An enzymic 'latch' on a global carbon store.
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Biodegradation, Environmental↗
Biomedical subjects
C Freeman
Publications and source records attributed to C Freeman.
The drugs-violence nexus among American and Canadian youth.
This paper examines the relationship between drug use and violence among representative samples of students in the United States and Ontario, Canada. Canada has significantly lower levels of violent crime than the United States, but students report similar rates of drug use. Using logistic regression analysis, we find a similar relationship between drug use and violence among adolescents in the two countries. All the drugs considered--cannabis, cocaine, and alcohol binge drinking--are significantly related to violent behavior; whether the perpetrator or the victim. The most noteworthy difference may be that in Ontario, drug use appears to be even more highly correlated with violence than in the United States.
Adolescent↗
Identification of active-site residues of the pro-metastatic endoglycosidase heparanase.
Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).
Amino Acid Sequence↗
Effect of Morphine and incision length on bowel function after colectomy.
PURPOSE: Return of bowel function remains the rate-limiting factor in shortening postoperative hospitalization of patients with colectomies. Narcotics are most commonly used in the management of postoperative pain, even though they are known to affect gut motility. Narcotic use has been felt to be proportional to the length of the abdominal incision. The aim of this study was to determine whether return of bowel function after colectomy is directly related to narcotic use and to evaluate the effect of incision length on postoperative ileus. METHODS: A prospective evaluation of 40 patients who underwent uncomplicated, predominantly left colon and rectal resections was performed. Morphine administered by patient controlled analgesia was the sole postoperative analgesic. The amount of morphine used before the first audible bowel sounds, first passage of flatus and bowel movement, and incision length were recorded. Spearman correlation coefficients were calculated between all variables. RESULTS: The strongest correlation was between time to return of bowel sounds and amount of morphine administered (r = 0.74; P = 0.001). There were also significant correlations between morphine use and time to report of first flatus (r = 0.47; P = 0.003) and time to bowel movement (r = 0.48; P = 0.002). There was no relationship between incision length and morphine use or incision length and return of bowel function in the total group. CONCLUSIONS: Return of bowel sounds, reflecting small-intestine motility after colectomy, correlated strongly with the amount of morphine used. Similarly, total morphine use adversely affects colonic motility. Because no relationship with incision length was found, efforts to optimize the care of patients with colectomies should be directed less toward minimizing abdominal incisions and more toward diminishing use of postoperative narcotics.
Adolescent↗
An update on community-acquired pneumonia in adults.
Community-acquired pneumonia (CAP) is a leading cause of morbidity and mortality, despite effective therapies. Guidelines for CAP management vary widely in their approach. Resistance of S pneumoniae to penicillins and other antibiotics has prompted evaluation of the new fluoroquinolones.
Adult↗
13-week drinking water toxicity study of hydrogen peroxide with 6-week recovery period in catalase-deficient mice.
A GLP OECD guideline study was conducted to evaluate the subchronic toxicity of hydrogen peroxide (HP) when administered continuously in the drinking water of catalase-deficient (C57BL/6N) mice and reversibility of toxic effects. Groups of mice (15/sex/group) received solutions of 0, 100, 300, 1000 or 3000 ppm HP in distilled water for 13 weeks; five/sex/group continued on untreated distilled water for an additional 6 weeks. Animals drinking 3000 ppm HP exhibited depressed water and food consumption and body weight. Females drinking 1000 ppm HP had reduced water consumption with intermittent effects on food consumption, but no body weight effects. HP administration did not produce any mortality, clinical signs, hematological effects or organ weight effects on brain, liver, kidneys, adrenals, testes, heart or spleen. Total protein and globulin were depressed among high dose males. Mild to minimal duodenal mucosal hyperplasia was noted in animals receiving 1000 and 3000 ppm HP and one male receiving 300 ppm for 13 weeks. There were no other histopathological findings. All effects noted during the treatment period, including the duodenal hyperplasia, were reversible during the 6-week recovery period. Females dosed with 300-3000 ppm HP during the treatment period showed increased water consumption during the recovery period. The no-observed-effect level (NOEL), based on duodenal mucosal hyperplasia, is 100 ppm in drinking water or 26 and 37 mg/kg/day HP, respectively, for males and females.
Acatalasia↗
Developmental toxicity study of Aquacoat ECD ethylcellulose aqueous dispersion administered orally to rats.
