Sexual assault. Effects of the research process on all the participants.
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Adolescent↗
Biomedical subjects
D M Templeton
Publications and source records attributed to D M Templeton.
Structure and metabolism of multiple heparan sulphate proteoglycans synthesized by the isolated rat glomerulus.
Metabolism of biosynthetically [35S]sulphate-labelled heparan sulphate proteoglycan (HSPG) was studied in the isolated glomerulus. Chromatography and electrophoresis resolved HS into 5 components, designated HS1a, HS1b, and HS2 to HS4 in order of increasing Kd. Both HS1a (250 kDa) and HS1b (130 kDa) are present in the glomerular basement membrane and have glycosaminoglycan chains of 25-45 kDa. Chemical analysis of glycosaminoglycan chains indicated a similar content of 50% N-sulphation and 30% 6-O-sulphation on the hexosamine residues of all HSs, with the remaining 20% of sulphate likely at the 2-O-position of uronic acid residues. By pulse-chase analysis, the basement-membrane fraction was found to have a half-life of residency in the glomerulus of 37 h. Both HS1a and HS1b are mainly released intact into the medium and are not further broken down in that compartment. In contrast, HS2 is almost completely released into the medium immediately after synthesis and is not normally recovered from the tissue. It is a 90-kDa HSPG with a hydrophobic core protein and glycosaminoglycan chains similar in size to those of HS1. In addition to these larger PGs, HS3 and HS4 represent glycosaminoglycan chains with little or no core protein. HS1a, HS1b and HS2 were iodinated and deglycosylated. Each has a 30-kDa core protein in addition to 18 kDa of chondroitinase ABC- and nitrous-acid-resistant O-linked carbohydrate. This suggests the possibility of a single core protein with variable glycosylation and destination. HS1a has 5-6 glycosaminoglycan chains, HS1b 2-3 and HS2 1-2. We propose that basement-membrane HSPG (HS1a and HS1b) and a related, underglycosylated secreted HSPG (HS2) are the major HSPGs synthesized by the isolated glomerulus. Other molecular species may represent discrete steps in the turnover of basement-membrane HSPG.
Animals↗
Reduction of tissue iron stores and normalization of serum ferritin during treatment with the oral iron chelator L1 in thalassemia intermedia.
In patients with thalassemia intermedia in whom hyperabsorption of iron may result in serious organ dysfunction, an orally effective iron-chelating drug would have major therapeutic advantages, especially for the many patients with thalassemia intermedia in the Third World. We report reduction in tissue iron stores and normalization of serum ferritin concentration after 9-month therapy with the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in a 29-year-old man with thalassemia intermedia and clinically significant iron overload (SF 2,174 micrograms/L, transferrin saturation 100%; elevated AST and ALT, abnormal cardiac radionuclide angiogram) who was enrolled in the study with L1 75 mg/kg/day after he refused deferoxamine therapy. L1-Induced 24-hour urinary iron excretion during the first 6 months of therapy was (mean +/- SD, range) 53 +/- 30 (11 to 109) mg (0.77 mg/kg), declining during the last 3 months of L1 to 24 +/- 14 (13-40) mg (0.36 mg/kg), as serum ferritin decreased steadily to normal range (present value, 251 micrograms/L). Dramatic improvement in signal intensity of the liver and mild improvement in that of the heart was shown by comparison of T1-weighted spin echo magnetic resonance imaging with images obtained immediately before L1 administration was observed after 9 months of L1 therapy. Hepatic iron concentration decreased from 14.6 mg/g dry weight of liver before L1 therapy to 1.9 mg/g liver after 9 months of therapy. This constitutes the first report of normalization of serum ferritin concentration in parallel with demonstrated reduction in tissue iron stores as a result of treatment with L1. Use of L1 as a therapeutic option in patients with thalassemia intermedia and iron overload appears warranted.
Adult↗
Speciation of tissue and cellular iron with on-line detection by inductively coupled plasma-mass spectrometry.
