Kinetics and mechanism for reduction of anticancer-active tetrachloroam(m)ine platinum(IV) compounds by glutathione

trans -[PtCl₄(NH₃)(thiazole)] (1), trans-[PtCl₄(cha)(NH₃)] (2), cis-[PtCl₄(cha)(NH₃)] (3) (cha =cyclohexylamine), and cis-[PtCl₄(NH₃)₂] (4) has been investigatedat 25 °C in a 1.0 M aqueous medium at pH 2.0–5.0 (1) and 4.5–6.8 (2–4) using stopped-flow spectrophotometry. The redox reactions follow the second-order rate law , where k is a pH-dependent rate constant and [GSH]_tot the total concentration of glutathione. The reduction takes place via parallel reactions between the platinum(IV) complexes and the various protolytic species of glutathione. The pH dependence of the redox kinetics is ascribed to displacement of these protolytic equilibria. The thiolate species GS⁻ is the major reductant under the reaction conditions used. The second-order rate constants for reduction of compounds 1–4 by GS⁻ are (1.43±0.01)×10⁷, (3.86±0.03)×10⁶, (1.83±0.01)×10⁶, and (1.18±0.01)×10⁶M⁻¹s⁻¹, respectively. Rate constants for reduction of 1 by the protonated species GSH are more than five orders of magnitude smaller. The mechanism for the reductive elimination reactions of the Pt(IV) compounds is proposed to involve an attack by glutathione on one of the mutually trans coordinated chloride ligands, leading to two-electron transfer via a chloride-bridged activated complex. The kinetics results together with literature data indicate that platinum(IV) complexes with a trans Cl-Pt-Cl axis are reduced rapidly by glutathione as well as by ascorbate. In agreement with this observation, cytotoxicity profiles for such complexes are very similar to those for the corresponding platinum(II) product complexes. The rapid reduction within 1 s of the platinum(IV) compounds with a trans Cl-Pt-Cl axis to their platinum(II) analogs does not seem to support the strategy of using kinetic inertness as a parameter to increase anticancer activity, at least for this class of compounds. Received: 8 December 1999 / Accepted: 15 February 2000     

Diabetes-induced decrease in rat brain microsomal Ca2+-ATPase activity

The Ca(2+)-ATPase activity of rat brain microsomes was studied in streptozotocin (STZ)-induced diabetes. Male rats, 200-250 g, were rendered diabetic by injection of STZ (45 mg kg(-1) body weight) via the teil vein. Brain tissues were collected at 1, 4 and 10 weeks after diabetes was induced for determination of Ca(2+)-ATPase activity, lipid peroxidation and tissue calcium levels. Diabetic rats had significantly elevated blood glucose levels compared to controls. Blood glucose levels were 92.92 +/- 1.22 mg dl(-1) (mean +/- SEM) for the control group, 362.50 +/- 9.61 mg dl(-1) at 1 week and >500 mg dl(-1) at 4, 8 and 10 weeks for the diabetics. Enzyme activities were significantly decreased at 1, 4, 8 and 10 weeks of diabetes relative to the control group (p < 0.001). Ca(2+)-ATPase activity was 0.084 +/- 0.008 U l(-1), 0.029 +/- 0.005 U l(-1), 0.029 +/- 0.006 U l(-1), 0.033 +/- 0.003 U l(-1) and 0.058 +/- 0.006 U l(-1) (mean +/- SEM) at control, 1, 4, 8 and 10 week of diabetes respectively. The change in calcium levels in diabetic rat brain at 8 and 10 weeks of diabetes was significantly higher than that of the control group (p < 0.05). On the other hand lipid peroxidation measured as TBARS (thiobarbituric acid reactive substances) was significantly higher at 8 and 10 weeks of diabetes (p < 0.05). The increase in lipid peroxidation observed in diabetic rat brain may be partly responsible for the decrease in calcium ATPase activity.     

Effects of triazolopyrimidine on lipid peroxidation and nitric oxide levels in the corticosteroid-impaired healing of rat tracheal anastomoses

