Exercise effects on methylation of ASC gene.

Chronic moderate exercise has been reported to reduce pro-inflammatory cytokines. To analyze the molecular mechanisms by which training exerts these effects, the epigenetic influences of age and exercise on the ASC gene, which is responsible for IL-1beta and IL-18 secretion, were investigated by ASC gene methylation. Further, the relationship between carcinogenesis and exercise, and methylation of the P15 tumor suppressive gene was also analyzed. High-intensity interval walking exercise, consisting of 3 min low-intensity walking at 40% of peak aerobic capacity followed by a 3 min high-intensity walking period above 70% of peak aerobic capacity, was continued for 6 months. Peripheral blood DNA extracts from young control (n=34), older control (n=153), and older exercise (n=230) groups were then analyzed by pyrosequencing for DNA methylation. Methylation of ASC decreased significantly with age (young control vs. older control, p<0.01), which is indicative of an age-dependent increase in ASC expression. Compared to the older control group, the degree of ASC methylation was higher in the older exercise group (older control vs. older exercise: p<0.01), and presumably lower ASC expression. Neither exercise nor age affected the methylation of the P15. In summary, chronic moderate exercise appears to attenuate the age-dependent decrease in ASC methylation, implying suppression of excess pro-inflammatory cytokines through reduction of ASC expression.

Reduction of Calponin h1 expression in human colon cancer blood vessels.

AIMS: Calponin h1 (CN) is a differentiation marker of smooth muscle cells that has been reported to be down-regulated in the blood vessels of several human tumors. In this study, we examined CN expression in blood vessels in relation to the clinical and pathological features of colon cancer tissue samples. METHODS: Fifty-six patients who had undergone colectomy for colon cancer were examined. To assess patients' disease-free survival, those who had metastasis at the time of surgical operation were excluded. Immunohistochemistry was performed by the indirect immunoperoxidase method, using serial sections made from formalin fixed and paraffin embedded tissue blocks. RESULTS: We found that the expression of vascular CN in the peripheral region of colon cancer tissues was significantly reduced in association with tumor progression, lymphatic invasion, vascular invasion and recurrence. This reduction of CN indicated not only a decrease of pericytes and/or smooth muscle cells in tumor vessels, but also the immaturity of those cells, since CN down-regulation occurred even in alpha-smooth muscle actin-positive cells. The down-regulation of CN in vessels in the peripheral region of tumor tissues was inversely associated with the expression of VEGF (vascular endothelial growth factor), seemingly advantageous to angiogenesis. CONCLUSION: The down-regulation of CN expression in colon cancer vasculature evaluated by immunohistochemistry may be useful in conjunction with conventional staging procedures to predict more reliable outcome and to select therapeutic treatment.

Carbon coated zirconium beads in beta-glucan gel and bovine glutaraldehyde cross-linked collagen injections for intrinsic sphincter deficiency: continence and satisfaction after extended followup.

PURPOSE: The durability of Durasphere (Carbon Medical Technologies, Saint Paul, Minnesota), used to treat stress urinary incontinence, has not been shown beyond 12 months of followup. Women treated with Durasphere and Contigen (Bard, Inc., Covington, Georgia) at 1 institution between 1996 and 2000 were compared to determine patient satisfaction and urinary continence after extended followup. MATERIALS AND METHODS: Between April 1996 and September 2000, 56 women were treated with Durasphere at this institution, of whom 43 were available for extended followup. Aged matched patients treated with Contigen were analyzed for comparison. Patient satisfaction and continence at last followup were subjectively assessed via telephone interview. Survival methods (Kaplan-Meier and Cox proportional hazards model) were used to analyze time to failure as a function of treatment group and other potential predictors. RESULTS: Treatment groups (Contigen and Durasphere) were similar with respect to all baseline factors. Treatment was initially effective in 63% of Durasphere and Contigen cases (p = 1.0). At 24 and 36 months Durasphere remained effective in 33% and 21% of patients compared with 19% and 9% for Contigen, respectively. At last followup only 9 patients (21%) treated with Durasphere and 2 (5%) treated with Contigen (median followup 51 and 62 months, respectively) claimed that treatment was still effective. After controlling for differences in followup time there was no significant difference in time to failure between the treatment groups (p = 0.25). A third of patients in each group believed that treatment was a success. CONCLUSIONS: Neither Contigen nor Durasphere provides durable improvement in continence. Despite this outcome a third of patients in the 2 groups were satisfied with the treatment outcome.