Iron accumulating to excess in tissues of humans and animal models occurs mainly as complexes with transferrin, ferritin, other hemoproteins, and insoluble hemosiderin particles. To determine the distribution of Fe amongst these molecular species, we have used inductively coupled plasma-mass spectrometry as a means of on-line, isotope-specific detection for their liquid chromatographic separation. The stable isotope 57Fe is a suitable isotope for monitoring the Fe content of each fraction, and its availability at high isotopic enrichment makes it an attractive choice for tracer studies when the use of a radioisotope is undesirable, e.g., in human subjects. The detection system offers the advantages of high sensitivity (detection limits in the parts per billion range), a wide dynamic range (linearity of the calibration curve over several orders of magnitude), and on-line analysis facilitating real-time evaluation of the chromatographic separation, in addition to isotope-specific information. The Fe distributions in healthy rat livers, liver and heart tissue from Fe-loaded human subjects, and human hepatocyte cultures are reported. The ferritin:hemosiderin ratio in these samples is shown to be an indicator of the degree of Fe loading and correlates well with that determined by Zeeman-corrected electrothermal atomic absorption as an alternative means of detection.
Animals↗
Effects of CdCl2 and Cd-metallothionein on cultured mesangial cells.
Cadmium is a potent nephrotoxin known to cause damage to the proximal tubular epithelium in vivo. The renal glomerulus is less frequently a target and is sensitive to Cd in vitro. We have previously presented evidence that the mesangial cell is a major target for Cd2+ toxicity in the isolated glomerulus (D. M. Templeton and N. Chaitu, Toxicology 61, 119-133, 1990). The present study was undertaken to investigate the sensitivity to Cd of rat mesangial cells grown in homogeneous culture. At a concentration of 1 microM, Cd2+ was a potent inducer of its binding protein metallothionein (MT). Cd2+ inhibited DNA synthesis in these cells with an EC50 of 5.4 +/- 0.4 microM, while preinduction of MT with Zn2+ was protective, raising the EC50 to 17.6 +/- 0.7 microM Cd2+. DNA synthesis in these cells is especially sensitive to Cd2+; only at concentrations of 20 microM Cd2+ and higher were significant effects on cell viability, attachment, and protein synthesis observed. Renal function depends in part on synthesis of specialized matrices by glomerular cells. Synthesis of both matrix and secreted proteoglycans was specifically affected by Cd2+ with an EC50 of about 10 microM for proteoglycan sulfation. We also investigated the effects of Cd-MT on these parameters. Contrary to observations that extracellular Cd-MT is a potent nephrotoxin in vivo, we were unable to demonstrate any effects of Cd-MT on DNA and protein synthesis at Cd concentrations below 60 microM in the cultured cells. Nor did Cd-MT at these concentrations affect DNA or protein synthesis in LLC-PK1 cells, a proximal tubule cell line. This was not due to failure of the cells to take up Cd because they accumulated comparable amounts of Cd whether it was provided as CdCl2 or Cd-MT. We conclude that ionic Cd2+ is the most toxic form of this metal to cultured mesangial cells. While these cells respond to micromolar concentrations of Cd2+ by increasing their content of metallothionein, presumably a protective response, only slightly higher levels may impair the regenerative capacity of mesangial cells, in addition to interfering with the specialized function of matrix synthesis.
Animals↗
Evaluation of the integrity of the blood-brain barrier after meningeal trauma.
RATIONALE AND OBJECTIVES: The authors used magnetic resonance imaging (MRI) techniques to examine the effect of meningeal trauma produced by cisterna magna puncture on the integrity of the blood-cerebrospinal fluid barrier (BCB) in a rat model. METHODS: Intravenous gadolinium-DTPA (Gd-DTPA), a relaxation rate modifier which normally does not cross the BCB, was used as a probe to follow leakage of fluid across the BCB. After Gd-DTPA injection, cerebrospinal fluid (CSF) serial samples were obtained through the needle used to create the experimental trauma. These samples were subsequently examined in vitro by MRI to obtain their T1 relaxation rates and assayed by mass spectrometry for gadolinium and elemental iron concentrations. RESULTS: The iron levels reflected the severity of puncture-related subarachnoid hemorrhage. Rats with ongoing meningeal damage showed significantly higher CSF levels of gadolinium and significantly higher CSF T1 relaxation rates than controls at all samples times over 1 hour after the puncture. Blood in the CSF could not explain these changes because the CSF iron levels did not significantly differ from control levels. CONCLUSIONS: Intravenously administered Gd-DTPA can gain access to the subarachnoid space through minor defects in the BCB and cause significant increases in CSF T1 relaxation rates.
Animals↗
Growth failure and bony changes induced by deferoxamine.