A developmental rat toxicity study of Aquacoat((R)) ECD was performed as part of a program to evaluate the safety of the product. Groups of 25 presumed-pregnant Charles River Sprague-Dawley CD rats received doses of 0, 903, 2709 and 4515mg/kg/day (dry weight basis) of Aquacoat ECD administered undiluted once daily via oral gavage on days 6-15 of gestation. All surviving dams underwent caesarean sectioning on day 20 of gestation. Foetuses were weighed, sacrificed and subject to external, visceral and skeletal evaluations. No test material-related maternal deaths occurred; one high-dose female died on day 14 due to gavage error. The only treatment-related clinical sign noted among dams receiving 2709mg/kg/day and greater was pale faeces which was attributed to the presence of the test material in the faeces. No statistically significant differences were noted among the measured maternal parameters. Foetal sex ratios and body weights were similar in all groups. The results of external and visceral foetal evaluations revealed no treatment-related alterations. The only statistically significant findings noted during the skeletal evaluation were increased litter incidences of incompletely ossified or wavy ribs noted among foetuses receiving 4515mg/kg/day, and a significant increase in the litter incidence of thickened ribs at doses of 2709 and 4515mg/kg/day. Given the nature of these findings and the lack of effects on any other parameter measured in this study, they were not considered adverse effects of treatment. Under the conditions of this study, the maternal and foetal no-observed-adverse-effect level (NOAEL) is in excess of 4515mg/mg/day.
Abnormalities, Drug-Induced↗
Evidence that platelet and tumour heparanases are similar enzymes.
In order to enter tissues, blood-borne metastatic tumour cells and leucocytes need to extravasate through the vascular basal lamina (BL), a process which involves a battery of degradative enzymes. A key degradative enzyme is the endoglycosidase heparanase, which cleaves heparan sulphate (HS), an important structural component of the vascular BL. Previously, tumour-derived heparanase activity (which has been shown to be related to the metastatic potential of murine and human melanoma cell lines) was reported to cleave HS and be inhibited by heparin, as distinct from human platelet heparanase, which cleaved both substrates [Nakajima, Irimura and Nicolson (1988) J. Cell Biochem. 36, 157-167]. We recently reported the purification of human platelet heparanase and showed that the enzyme is a 50-kDa endoglucuronidase [Freeman and Parish (1998) Biochem. J. 330, 1341-1350]. We now report the purification and characterization of heparanase activity from highly metastatic rat 13762 MAT mammary adenocarcinoma and human HCT 116 colonic carcinoma cells and from rat liver using essentially the same procedure that was reported for purification of the human platelet enzyme. The rat 13762 MAT tumour enzyme, which has a native M(r) of 45 kDa when analysed by gel-filtration chromatography and by SDS/PAGE, was observed to be an endoglucuronidase that degraded heparin and HS to fragments of the same sizes as the human platelet enzyme does. N-deglycosylation of both the human platelet and rat 13762 MAT tumour enzymes gave, in each case, a 41-kDa band by SDS/PAGE analysis, demonstrating that the observed difference in M(r) between the platelet and tumour enzymes may have been due largely to differences in the relative amounts of N-glycosylation. Two peptides were isolated following Endoproteinase Lys-C digestion of both the human platelet and rat 13762 MAT tumour heparanases and were shown to be highly similar. Both the rat liver and human colonic carcinoma heparanases also degraded both heparin and HS to fragments of the same sizes as the human platelet enzyme does. Western-blot analysis of an SDS/PAGE gel using antibodies raised against human platelet heparanase demonstrated that human platelet, human tumour and rat tumour heparanases were immunochemically cross-reactive. In conclusion, because of the similarities in their sizes, substrate specificities, peptide sequences and immunoreactivities, we propose that heparanase activities present in human platelets, rat liver and in rat and human tumour cells are, in fact, mediated by a similar enzyme.
Amino Acid Sequence↗
Identification of sulfated oligosaccharide-based inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity.