Electron beam computerized tomography assessment of in vivo single kidney glomerular filtration rate and tubular dynamics during chronic partial unilateral ureteral obstruction in the pig.

PURPOSE: The assessment of hydronephrosis due to chronic partial ureteral obstruction is controversial. We determined whether a new radiographic technique for assessing kidney function, electron beam computerized tomography (CT), can detect altered renal physiology due to chronic partial ureteral obstruction. We also compared and contrasted electron beam CT with standard well tempered diuretic mercaptoacetyltriglycine (MAG-3) urography. MATERIALS ANDS METHODS: Six pigs underwent creation of unilateral partial ureteral occlusion or sham operation. Three weeks after surgery diuretic enhanced MAG-3 renal scan was done and 48 hours later contrast enhanced electron beam CT was performed. RESULTS: Mean differential function plus or minus standard error of mean of the obstructed kidney was 5.6% +/- 2.4% on MAG-3 renography. In contrast, electron beam CT revealed significantly preserved mean renal function at 24.5% +/- 2.7% (p <0.01). Electron beam CT analysis of tubular function revealed persistent glomerular filtration and filtrate flow through the proximal tubules and loop of Henle with a selective decrease in distal tubular flow, which were findings suggestive of proximal tubular sparing that were not demonstrated by nuclear renography. CONCLUSIONS: Renal function on MAG-3 renography is primarily determined by measuring kidney perfusion and tubular secretion of the isotope. In contrast, electron beam CT determines renal function via quantifying the in vivo single kidney glomerular filtration rate and by assessing renal tubular function. This study documents that electron beam CT of differential renal function is significantly different from that of MAG-3 renography. To our knowledge which of these 2 radiographic studies is most clinically applicable is unknown to date.

Eosinophilic cystitis in adults.

PURPOSE: We describe the largest clinical experience with the diagnosis and management of largely anecdotally reported eosinophilic cystitis. MATERIALS AND METHODS: Five women and 12 men 18 to 84 years with proved eosinophilic cystitis were treated in a 23-year period. Some combination of hematuria, irritative voiding, dysuria and suprapubic pain was present in 14 cases (82%). The remaining 3 patients (18%) were asymptomatic and the diagnosis was made by cystoscopy done because of a history of bladder carcinoma. Available data included no peripheral eosinophilia in 10 of 10 patients studied, pyuria in 12 (92%), microhematuria in 11 of 13 (84%), sterile urine in all 17, abnormal urine cytology in 2 of 17 (12%), bilateral hydronephrosis in 1 and a bladder mass or thickening in 2. Cystoscopy showed erythema in all cases and tumor-like lesions or edema in 3 (17.6%). Histological studies revealed eosinophilic cystitis in all 17 patients, while in 1 with no history of bladder carcinoma eosinophilic cystitis coexisted with carcinoma. RESULTS: Two patients were lost to followup and the remaining 15 were followed 1 to 37 months. After biopsy and fulguration of the lesions 10 patients received no further treatment, including 6 with complete symptom resolution and 1 with improvement. The 3 asymptomatic patients with a history of bladder carcinoma remained asymptomatic and disease-free. Another 4 patients underwent medical therapy and improved, of whom 1 had recurrence that was successfully re-treated medically. The remaining patient, who was symptomatic, underwent cystoprostatectomy for end stage bladder disease. CONCLUSIONS: Manifestations of eosinophilic cystitis indistinguishably mimic those of other inflammatory and malignant bladder disorders that may precede or coexist with it. The condition usually follows a benign course in most cases but occasionally its relentless progression causes crippling disease.

Outcome of isolated renal cell carcinoma fossa recurrence after nephrectomy.

