An improved method for the structural profiling of keratan sulfates: analysis of keratan sulfates from brain and ovarian tumors.
A previously developed method for the structural fingerprinting of keratan sulfates (Brown et al., Glycobiology, 5, 311-317, 1995) has been adapted for use with oligosaccharides fluorescently labeled with 2-aminobenzoic acid following keratanase II digestion. The oligosaccharides are separated by high-pH anion-exchange chromatography on a Dionex AS4A-SC column. This methodology permits quantitative analysis of labeled oligosaccharides which can be detected at the sub-nanogram ( approximately 100 fmol) level. Satisfactory calibration of this method can be achieved using commercial keratan sulfate standards. Keratan sulfates from porcine brain phosphocan and human ovarian tumors have been examined using this methodology, and their structural features are discussed. (+info)
Effect of fasting on temporal variation in the nephrotoxicity of amphotericin B in rats.
Evidence for temporal variation in the nephrotoxicity of amphotericin B was recently reported in experimental animals. The role of food in these variations was determined by studying the effect of a short fasting period on the temporal variation in the renal toxicity of amphotericin B. Twenty-eight normally fed and 28 fasted female Sprague-Dawley rats were used. Food was available ad libitum to the fed rats, while the fasted animals were fasted 12 h before and 24 h after amphotericin B injection to minimize stress for the animals. Water was available ad libitum to both groups of rats, which were maintained on a 14-h light, 10-h dark regimen (light on at 0600 h). Renal toxicity was determined by comparing the levels of excretion of renal enzyme and the serum creatinine and blood urea nitrogen (BUN) levels at the time of the maximal (0700 h) or the minimal (1900 h) nephrotoxicity after the intraperitoneal administration of a single dose of dextrose (5%; control group) or amphotericin B (50 mg/kg of body weight; treated group) to the rats. The nephrotoxicities obtained after amphotericin B administration at both times of day were compared to the nephrotoxicities observed for time-matched controls. In fed animals, the 24-h urinary excretion of N-acetyl-beta-D-glucosaminidase and beta-galactosidase was significantly higher when amphotericin B was injected at 0700 and 1900 h. The excretion of these two enzymes was reduced significantly (P < 0.05) in fasting rats, and this effect was larger at 0700 h (P < 0.05) than at 1900 h. The serum creatinine level was also significantly higher (P < 0.05) in fed animals treated at 0700 h than in fed animals treated at 1900 h. Fasting reduced significantly (P < 0.05) the increase in the serum creatinine level, and this effect was larger in the animals treated at 0700 h. Similar data were obtained for BUN levels. Amphotericin B accumulation was significantly higher (P < 0.05) in the renal cortexes of fed rats than in those of fasted animals, but there was no difference according to the time of injection. These results demonstrated that fasting reduces the nephrotoxicity of amphotericin B and that food availability is of crucial importance in the temporal variation in the renal toxicity of amphotericin B in rats. (+info)
Endometrial lysosomal enzyme activity in normal cycling endometrium.
The objective of this study was to evaluate the possible role of four lysosomal enzymes in endometrial function and remodelling during the normal menstrual cycle by fluorimetric measurement (acid phosphatase, N-acetyl-beta-D-glucosaminidase, alpha-L-fucosidase and alpha-D-mannosidase). A prospective study was conducted of 45 endometrial biopsies obtained from women with normal menstrual cycles. Activity of all four enzymes was identified in human endometrium. Activity of acid phosphatase and N-acetyl-beta-D-glucosaminidase was relatively high, whilst that of alpha-L-fucosidase and alpha-D-mannosidase was low. There was no significant change in the activity of any of the four enzymes from the proliferative to the secretory phase of the cycle. This study suggests that the activity of these enzymes remains constant throughout a major portion of the normal cycle. (+info)
Immediate and early renal function after living donor transplantation.
BACKGROUND: In order to assess the immediate renal function after living donor transplantation, renal function was compared in eight renal allograft recipients and their living related kidney donors during the first 24 h after transplantation. METHODS: Substantial and comparable intraoperative volume loading with Ringer's acetate and mannitol was performed together with the administration of frusemide. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were estimated by the clearances of inulin and p-aminohippurane, respectively. Tubular reabsorptive function and injury were estimated from the clearance of lithium, the fractional excretion of sodium and the urinary excretion of N-acetyl-beta-glucosaminidase. RESULTS: One hour after completion of surgery, GFR (54 +/- 7 ml/min) and ERPF (294 +/- 35 ml/min) were only 30% lower in the grafts than in the remaining donor kidneys, increasing to similar levels within 3 h. Only minor tubular dysfunction and injury were revealed in the grafted kidneys, and these tended to normalize within 24 h. CONCLUSIONS: By the present transplantation procedure comprising short ischaemia time and substantial volume expansion combined with mannitol and frusemide administration, kidneys from living donors regain nearly normal function within a few hours after transplantation. (+info)
Active site characterization of the exo-N-acetyl-beta-D- glucosaminidase from thermotolerant Bacillus sp. NCIM 5120: involvement of tryptophan, histidine and carboxylate residues in catalytic activity.