We reviewed the linear growth and growth plate morphology in all children with homozygous beta thalassemia followed in Toronto, for whom monthly height percentiles were available before, and for a 36-month period after, the initiation of nightly subcutaneous deferoxamine therapy. All patients were less than 7 years of age when begun on deferoxamine, and had received nightly deferoxamine for a minimum of 36 months. Marked abnormalities of the metaphyseal growth plate were readily observed in the distal ulnar, radial, and tibial metaphyses in 11 of 37 patients in whom a significant decline in mean height percentile was also noted. (In 10 of these 11 patients, height was less than the 15th percentile after 36 months.) These 11 patients had received a significantly greater (p less than 0.025) initial and average daily dose of deferoxamine, and had maintained a significantly lower (p less than 0.025) mean serum ferritin concentration over the 36 months, than the remainder of the cohort. To determine whether deferoxamine played a causative role in growth failure, growth in patients who began deferoxamine before the age 2 years was compared to that of patients who began after age 5 years, for the period between 2 and 5 years of age. Only patients begun on deferoxamine prior to age 2 years demonstrated a significant (p less than 0.01) decline in height percentile by the third year, implicating deferoxamine therapy as the cause of growth failure. We conclude that both the decline in height percentile and the bony changes observed in well-chelated patients are directly related to deferoxamine therapy.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkaline Phosphatase↗
Conserved charge of glomerular and mesangial cell proteoglycans: possible role of amino acid-derived sulphate.
Sulphation of proteoglycans was studied in isolated glomeruli and cultured rat mesangial cells. Both preparations produced heparan, dermatan, and chondroitin sulphates, recoverable both from the tissue layers and the conditioned media. The proportion of heparan sulphate made by mesangial cells was independent of the age of the culture, but declined in later passage. These preparations differed from several other nontransformed cell types studied to date in that the degree of proteoglycan sulphation was independent of the concentration of inorganic sulphate provided. Even when no exogenous sulphate was added, sulphation-dependent charge density of newly synthesized proteoglycans was conserved. Both isolated glomeruli and cultured mesangial cells produced proteoglycans with 35S-labelled sulphate esters when [35S]methionine was provided as the sole source of labelled sulphate. Conversion of methionine to cysteine and subsequent oxidation of organic sulphate via the sulphinyl pyruvate pathway is the only mechanism known in eukaryotic cells that can account for this observation. We conclude that facile oxidation of sulphur-containing amino acids can contribute to sulphation of glomerular proteoglycans and may serve to sustain the charge density of these multifunctional molecules when the supply of inorganic sulphate is otherwise compromised.
Amino Acids↗
Increased microalbuminuria in diabetic rats is independent of angiotensin II or glomerular proteoglycan synthesis.
Increased microalbuminuria is seen early in rats with both streptozotocin-induced and genetic (Bio-Breeding) diabetes. This study examines the roles of angiotensin II-dependent mechanism(s) and sulfation of glomerular proteoglycans in this phenomenon, as both processes have been implicated by several lines of circumstantial evidence. Anionic sites in the glomerular basement membrane, attributed to the presence of heparan sulfate, were quantitated by polyethyleneimine staining at 15, 21, and 70 days of diabetes in rats treated with streptozotocin, with or without insulin, and at 70 days in the Bio-Breeding rats. All diabetic rats developed increased microalbuminuria: control, 0.08 +/- 0.03 microgram/mL glomerular filtration rate, mean +/- SD; streptozotocin without insulin at 15 days, 0.92 +/- 0.06 microgram/mL (p < 0.05); streptozotocin with insulin at 21 days, 0.61 +/- 0.37 microgram/mL (p < 0.05 vs. control). At 70 days, both the Bio-Breeding and the streptozotocin rats sustained their microalbuminuria to the same degree (p < 0.05 vs. control). Enalapril (250 mg/L) in the drinking water of diabetic animals did not reduce the microalbuminuria. Although the polyethyleneimine-stained heparan sulfate sites decreased significantly in the streptozotocin rats, they remained unchanged in the Bio-Breeding rats. To determine the cause of reduced heparan sulfate staining, the in vitro synthesis and degree of sulfation of proteoglycans by glomeruli isolated from control and streptozotocin diabetic rat kidneys were compared. The amount of heparan sulfate synthesis and degree of sulfation were unchanged in diabetic glomeruli, although lower incorporation into the extracellular matrix and greater secretion into the medium were noted.(ABSTRACT TRUNCATED AT 250 WORDS)
Albuminuria↗
Proteoglycans in cell regulation.
