fluids in estrous bitches are associated with in vivo sperm capacitation.

A 35-kDa protein is the basic unit of the core from the 2 x 10(4)-kDa aggregation factor responsible for species-specific cell adhesion in the marine sponge Microciona prolifera.

Dissociated sponge cells quickly reaggregate in a species-specific manner, differentiate, and reconstruct tissue, providing a very handy system to investigate the molecular basis of more complex intercellular recognition processes. Species-specific cell adhesion in the marine sponge Microciona prolifera is mediated by a supramolecular complex with a Mr = 2 x 10(7), termed aggregation factor. Guanidinium hydrochloride/cesium chloride dissociative gradients and rhodamine B isothiocyanate staining indicated the presence of several proteins with different degrees of glycosylation. Hyaluronate has been found to be associated with the aggregation factor. Chemical deglycosylation revealed a main component accounting for nearly 90% of the total protein. The cDNA-deduced amino acid sequence predicts a 35-kDa protein (MAFp3), the first sponge aggregation factor core protein ever described. The open reading frame is uninterrupted upstream from the amino terminus of the mature protein, and the deduced amino acid sequence for this region has been found to contain a long stretch sharing homology with the Na+-Ca2+ exchanger protein. A putative hyaluronic acid binding domain and several putative N- and O-glycosylation signals are present in MAFp3, as well as eight cysteines, some of them involved in intermolecular disulfide bridges. Northern blot data suggest variable expression, and Southern blot analysis reveals the presence of other related gene sequences. According to the respective molecular masses, one aggregation factor molecule would contain about 300 MAFp3 units, suggesting that sponge cell adhesion might be based on the assembly of multiple small glycosylated protein subunits.

Hyaluronate and large molecular weight proteoglycans in synovial fluid from horses with various arthritides.

OBJECTIVE: To investigate the presence of large molecular weight (MW) proteoglycans (PG) and hyaluronate (HA) in synovial fluid (SF) from horses with various arthritides and from control joints. DESIGN: Horses with acute (< 2 weeks) or chronic (> 4 weeks) lameness were examined by clinical examination, intrasynovial anesthesia, radiography, arthroscopy, and SF analysis. Joints were grouped on the basis of diagnosis: acute traumatic arthritis, chronic traumatic arthritis (with a subgroup of degenerative joint disease), intra-articular fracture, and infectious arthritis. ANIMALS: 31 horses with arthritis and 9 control horses; altogether 43 SF samples were analyzed. PROCEDURE: High-performance liquid chromatography was used to assess HA and large MW PG in SF samples. RESULTS: A PG peak was identified in 8 of 23 SF samples of joints with chronic traumatic arthritis, 4 of which had no or minimal abnormal radiographic findings but mild articular cartilage fibrillation detected by arthroscopy, and in 3 joints with intra-articular fracture and 1 with resolving infectious arthritis, but not in joints with acute traumatic arthritis or in control joints. There was significant difference (P < 0.01) in mean (+/- SEM) HA concentration between control joints and joints with chronic traumatic arthritis (0.32 +/- 0.04 g/L; n = 9 vs 0.18 +/- 0.01 g/L; n = 23). CONCLUSION: Large MW PG fragments are released into equine SF in the course of articular disease and can be detected simultaneously with HA by high-performance liquid chromatography. CLINICAL RELEVANCE: The SF HA concentration can be used as diagnostic marker for chronic traumatic arthritis. However, SF PG or other marker cannot be used for diagnosing or monitoring degenerative joint disease.