Inhibitors of tumor angiogenesis and metastasis are rapidly emerging as important new drug candidates for cancer therapy. To facilitate the identification of such drugs, we recently developed novel and rapid in vitro assays for human angiogenesis and for the extracellular matrix-degrading enzyme heparanase, which has been implicated in tumor metastasis. In this study, sulfated oligosaccharides, which are structural mimics of heparan sulfate, were investigated as drug candidates because these compounds may interfere with heparan sulfate recognition by many angiogenic growth factors and may inhibit cleavage of heparan sulfate by heparanase. In the preliminary screening studies, it was found that inhibitory activity in both assay systems was critically dependent on chain length and degree of sulfation, highly sulfated linear oligosaccharides of five or more monosaccharides in length being the most active. However, two sulfated oligosaccharides stood out as potential antitumor drugs, phosphomannopentaose sulfate (PI-88) and maltohexaose sulfate, both of these compounds having the important property of simultaneously being potent inhibitors of in vitro angiogenesis and heparanase activity. Due to the ease of manufacture of the starting material, phosphomannopentaose, PI-88 was studied in more detail. PI-88 was shown to inhibit the primary tumor growth of the highly invasive rat mammary adenocarcinoma 13762 MAT by approximately 50%, inhibit metastasis to the draining popliteal lymph node by approximately 40%, and reduce the vascularity of tumors by approximately 30%, all of these effects being highly significant. Acute hematogenous metastasis assays also demonstrated that PI-88 was a potent (>90%) inhibitor of blood-borne metastasis. Thus, by the use of novel in vitro screening procedures, we have identified a promising antitumor agent.
Animals↗
There is no role for hyperfractionated radiotherapy in the management of children with newly diagnosed diffuse intrinsic brainstem tumors: results of a Pediatric Oncology Group phase III trial comparing conventional vs. hyperfractionated radiotherapy.
PURPOSE: In June 1992, POG began accrual to a phase III study, POG-9239, designed to compare the time to disease progression, overall survival, and toxicities observed in children with newly diagnosed brainstem tumor treated with 100 mg/m2 of infusional cisplatin and randomized to either conventional vs. hyperfractionated radiotherapy. METHODS AND MATERIALS: Patients eligible for study were those between 3 and 21 years of age with previously untreated tumors arising in the pons. Histologic confirmation of diagnosis was not mandatory, provided that the clinical and MRI scan findings were typical for a diffusely infiltrating pontine lesion. Treatment consisted of a six-week course of local field radiotherapy with either once a day treatment of 180 cGy per fraction to a total dose of 5400 cGy (arm 1) or a twice a day regimen of 117 cGy per fraction to a total dose of 7020 cGy (the second of the three hyperfractionated dose escalation levels of POG-8495) (arm 2). Because of previously reported poor results with conventional radiotherapy alone, cisplatin was included as a potential radiosensitizer in an attempt to improve progression-free and ultimate survival rates. Based on results of the phase I cisplatin dose escalation trial, POG-9139, 100 mg/m2 was chosen for this trial and was delivered by continuous infusion over a 120-hour period, beginning on the first day of radiotherapy and repeated during weeks 3 and 5. One hundred thirty eligible patients were treated on protocol, 66 on arm 1 and 64 on arm 2. RESULTS: The results we report are from time of diagnosis through October 1997. For patients treated on arm 1, the median time to disease progression (defined as time to off study) was 6 months (range 2-15 months) and the median time to death 8.5 months (range 3-24 months); survival at 1 year was 30.9% and at 2 years, 7.1%. For patients treated on arm 2, the corresponding values were 5 months (range 1-12 months) and 8 months (range 1-23 months), with 1- and 2-year survival rates at 27.0% and 6.7%, respectively. Evaluation of response by MRI at 4 or 8 wks post treatment was available in 108 patients and revealed a complete response in 1 patient of each Rx arm, a partial response (> 50% decrease in size) in 18 patients of arm 1 and 15 patients of arm 2, minimal to no response (stable) in 25 patients of arm 1 and 23 patients of arm 2, and progressive disease in 13 patients of arm 1 and 12 patients of arm 2. The pattern of failure was local in all patients. Morbidity of treatment was similar in both Rx arms, with no significant toxicity (including hearing loss) reported. Autopsy was performed in 6 patients, and confirmed the presence of extensive residual tumor in these cases. CONCLUSION: The major conclusion from this trial is that the hyperfractionated method of Rx 2 did not improve event-free survival (p = 0.96) nor did it improve survival (p = 0.65) over that of the conventional fractionation regimen of Rx 1, and that both treatments are associated with a poor disease-free and survival outcome.
Adolescent↗
Human HPA endoglycosidase heparanase. Map position 4q21.3.
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Base Sequence↗
Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis.