Corticosteroids are used to reduce the oedema and prevent scar tissue formation of the upper airways by their ability to inhibit influx of inflammatory cells, limit capillary permeability and block collagen synthesis in the early stages of wound healing. Triazolopyrimidine (Trapidil) is an antiplatelet agent that acts in part as a phosphodiesterase inhibitor and as a competitive inhibitor of the platelet-derived growth factor (PDGF) receptor. Trapidil, with its vasodilator and NO releasing effect may have some potential to diminish the tissue injury. This study was carried out to evaluate the effects of trapidil (triazolopyrimidine) on lipid peroxidation and nitric oxide in the corticosteroid-impaired healing of tracheal anastomoses. Thirty-four adult Wistar rats were divided into five groups. The animals underwent tracheal transection and primary anastomoses. The groups were assigned as follows: group I, control, (GI, n = 6); group II, sham, (GII, n = 6); group III, dexamethasone, 0.1 mg kg(-1) twice daily intramuscularly, (GIII, n = 8); group IV, trapidil, 6 mg kg(-1) twice daily intraperitoneally (GIV, n = 7); group V, dexamethasone, 0.1 mg kg(-1) plus trapidil, 6 mg kg(-1) twice daily (GV, n = 7), for 1 week. After 1 week, anastomotic healing was assessed by measurement of bursting pressure, evaluation of histopathology, measurement of MDA and nitrite/nitrate levels. In GIII, GIV and GV bursting pressures resulted in significantly reduced anastomotic strength compared to the controls (p < 0.001 for all groups). The difference between bursting pressures of GIII and GIV was not found to be statistically significant (p = 0.966). In regard to fibroblast proliferation and collagen content, a significant difference was found between GIII and GI (p < 0.01), A significant difference was also found when GIV and GV were compared to GIII (p < 0.01). MDA and nitrite/nitrate levels were found to be higher in GIII when compared to all other groups. MDA levels of GIV and GV rats were found to be lower than GIII (p < 0.001, for both groups). The nitrite/nitrate levels of GIV and GV rats were found to be lower than GIII (p < 0.05), and higher than GI (p < 0.001). Trapidil may be useful for its preventive effects on lipid peroxidation and possible increases in NO in cases with corticosteroid-impaired healing of trachea anastomoses.     

Exaggerated TSH responses to TRH in patients with goiter and 'normal' basal TSH levels

BACKGROUND/AIM: The availability of sensitive thyrotropin (TSH) assays decreased the diagnostic value of thyrotropin-releasing hormone stimulation tests (TRH-ST) in subclinical hypothyroidism. In this study we aimed to evaluate the relation between basal and stimulated serum TSH levels on TRH-ST and to determine the prevalence of patients with normal basal serum TSH and exaggerated TSH responses. METHODS: 179 patients (117 girls, 123 pubertal) with a median age of 12 (2.7-21.4) years who presented with goiter were enrolled and evaluated for their pubertal stage, height, thyroid autoimmunity, ultrasonography, thyroid function, and TRH-ST. Serum TSH concentrations were determined by sensitive assays. At TRH-ST, a peak serum TSH level >25 mIU/l was considered as an exaggerated response. RESULTS: 30 (17%) patients had an exaggerated TSH response. In patients with serum TSH levels between 2 and 4.68 mIU/l (upper half the normal range), an exaggerated TSH response was observed in 19.5%. A positive correlation between basal and TRH-stimulated TSH levels was determined (r = 0.536, p < 0.01). In patients with an exaggerated TSH response, 23 had normal (discordant) and 7 had high basal TSH levels (concordant). The mean basal serum TSH level was lower in the discordant group compared to the concordant group (p < 0.01). CONCLUSION: Basal serum TSH levels might not be sufficient for diagnosing subclinical hypothyroidism. Stimulated TSH levels on TRH-ST are valuable, especially when serum TSH concentrations are in the upper half of the normal range.     

Glucose stimulates the expression and activities of nitric oxide synthases in incubated rat islets: an effect counteracted by GLP-1 through the cyclic AMP/PKA pathway

We have examined the expression and activity of inducible nitric oxide synthase (iNOS) and the activity of neuronal constitutive NOS (ncNOS) in isolated rat pancreatic islets, stimulated by a hyperglycaemic concentration of glucose, and whether the NOS activities could be modulated by activation of the cyclic AMP/protein kinase A (cyclic AMP/PKA) system in relation to the insulin secretory process. Here, we show that glucose stimulation (20 mmol/l) induces iNOS and increases ncNOS activity. No iNOS is detectable at basal glucose levels (3.3 mmol/l). The addition of glucagon-like-peptide 1 (GLP-1) or dibutyryl-cAMP to islets incubated with 20 mmol/l glucose results in a marked suppression of iNOS expression and activity, a reduction in ncNOS activity and increased insulin release. The GLP-1-induced suppression of glucose-stimulated iNOS activity and expression and its stimulation of insulin release is, at least in part, PKA dependent, since the PKA inhibitor H-89 reverses the effects of GLP-1. These observations have been confirmed by confocal microscopy showing the glucose-stimulated expression of iNOS, its suppression by GLP-1 and its reversion by H-89 in -cells. We have also found that the NO scavenger cPTIO and the NOS inhibitor L-NAME potentiate the insulin response to glucose, again suggesting that NO is a negative modulator of glucose-stimulated insulin release. We conclude that the induction of iNOS and the increase in ncNOS activity caused by glucose in rat islets is suppressed by the cyclic AMP/PKA system. The inhibition of iNOS expression by the GLP-1/cyclic AMP/PKA pathway might possibly be of therapeutic potential in NO-mediated -cell dysfunction and destruction.     