PURPOSE: Local recurrence of renal cell carcinoma in the renal fossa after complete radical nephrectomy is uncommon. We characterize and determine outcome in a small subset of patients. MATERIALS AND METHODS: From 1970 to 1998 the incidence of isolated renal bed recurrence among 1,737 T1-3N0M0 unilateral nephrectomy cases was 1. 8% (standard error [SE] 0.4) at 5 years. There were 30 patients in whom isolated local fossa carcinoma recurred after complete radical nephrectomy without evidence of metastatic disease. Patients with any nodal involvement at radical nephrectomy were excluded from study as were those who had undergone any form of partial nephrectomy. Patient charts were reviewed for clinical presentation, stage, treatment, development of metastatic disease and survival. Pathological stage was assigned according to the 1997 TNM staging system. Recurrence was identified in 12 (40%) patients during routine followup and the remaining 18 (60%) presented with symptoms related to the recurrent tumor. Patients were divided into 3 treatment groups of observation (9), therapy excluding surgical extirpation (11) and complete surgical resection alone or in conjunction with additional therapy (10). Mean time from local recurrence to development of metastatic disease was calculated. Survival from local recurrence to overall death and disease specific death was estimated using the Kaplan-Meier method. Survival curves for the different treatment groups were then compared. RESULTS: There were 30 patients identified with an ipsilateral renal fossa recurrence of renal cell carcinoma after complete nephrectomy in the absence of disseminated disease. Mean followup was 3.3 years (range 0.006 to 14.8) and no patient was lost to followup. The T stage of the primary tumor was T1/T2 in 13 cases, T3a in 4, T3b in 12, and T3c in 1, and all were node negative. Mean time to metastasis was 1. 6 years (range 0.006 to 7.3) in the 19 patients who had documented interval metastatic disease after local recurrence. There were 26 deaths, of which 25 were disease specific. Estimated overall crude and cause specific survival at 1 and 5 years was 66% and 28%, respectively. Calculating survival among symptomatic and asymptomatic patients revealed no discernible difference in outcome (p = 0.94). The 5-year survival rate with surgical resection was 51% (SE 18) compared to 18% (12) treated with adjuvant medical therapy and only 13% (12) with observation alone. The differences in cause specific survival were significant (p

In vitro synthesis of hyaluronan by a single protein derived from mouse HAS1 gene and characterization of amino acid residues essential for the activity.

HAS1 was expressed as a FLAG-tagged HAS1 fusion protein in COS-1 cells. This recombinant protein was extracted with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) from the membrane fraction and purified by anti-FLAG affinity chromatography and subsequent SDS-polyacrylamide gel electrophoresis. A protein solubilized from the one single band on the gel was able to synthesize hyaluronan when incubated with UDP-GlcNAc and UDP-GlcA as donor substrates without any further additions. The detergent-solubilized and purified HAS1 protein, however, exhibited quite different kinetic properties from the membrane-bound protein. When assayed under the reconstitutive conditions where the reaction mixture was layered onto the buffer containing high concentration of CHAPS, the activity was enhanced and the kinetic properties became similar to those of the membrane-bound protein. In addition, a HAS1 gene product by an in vitro transcription/translation system also showed HAS1 activity under the reconstitutive conditions. To our surprise, when incubated with UDP-GlcNAc alone, the protein was found to synthesize chito-oligosaccharide. Taking advantage of these enzyme reaction properties, active sites on the protein involved in for hyaluronan and chito-oligosaccharide synthesis were characterized. Site-directed mutagenesis induced in the cytoplasmic central loop domain of the protein revealed that several amino acid residues conserved among those domains of various proteins of a HAS family were essential for both hyaluronan and chito-oligosaccharide syntheses but one of them was not for chito-oligosaccharide synthesis. The substitutions that caused partial or severe loss of the activity gave no significant changes of the K(m) values of the mutated proteins, suggesting that no conformational or other indirect changes were involved in the effect. Taken together, the results suggest that the HAS1 protein alone is able to synthesize hyaluronan and different amino acid residues on the cytoplasmic central loop domain are involved in transferring GlcNAc and GlcA residues, respectively.

Increased synthesis of hyaluronate enhances motility of human melanoma cells.