The endoglycosidase heparanase is an important in the degradation of the extracellular matrix by invading cells, notably metastatic tumor cells and migrating leukocytes. Here we report the cDNA sequence of the human platelet enzyme, which encodes a unique protein of 543 amino acids, and the identification of highly homologous sequences in activated mouse T cells and in a highly metastatic rat adenocarcinoma. Furthermore, the expression of heparanase mRNA in rat tumor cells correlates with their metastatic potential. Exhaustive studies have shown only one heparanase sequence, consistent with the idea that this enzyme is the dominant endoglucuronidase in mammalian tissues.
Adenocarcinoma↗
Human platelet heparanase: purification, characterization and catalytic activity.
Heparan sulphate (HS) is an important component of the extracellular matrix (ECM) and the vasculature basal lamina (BL) which functions as a barrier to the extravasation of metastatic and inflammatory cells. Platelet-tumour cell aggregation at the capillary endothelium results in activation and degranulation of platelets. Cleavage of HS by endoglycosidase or heparanase activity produced in relatively large amounts by the platelets and the invading cells may assist in the disassembly of the ECM and BL, and thereby facilitate cell migration. Using a recently published rapid, quantitative assay for heparanase activity towards HS [Freeman, C. and Parish, C.R. (1997), Biochem. J., 325, 229-237], human platelet heparanase has now been purified 1700-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The enzyme, which was shown to be an endoglucuronidase that degrades both heparin and HS, has a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS/PAGE. Platelet heparanase degraded porcine mucosal HS in a stepwise fashion from a number average molecular mass of 18.5 to 13, to 8 and finally to 4.5 kDa fragments as determined by gel filtration analysis. Bovine lung heparin was degraded from 8.9 to 4.8 kDa while porcine mucosal heparin was degraded from 8.1 kDa to 3.8 and finally to 2.9 kDa fragments. Studies of the enzyme's substrate specificity using modified heparin analogues showed that substrate cleavage required the presence of carboxyl groups, but O- and N-sulphation were not essential. Inhibition studies demonstrated an absolute requirement for the presence of O-sulphate groups. Platelet heparanase was inhibited by heparin analogues which also inhibited tumour heparanase, suggesting that sulphated polysaccharides which inhibit tumour metastasis may act to prevent both tumour cell and platelet heparanase degradation of endothelial cell surface HS and the basal laminar.
Animals↗
Para-aortic irradiation only appears to be adequate treatment for patients with Stage I seminoma of the testis.
PURPOSE: Results of treatment of patients with Stage I seminoma with orchiectomy and radiotherapy are excellent. Even without adjuvant radiotherapy, the relapse rate is only 15-20%; most of the patients fail in the retroperitoneum, with rare failures observed in the pelvis (0.5-2%). In 1991, we began a prospective study evaluating para-aortic lymph node radiation as the only adjuvant treatment for such patients. This paper reports our preliminary results. MATERIALS & METHODS: Between March 1991 and January 1996, 35 patients with histologically proven Stage I seminoma were entered in the study. Median age was 37.9 years (range: 27-65 years). A radical inguinal orchiectomy was performed in all patients. Staging workup consisted of a chest X-ray; B-HCG, alpha-fetoprotein, and CT scan of the abdomen and pelvis in all patients. Lymphangiogram was done in 23 (66%) of 35 patients for further evaluation of the retroperitoneal lymph nodes. Radiotherapy consisted of treatment to the para-aortic region only. Parallel opposed fields extending from the top of T11 to the bottom of L5 were used. The median field size was 8.7 x 21.8 cm (range: 7-11 x 18-26 cm). The median total dose, prescribed at midpoint, was 25 Gy given in 15 daily fractions of 1.66 Gy. Follow-up was performed every 3 months for the first year, every 4-5 months for the second and third years, and every 6 months thereafter. Chest X-ray, tumor markers, and CT scan of the pelvis were performed routinely as part of the follow-up investigation. RESULTS: At a median follow-up of 39.7 months (range: 16-74 months), 34 (97.1%) of 35 patients are alive with no evidence of disease for an overall actuarial survival rate of 97.1% at 5 years and a cause-specific actuarial survival rate of 100%. Treatment morbidity was limited to Grade I-II acute side effects in 18 (51.4%) of 35 patients. No late side effects were seen. CONCLUSION: From our preliminary results, adjuvant radiation treatment limited to the para-aortic lymph node region, without ipsilateral pelvic irradiation, appears to be adequate treatment for Stage I seminoma. Such an approach in our patients resulted in minimal toxicity and excellent disease-free survival.
Adult↗
Subchronic oral toxicity study of Aquacoat ECD ethylcellulose aqueous dispersion in the rat.