Proteoglycans are a diverse group of proteins carrying one or more glycosaminoglycan side chains linked to the protein as O-glycosides. Our appreciation of these structures has matured from a curiosity about unusual structural glycoproteins, to confer upon them a central role in cell biology. The major classes of glycosaminoglycans are heparan sulfate and heparin, chondroitin and dermatan sulfates, keratan sulfate and hyaluronic acid. The latter is unique in that it does not contain sulfate residues, and appears to be synthesized, at least sometimes, free of a carrier protein. There is now a wealth of information on the ability of these structures to influence the growth and development of cells and tissues. Many direct and specific effects of proteoglycans will undoubtedly be found, and there are likely to be indirect effects of the glycosaminoglycans relating to their polyelectrolyte nature. Convincing arguments that biological activity resides in certain proteoglycan core proteins are also appearing. The following discussion concerns the role of proteoglycans in the regulation and action of autocrine and polypeptide growth factors, direct mitogenic and antimitogenic actions of glycosaminoglycans, the role of these structures in regulating gene expression, and the biological activities of proteoglycan core proteins. The probable role of proteoglycans in normal glomerular cell function, and in progressive renal disease, will be presented as a harbinger of the significant role we can expect them to play in diagnosis and therapy in the near future.
Amino Acid Sequence↗
Quantifying cadmium and lead in whole blood.
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Cadmium↗
Toxicological significance of metallothionein.
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Alkylating Agents↗
Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry. II. Rapid survey method for profiling trace elements in body fluids.
A rapid survey of the elements in biological materials, covering most of the elements in the periodic table, is possible by using available software for semi-quantitative analysis (SEMI-QUANT) by inductively coupled plasma-mass spectrometry. The procedure takes 5 min after sample preparation and gives results with a precision (CV) of approximately 20%. At a 10-fold dilution, 13 elements can be consistently and reliably detected in serum and 15 elements in whole-blood samples. At present the most important limitation of this method is mass overlap by polyatomic species for some elements of interest (e.g., Cr, Mn, and V). However, for the set of elements that can be reliably determined at endogenous concentrations, including Li, B, Mg, Fe, Cu, Zn, Rb, and Sr, the rapid scanning capability may be useful. Although matrix effects limit the direct interpretation of the semi-quantitative output, reasonable estimates of concentration are attainable by using matrix-matched standards or by adding a multielement standard to an aliquot from one sample in the set. We also present an example of determination of 25 elements in saliva from a patient with extensive dental work: Components of many of his dental alloys were readily identified. The method may also prove useful for screening multiple toxic exposures to heavier elements, such as Pb, Tl, Cd, and Hg.
Adult↗
Cadmium uptake by cells of renal origin.
We compared the ability of rat glomerular mesangial cells and LLC-PK1 cells to take up Cd2+ from solution. The former are smooth muscle-like cells of mesenchymal origin, the latter an established line of proximal tubular epithelium. Both cells, as well as primary glomerular epithelia, accumulated Cd2+ against a concentration gradient in a time-dependent manner. Uptake by mesangial cells obeyed a Michaelis model with an apparent Km of 19 microM and could be described by an initial rapid step of surface binding followed by rate-limiting internalization. In contrast, uptake by LLC-PK1 cells was non-saturable under accessible concentrations of Cd2+ and internalization was not a necessary consequence of association with the cell surface. In several other cell types, Cd2+ uptake has been shown to be inhibited by blockage of cell-surface sulfhydryl groups. In contrast, uptake by neither mesangial nor LLC-PK1 cells was depressed by N-ethylmaleimide, which actually enhanced the surface binding and to a lesser extent the uptake by the LLC-PK1 cell line. Neither depended on metabolic energy for uptake or utilized Ca2+ channels. The internalization process was temperature dependent and was obliterated at 2 degrees C. In mesangial cells, this allowed direct observation of the internalization event from a presaturated surface pool. The rate of this process was consistent with the Vmax calculated from the Michaelis model. Surface binding and uptake were decreased by binding of Cd2+ to serum proteins and albumin and were much less dependent on the presence of low molecular weight components of serum. Therefore, these cells may be especially sensitive to Cd2+ at concentrations encountered in vivo because of the low protein content of the plasma ultrafiltrate. Surface binding of Cd2+ to mesangial cells was suppressed by competing divalent ions following the order of the Irving-Williams series (Mn less than Co less than Ni less than Cu greater than Zn), although Zn2+ showed the greatest effect on internalization. In LLC-PK1 cells, Zn2+ and Cu2+ were both effective in decreasing Cd2+ uptake. We conclude that Cd2+ uptake by the tubular epithelial cells is rapid and independent of specific cell surface interactions, whereas uptake by rat mesangial cells follows binding to a specific surface ligand saturating at about 1.5 x 10(7) copies/cell. In both types of cells the uptake appears quite specific for Cd2+ and shows some cross-reactivity with other metal cations explicable by competitive ligand binding.