Hyaluronate plays a unique role in the cancer cell microenvironment. In particular, melanoma is the tumor type in which hyaluronate and hyaluronate recognition have been most closely linked to malignancy. In this study we show that a human melanoma cell line stably transfected with hyaluronate synthase cDNA displays enhanced motility. We used a fixed erythrocyte exclusion assay to isolate subsets of the WM793 human melanoma cell line that expressed either high or low amounts of hyaluronate. A cell line with a high level of hyaluronate on its surface (WM793H) displayed significantly higher cell motility on colloidal-gold-coated coverslips than did a line with a low level (WM793L). Next, in order to directly investigate the effects of hyaluronate on melanoma cell migration, we transfected cDNA encoding mouse hyaluronate synthase HAS1 or HAS2 into the re-cloned human melanoma cell line that produced a low amount of hyaluronate (WM793L) by the lipofection method. Several clonal transfectants differentially producing hyaluronate were obtained. There was a positive correlation between total hyaluronate synthesis and formation of the pericellular hyaluronate-rich matrix. We observed an increase in the migration ability of hyaluronate cDNA (HAS1 or HAS2)-transfected cells compared with control cells on glass plates covered with colloidal gold particles. A migration-inhibition assay with anti-CD44 monoclonal antibody showed blocking of the cell motility. It is speculated that the tumor cells might migrate through a hyaluronate-rich extracellular environment when they invade nearby host tissues and that hyaluronate production by the tumor cells could increase this migration. These results suggest that hyaluronate may play a role in the aggressiveness of human melanoma cells.

[Cancer metastasis and extracellular matrix].

We investigated the roles of the extracellular matrix, especially of hyaluronan, in cancer metastasis. A positive relationship was shown between hyaluronan synthetic activity and the metastatic ability of cancer cells by manipulating regulation of gene expression of hyaluronan synthases. These techniques may provide future therapeutic methods for cancer metastasis.

Treatment options for women with stress urinary incontinence.

About one-quarter million surgical procedures are performed each year in the United States for stress urinary incontinence. After outlining the presentation and diagnostic evaluation of stress urinary incontinence, this review concentrates specifically on the numerous conservative management strategies and minimally invasive surgical options for women with this common complaint. In the evaluation of nursing home residents with incontinence, the Minimum Data Set and Resident Assessment Protocol facilitate nonspecialist evaluation and management. In healthy adults, the therapeutic implications of the physical examination of the pelvic floor, assessing for the presence and strength of the voluntary contraction of the pelvic floor muscles, are detailed as the basis for all conservative management strategies. Reports on the effectiveness of pelvic floor muscle reeducation and pelvic floor electrical stimulation vary substantially, as do long-term results of surgical interventions. Surgical management is highly effective in the appropriate candidate. The current theory and practice of surgical treatment of stress urinary incontinence are outlined, with certain caveats regarding the lack of long-term follow-up for newer less invasive techniques.

Receptor for hyaluronan-mediated motility and CD44 expressions in colon cancer assessed by quantitative analysis using real-time reverse transcriptase-polymerase chain reaction.

Receptor for hyaluronan (HA)-mediated motility (RHAMM) is a receptor for HA-mediated motility and its expression is correlated with malignancy of ras-transformed cells in that binding of HA to this receptor activates their migratory ability. CD44, a cell surface receptor for HA is also implicated in metastatic behavior of some cancer cells. In this study we examined the relationships of cancer progression with mRNA levels of RHAMM, CD44 (all forms), and exon 6 of CD44 using the real-time reverse transcriptase-polymerase chain reaction method in specimens of colon cancers at different diagnostic stages from 30 patients. Increased mRNA levels of RHAMM were observed in 29 specimens (97%), CD44s (all forms) in 21 specimens (70%), and its exon 6 in 19 specimens (63%) in comparison with those in the corresponding noncancerous tissue specimens. A statistically significant correlation between RHAMM expression and cancerous specimens at any of Dukes' stages A, B, and C was found, and the overexpression of CD44 mRNAs was confirmed in specimens at Dukes' stage C. Thus, our present study for the first time suggests that RHAMM expression may be a clinically useful indicator of colon cancer.

Metabolic activities of partially degenerated hypertrophic chondrocytes: gene expression of hyaluronan synthases.

Ultrastructural aspects of hypertrophic chondrocytes in hamster and mouse epiphysial cartilage were examined in relation to their metabolic activities. With the hypertrophic change, cytoplasmic vacuolization proceeded leaving the partially intact endoplasmic reticulum (ER). In the hypertrophic cells, cytoplasmic hyaluronan was stained with the biotinylated hyaluronan-binding region (b-HABR) of aggrecan, and mRNAs of hyaluronan synthase (Has 1, Has 2 and Has 3) were detected by in situ hybridization. When the epiphysial cartilage was cultured in the presence of 35S, 3H-GlcNAc, 3H-proline or 14C-palmitic acid, vacuolated late hypertrophic chondrocytes were labeled with these radioactive precursors. The evidence indicates that late hypertrophic chondrocytes are metabolically active, which appears to be essential for the enlargement of chondrocytes.

Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.

Relationship between hyaluronan production and metastatic potential of mouse mammary carcinoma cells.

To investigate the roles of hyaluronan produced by cancer cells in cancer metastasis, the metastatic potential of the highly metastatic mouse mammary carcinoma FM3A HA1 cell line was compared with those of hyaluronan-deficient mutant cells. Five different mutant clones showed markedly reduced hyaluronan production and lacked the ability to form hyaluronan-rich pericellular coats. These mutant clones displayed significant decreases in metastatic ability compared with the parental cells after i.v. injection into syngeneic mice. These results suggested that the decreased hyaluronan production caused not only the lack of matrix formation but also decreased metastatic potential of the cancer cells. Expression of mouse hyaluronan synthase 1 (HAS1) by transfection into HAS- cells defective in hyaluronan synthase activity rescued hyaluronan matrix formation as well as hyaluronan production. Lung metastasis after i.v. injection of HAS1 transfectants was also recovered significantly. The results provide direct evidence for the involvement of hyaluronan in cancer metastasis.

The gene structure and promoter sequence of mouse hyaluronan synthase 1.

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of lambda phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon-intron boundaries and all of the exons. This gene spans about 11kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon-intron organization was found between the genes of mouse HAS1 and Xenopus laevis DG42 which was recently identified as Xenopus hyaluronan synthase. The transcription initiation site was determined by rapid amplification of the cDNA ends (5'-RACE). Position +1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1.4 kb of the 5' flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.

Identification of hyaluronan-binding domains of aggrecan.

Aggrecan, a large cartilage proteoglycan, interacts with hyaluronan (HA), to form aggregates which function to resist compression in joints. The N-terminal region of aggrecan contains two structurally related globular domains, G1 and G2 separated by IGD domain. The G1 domain consists of three subdomains, A, B, and B', structural features characteristic to many other HA-binding proteoglycans. Here, we studied the interaction of aggrecan domains with HA using recombinant proteins expressed in 293 cells, an embryonal kidney cell line. Deglycosylation of the recombinant aggrecan fragment reduced the HA binding activity. We found that both the B and B' subdomains were required for HA binding and that a single module of A, B, or B' was unable to bind HA. The A subdomain increased the HA binding activity of the B-B' region. The G2 domain had no HA binding activity confirming previous reports. Studies of HA-binding properties using a BIAcoreTM biosensor system revealed that the KD of recombinant aggrecan fragment (AgW) consisting of G1, IGD, and G2 was 0.226 microM, whereas the KD of another HA-binding protein, native bovine link protein, is 0.089 microM. In contrast, AgMut11 which lacked subdomain A showed little HA binding activity. AgMut12 consisting of only B-B' had a 3.4-fold lower affinity and AgMut13 containing A-B-B' was 1.5-fold lower than AgW. These results suggest that carbohydrates are essential for high level aggrecan binding to HA and that the A subdomain of aggrecan functions in a cooperative manner with subdomains B and B'.

Chromosomal localization of the human and mouse hyaluronan synthase genes.

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17.HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.

Hyaluronic acid of wound fluid in adult and fetal rabbits.

Fetal wound healing proceeds without fibrosis or scar formation in contrast to adult wound healing. The mechanisms responsible for this remarkable process are mediated in part through a fetal wound extracellular matrix rich in hyaluronic acid (HA). Polyvinylalcohol sponge (PVA) wound implants were placed pervertebrally at 24 days' gestation in fetal (N = 118) rabbits and in adult (N = 44) rabbits, and then harvested at 1, 2, 3, 4, 5, and 7 days postwounding. To analyze the fetal and adult wound matrix, the HA concentration of wound fluid within the PVA sponge was quantitated using a newly developed assay. A significantly increased (P < .05) HA deposition on days 1 through 7 in the fetal wounds was found compared with the adult wound. These observations may suggest an important physiologic role in fetal wound healing by providing a more fluid and malleable matrix. These results, coupled with earlier findings of the lack of an acute inflammatory response in the fetus, further support the hypothesis that fetal response to injury is significantly different from adult response in this prescience of an implanted PVA sponge.