Groups of 20 male and 20 female Sprague-Dawley rats were administered undiluted Aquacoat ECD ethylcellulose aqueous dispersion by oral gavage at doses of 903, 2709 or 4515 mg/kg body weight/day (dry weight basis) for 90 days. Control animals received water at the same dosage volume as the high-dose group. Body weights and food consumption were recorded weekly. Blood was collected prior to study termination for haematology and clinical chemistry measurements. Survivors underwent complete necropsies on days 91 94. Selected organs were weighed and histologically examined. The only treatment-related clinical sign observed was pale faeces which was noted among males and females receiving 2709 and 4515 mg/kg/day Aquacoat ECD. No statistically significant differences in body weights, body weight gains, food consumption and organ weights were noted among males and females when compared with controls. No treatment-related effects in haematology parameters were noted. Significantly decreased total protein and globulin levels and increases in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in male rats receiving 2709 and 4515 mg Aquacoat ECD/kg/day were considered to be treatment related. No gross or microscopic lesions were attributed to Aquacoat ECD treatment. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for female rats is in excess of 4515 mg/kg/day: the NOAEL for male rats is 903 mg/kg/day.
Administration, Oral↗
Drug treatment for bulimia nervosa.
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Anticonvulsants↗
A rapid quantitative assay for the detection of mammalian heparanase activity.
Heparan sulphate (HS) is an important component of the extracellular matrix and the vasculature basal laminar which functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of HS by endoglycosidase or heparanase activity produced by invading cells may assist in the disassembly of the extracellular matrix and basal laminar, and thereby facilitate cell migration. Heparanase activity has previously been shown to be related to the metastatic potential of murine and human melanoma cell lines [Nakajima, Irimura and Nicolson (1988) J. Cell. Biochem. 36, 157-167]. To determine heparanase activity, porcine mucosal HS was partially de-N-acetylated and re-N-acetylated with [3H]acetic anhydride to yield a radiolabelled substrate. This procedure prevented the masking of, or possible formation of, new heparanase-sensitive cleavage sites as has been observed with previous methods of radiolabelling. Heparanase activity in a variety of tissues and cell homogenates including human platelets, colonic carcinoma cells, umbilical vein endothelial cells and rat mammary adenocarcinoma cells (both metastatic and non-metastatic variants) and liver homogenates all degraded the substrate in a stepwise fashion from 18.5 to approximately 13, 8 and finally to 4.5 kDa fragments, as assessed by gel-filtration analysis, confirming the substrate as suitable for the detection of heparanase activity present in a variety of cells and tissues. A rapid quantitative assay was developed with the HS substrate using a novel method for separating degradation products from the substrate by taking advantage of the decreased affinity of the heparanase-cleaved products for the HS-binding plasma protein chicken histidine-rich glycoprotein (cHRG). Incubation mixtures were applied to cHRG-Sepharose columns, with unbound material corresponding to heparanase-degradation products. Heparanase activity was determined for a variety of human, rat and murine cell and tissue homogenates. The highly metastatic rat mammary adenocarcinoma and murine lung carcinoma cell lines had four to ten times the heparanase activity of non-metastatic variants, confirming the correlation of heparanase activity with metastatic potential. Human cancer patients had twice the serum heparanase levels of normal healthy adults. The assay will be valuable for the determination of heparanase activity from a variety of tissue and cell sources, as a diagnostic tool for the determination of heparanase potential, and for the development of specific inhibitors of heparanase activity and metastasis.
Adult↗
Comparison of a monoclonal antibody-based IgM capture ELISA with a neutralization assay for assessing response to trivalent oral poliovirus vaccine.
Monoclone-based IgM capture ELISAs were developed for each of the three poliovirus serotypes and compared with a neutralization assay for detecting response to trivalent oral poliovirus vaccine among 224 infants. The IgM-based response rates were significantly higher than the neutralizing antibody-based rates: 95% versus 83% to poliovirus type 1, 99% versus 94% to poliovirus type 2, and 89% versus 59% to poliovirus type 3. IgM responses to the first vaccine dose were significantly associated between serotypes, suggesting that some of the discordance may reflect a heterotypic IgM response. When the response rates in 4 vaccine formulation groups were compared, group differences using the two assays were similar for poliovirus types 1 and 2 but not for type 3. Therefore, IgM results using these assays may not be adequate substitutes for neutralizing antibody results when determining vaccine response.
Animals↗