The exo-N-acetyl-beta-d-glucosaminidase (EC 126.96.36.199) from thermotolerant Bacillus sp. NCIM 5120 is a homotetramer with a molecular mass of 240000 kDa. Chemical modification studies on the purified exo-N-acetyl-beta-d-glucosaminidase revealed the involvement of a single tryptophan, histidine and carboxylate, per monomer, in the catalytic activity of the enzyme. Spectral analysis and maintenance of total enzyme activities indicated that N-acetylglucosamine (competitive inhibitor) and p-nitrophenyl-N-acetyl-beta-d-glucosaminide (substrate) prevented the modification of a single essential tryptophan, histidine and carboxylate residue. Kinetic parameters of partially inactivated enzyme (by NBS/HNBB) showed the involvement of tryptophan in substrate binding while that of histidine (by photooxidation/DEPC) and carboxylate (by EDAC/WRK) in catalysis. The Bacillus sp. NCIM 5120 exo-N-acetyl-beta-d-glucosaminidase deviates from the reported N-acetyl-beta-d-glucosaminidases and beta-hexosaminidases that utilize anchimeric assistance in their hydrolytic mechanism. (+info)
Sanfilippo type B syndrome (mucopolysaccharidosis III B): allelic heterogeneity corresponds to the wide spectrum of clinical phenotypes.
Sanfilippo B syndrome (mucopolysaccharidosis IIIB, MPS IIIB) is caused by a deficiency of alpha-N-acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes leads to degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. Age of onset and rate of progression vary considerably, whilst diagnosis is often delayed due to the absence of the pronounced skeletal changes observed in other mucopolysaccharidoses. Cloning of the gene and cDNA encoding alpha-N-acetylglucosaminidase enabled a study of the molecular basis of this syndrome. We were able to identify 31 mutations, 25 of them novel, and two polymorphisms in the 40 patients mostly of Australasian and Dutch origin included in this study. The observed allellic heterogeneity reflects the wide spectrum of clinical phenotypes reported for MPS IIIB patients. The majority of changes are missense mutations; also four nonsense and nine frameshift mutations caused by insertions or deletions were identified. Only five mutations were found in more than one patient and the observed frequencies are well below those observed for the common mutations in MPS IIIA. R643C and R297X each account for around 20% of MPS IIIB alleles in the Dutch patient group, whilst R297X, P521L, R565W and R626X each have a frequency of about 6% in Australasian patients. R643C seems to be a Dutch MPS IIIB allele and clearly confers the attenuated phenotype. One region of the gene shows a higher concentration of mutations, probably reflecting the instability of this area which contains a direct repeat. Several arginine residues seem to be 'hot-spots' for mutations, being affected by two or three individual base pair exchanges. (+info)
Effect of age on urinary excretion of N-acetyl-beta-D-glucosaminidase.
To examine the relationship between the concentrations of urinary NAG and age, we measured ratios of urinary N-acetyl-beta-D-glucosaminidase (NAG) to urinary creatinine (NAG index) in 137 healthy subjects, aged from 19 to 88 years. The study is also designed to evaluate the relationship between urinary NAG and blood pressure. The subjects were divided into 7 subgroups, according to their age (< 30, 30-39, 40-49, 50-59, 60-69, 70-79, 80 or more years). There was a positive correlation between NAG index and age (r = 0.36; P < 0.001). The regression equation relating NAG index (y) to age (x) was y = 0.065x + 0.97. The mean NAG indexes for the 7 subgroups divided by age were significantly different (P < 0.01). There was a positive correlation between NAG index and systolic blood pressure (r = 0.18; P < 0.05), but was not between diastolic blood pressure and NAG index. In multiple regression analysis, age and BUN significantly correlated with NAG index (r = 0.32; P < 0.01, r = 3.3; P = 0.07, respectively), although there was no correlation between systolic blood pressure and NAG index. This cross-sectional study showed a clear elevation in NAG index with age. The rate of elevation was 0.65 per decade. Urinary excretion of NAG may be unrelated to blood pressure. (+info)
A quantitative study of pinocytosis and lysosome function in experimentally induced lysosomal storage.
The highly pinocytic epithelial cells of the visceral yolk sac from 17.5-day rat conceptuses were used as a model in which to induce engorgement of the vacuolar system by direct accumulation of substances that are not hydrolysed by lysosomal enzymes. The ultra-structural appearances of these cells in pregnant animals that 24-48h before had received intraperitoneal injections of Triton WR-1339, polyvinylpyrrolidone, dextran or sucrose revealed gross abnormalities that were confined to the vacuolar system; in comparison with normal tissue the number, and in some cases the size, of vacuoles was increased, leading to close packing within the apical cytoplasm and distortion of the normal rounded shape. By culturing yolk sacs in vitro, rates of ingestion of 125I-labelled polyvinylpyrrolidone and of 125I-labelled bovine serum albumin were determined, together with the rate of digestion of the labelled protein. The rates of exocytosis of 125I-labelled polyvinylpyrrolidone and of lysosomal enzymes were also determined. No significant differences between normal and highly vacuolated tissues were found. Apparently marked vacuolation of these cells by these agents is without significant effect on pinocytosis, exocytosis or intralysosomal proteolysis. (+info)