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-L-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K(m) and V(max) values for the same substrate by Lineweaver-Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K(m) values at optimum pH and 20 degrees C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective V(max) values at optimum pH and 20 degrees C were 0.875 micromol/L min, 0.186 micromol/L min, 0.214 micromol/L min and 0.253 micromol/L min with the same substrate. The K(i) and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations.     

Population differentiation in the redshank (<Emphasis Type="Italic">Tringa totanus</Emphasis>) as revealed by mitochondrial DNA and amplified fragment length polymorphism markers

The redshank (Tringa totanus) is declining throughout Europe and to implement efficient conservation measures, it is important to obtain information about the population genetic structure. The aim of the present study was two-fold. First, we analysed the genetic variation within and between populations in the Baltic region in southern Scandinavia. Evidence of genetic structure would suggest that different populations might require separate management strategies. Second, in an attempt to study large-scale genetic structure we compared the Baltic populations with redshanks from northern Scandinavia and Iceland. This analysis could reveal insights into phylogeography and long-term population history. DNA samples were collected from six breeding sites in Scandinavia presumed to include two subspecies (totanus and britannica) and a further sample from Iceland (subspecies robusta). Two methods were used to study the population genetic structure. Domain II and III of the mitochondrial control region was analysed by DNA sequencing and nuclear DNA was analysed by screening amplified fragment length polymorphism (AFLP) markers. Mitochondrial DNA showed no variation between individuals in domain II. When analysing an 481 bp fragment of domain III seven haplotypes were found among birds. On the basis of mtDNA sequences, redshanks showed some evidence of a recent expansion from a bottlenecked refugial population. Bayesian analyses of AFLP data revealed a significant genetic differentiation between suggested subspecies but not between populations within the Baltic region. Our results indicate that populations of redshanks in Europe constitute at least three separate management units corresponding to the recognised subspecies.     

Isolation and characterization of a white rot fungus <Emphasis Type="Italic">Bjerkandera</Emphasis> sp. strain capable of oxidizing phenanthrene

Strain BOL13 was selected from 18 fungal strains isolated from an oil-spill contaminated site in Oruro, Bolivia. It was identified as a basidiomycete with high homology to Bjerkandera. The fungus degraded 100 mg phenanthrene l⁻¹ at 0.17 mg l⁻¹ d⁻¹ at 30 °C at pH 7. During phenanthrene degradation, a maximum manganese peroxidase activity of 100–120 U l⁻¹ was measured after 10 days of incubation. The ability of Bjerkandera sp. to produce lignin-modifying enzymes and to oxidize phenanthrene under various pH and temperature conditions was confirmed.     

The effect of selenium and/or vitamin E treatments on radiation-induced intestinal injury in rats

Cytotoxic effects of ionizing radiation on gastrointestinal epithelium may be related to oxidative stress. In this study, we wanted to investigate the effects of selenium, vitamin E and selenium plus vitamin E pretreatments prior to whole abdominal irradiation on intestinal injury. Irradiation caused increased lipid peroxide and decreased GSH levels in the intestine. Intestinal superoxide dismutase and glutathione peroxidase activities were increased, but glutathione transferase activity decreased following irradiation. Selenium and/or vitamin E pretreatments ameliorated these disturbances in prooxidant-antioxidant balance. This amelioriation has been verified with histopathological findings. These results indicate that antioxidant pretreatments prior to irradiation may have some beneficial effects against irradiation-induced intestinal injury.     

Lymphatic malformation causing intractable chylorrhagia

Lymphatic malformation is a developmental error usually noticed at birth or in early childhood. Lesions of the upper leg and lower trunk are the most difficult to remove, because they are often the largest ones encountered and also because they tend to extend proximally into the retroperitoneal tissues. Chyle reflux, usually associated with lymphedema of the extremity, has not been reported to be caused by lymphatic malformation. We report a case of intrapelvic retroperitoneal lymphatic malformation with an extension of gluteal-thigh soft-tissue involvement causing intractable chylorrhagia. The tumor was subtotally excised, and the defect was closed by a distally based, peninsular latissimus dorsi myocutaneous flap. The flap served both as a filling material and as a "bridge" between the residual tumor, including abnormal lymphatics, and normal lymph flow.