Animals↗
Comparison of oral iron chelator L1 and desferrioxamine in iron-loaded patients.
The efficacy of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) was compared with that of subcutaneous desferrioxamine in 26 patients with transfusional iron overload. Immediately after red-cell transfusion, 20 patients were randomised to receive either desferrioxamine (50 mg/kg daily as a 12 h subcutaneous infusion), or L1 (50 mg/kg daily by mouth). Patients were evaluated during treatment with the other drug after transfusion the next month. Mean (SD) daily urinary iron excretion was lower during L1 than during desferrioxamine (12.3 [6.7] vs 18.2 [15.3] mg/day). In 5 patients the dose of L1 was raised from 50 to 75 mg/kg daily; mean urinary iron excretion rose from 13.8 (7.0) mg/day to 26.7 (17.8) mg/day, comparable with that during desferrioxamine (24.9 [24.3] mg/day). Faecal iron excretion rose slightly over baseline in 6 patients studied during L1 administration (from 8.5 [0.9] mg/day to 12.2 [0.9] mg/day). Pharmacokinetic studies showed an elimination half-life for L1 of 117-237 min. Studies in dogs and in volunteers showed no absorption of the L1-iron complex, excluding a contribution of absorption of intraluminal complexes of L1 and food iron to urinary iron excretion. Further animal toxicity testing is needed before L1 can be studied in a broader group of patients.
Adolescent↗
Effects of divalent metals on the isolated rat glomerulus.
Glomerular changes are prominent in metal-induced nephrotoxicity, and the glomerulus may be a primary site of damage in some cases. In order to separate direct effects of metal ions from those occurring secondary to systemic or post-glomerular toxicity, we have studied the effects of divalent salts of Hg, Cd, Cu, Zn, Mn, Ni and Co on several metabolic parameters in freshly isolated rat glomeruli. The concentration of the metal ion causing a 50% reduction in the incorporation of [3H]leucine into protein over 16 h varied from 30 microM with Cd to about 2 mM with Ni and Mn. The log of the concentration was significantly correlated with the softness of the metal ion, indicating greater toxicity of ions such as Cd2+ and Hg2+ that prefer sulphur as a ligand. Incorporation of [3H]sulphate into proteoglycans was affected in a comparable manner to total protein synthesis. However, the softer metals caused a preferential decrease in the production of more highly charged dermatan sulphate, indicative of an effect on mesangial cells. Glomeruli were found to contain about 50 fmol each of glutathione upon isolation, and this value decreased with time. However, Cd and Zn caused increases in total glomerular glutathione, and this phenomenon was inhibited by buthionine sulphoximine, indicating that synthesis of new glutathione was responsible for the increases. These studies demonstrate the usefulness of the isolated glomerulus for investigating mechanisms of nephrotoxicity, and indicate that the mesangial cell may be a preferred target for some metal ions.
Animals↗
Variability of proteoglycan expression in the isolated rat glomerulus.
The sulphation of proteoglycans in freshly isolated rat glomeruli was studied by biosynthetic labelling with [35S]sulphate. At least 75% of the observed sulphation requires de novo synthesis of core protein and proceeds at a constant rate over at least 40 h. Heparan and dermatan sulphate proteoglycans (HSPG and DSPG, respectively) are the two major species produced, with only minor amounts (less than 5%) of chondroitin sulphate labelled under these conditions. Several factors affect the population distribution of labelled material. When glomeruli were obtained from rats 6 weeks of age, HSPG accounted for 75 +/- 9% of tissue proteoglycan sulphated over 16 h. When older rats (12-14 weeks) were used, only 32 +/- 10% of label was associated with HSPG, DSPG accounting for the remainder. Production of HSPG is sulphate-dependent, increasing relative to DSPG with increasing sulphate, up to physiological concentrations. However, the net charge-density of sulphated material is conserved even at the lowest concentrations of sulphate. This may reflect the importance of electrostatic properties in the function of glomerular proteoglycans. The production of HSPG increases relative to DSPG with time following isolation and this effect is more dramatic in glomeruli from younger rats. However, reciprocal changes in production of HSPG and DSPG sustain a constant rate of sulphation. This phenomenon may arise from interdependency of the glomerular epithelial and mesangial cells with respect to regulation of proteoglycan synthesis.
Age Factors↗
Evaluation of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in iron-loaded patients.
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Administration, Oral↗