Rescue Agent Research Articles

Substitution reactions of [Pt(terpy)X]2+ with some biologically relevant ligands. Synthesis and crystal structure of [Pt(terpy)(cyst-S)](ClO4)2.0.5H2O and [Pt(terpy)(guo-N7)](ClO4)2.0.5guo.1.5H2O.

Substitution reactions of the complexes [Pt(terpy)(H(2)O)](2+), [Pt(terpy)(cyst-S)](2+) and [Pt(terpy)(guo-N(7))](2+), where terpy = 2,2':6',2"-terpyridine, cyst = L-cysteine and guo = guanosine, with some biologically relevant ligands such as inosine (INO), inosine-5'-monophosphate (5'-IMP), guanosine-5'-monophosphate (5'-GMP), l-cysteine, glutathione, thiourea, thiosulfate and diethyldithiocarbamate (DEDTC), were studied in aqueous 0.10 M NaClO(4) at pH 2.5 and 6.0 using variable-temperature and -pressure stopped-flow spectrophotometry. The reactions of [Pt(terpy)(H(2)O)](2+) with INO, 5'-IMP and 5'-GMP showed that these ligands are very good nucleophiles. The second order rate constants varied between 4 x 10(2) and 6 x 10(2) M(-1) s(-1) at 25 degree C. The [Pt(terpy)(cyst-S)](2+) complex is unreactive towards nitrogen donor nucleophiles, and cysteine cannot be replaced by N(7) from INO, 5'-IMP and 5'-GMP. However, sulfur donor nucleophiles such as thiourea, thiosulfate and diethyldithiocarbamate could displace the Pt-cysteine bond. Diethyldithiocarbamate is the best nucleophile and the order of reactivity is: thiourea < thiosulfate < DEDTC with rate constants of 0.936 +/- 0.002, 5.99 +/- 0.02 and 8.88 +/- 0.07 M(-1) s(-1) at 25 degree C, respectively. The reactions of [Pt(terpy)(guo-N(7))](2+) with sulfur donor ligands showed that these nucleophiles could substitute guanosine from the Pt(ii) complex, of which diethyldithiocarbamate and thiosulfate are the strongest nucleophiles. The tripeptide glutathione is also a very efficient nucleophile. Activation parameters (Delta H(++), Delta S(++) and Delta V(++)) were determined for all reactions. The crystal structures of [Pt(terpy)(cyst-S)](ClO(4))(2).0.5H(2)O and [Pt(terpy)(guo-N(7))](ClO(4))(2).0.5guo.1.5H(2)O were determined by X-ray diffraction. Crystals of [Pt(terpy)(cyst-S)](ClO(4))(2).0.5H(2)O are orthorhombic with the space group P2(1)2(1)2(1), whereas [Pt(terpy)(guo-N(7))](ClO(4))(2).0.5guo.1.5H(2)O crystallizes in the orthorhombic space group P2(1)2(1)2. A typical feature of terpyridine complexes can be found in both molecular structures: the Pt-N (central) bond distance, 1.982(7) and 1.92(2) A, respectively, is shorter than the other two Pt-N distances, being 2.043(7) and 2.034(7) A in [Pt(terpy)(cyst-S)](ClO(4))(2).0.5H(2)O and 2.03(2) and 2.04(2) A in [Pt(terpy)(guo-N(7))](ClO(4))(2).0.5guo.1.5H(2)O, respectively. In both crystal structures two symmetrically independent cations representing different conformers are present in the asymmetric unit. The results are analysed in reference to the antitumour activity of Pt(II) complexes, and the importance of the rescue agents are discussed.

Metabolism of dexrazoxane (ICRF-187) used as a rescue agent in cancer patients treated with high-dose etoposide.

The study was undertaken to determine the metabolism of dexrazoxane (ICRF-187) to its one-ring open hydrolysis products and its two-rings opened metal-chelating product ADR-925 in cancer patients with brain metastases treated with high-dose etoposide. In this phase I/II trial dexrazoxane was used as a rescue agent to reduce the extracerebral toxicity of etoposide. Dexrazoxane and its one-ring open hydrolysis products were determined by HPLC and ADR-925 was determined by a fluorescence flow injection assay. The two one-ring open hydrolysis intermediates of dexrazoxane appeared in the plasma at low levels upon completion of dexrazoxane infusion and then rapidly decreased with half-lives of 0.6 and 2.5 h. A plasma concentration of 10 micro M ADR-925 was also detected at the completion of the dexrazoxane i.v. infusion period, indicating that dexrazoxane was rapidly metabolized in vivo. A plateau level of 30 micro M ADR-925 was maintained for 4 h and then slowly decreased. The pharmacokinetics of dexrazoxane were found to be similar to other reported data in other settings and at lower doses. The rapid appearance of ADR-925 in plasma may make ADR-925 available to be taken up by heart tissue and bind free iron. These results suggest that the dexrazoxane intermediates are enzymatically metabolized to ADR-925 and provide a pharmacodynamic basis for the antioxidant cardioprotective activity of dexrazoxane.

Lipoic acid as a rescue agent for adriamycin-induced hyperlipidemic nephropathy in rats

Adriamycin [ADR] is a cell independent model of glomerular injury. Hyperlipidemia associated with nephrotic syndrome may play a role in the deterioration of renal function. The study was carried out with adult male albino rats of Wistar strain comprising six rats in each group. Rats were fed with a standard rat pellet diet and were given access to water ad libitum. Group I rats served as controls (0.5 ml/day/rat i.p). Group II rats received ADR (1 mg/kg body weight/day) intravenously through the tail vein. Group III rats were given lipoic acid [LA] (35 mg/kg body weight/day) intraperitoneally. Group IV rats received LA 24 hours prior to the administration of ADR. ADR administered rats showed a significant increase ( p < 0.05) in serum lipids and lipoprotein fractions. However, the kidney of ADR administered rat showed a decline ( p < 0.05) in the content of phospholipids and free fatty acids. Significant ( p < 0.05) alterations were also observed in the activities of lipid metabolizing enzymes Pre-treatment with LA reverted the levels of lipid profiles and serum lipoproteins to near normal. The activities of lipid metabolizing enzymes also showed considerable changes on LA pre-treatment. This study highlights the antilipemic action of LA and its role as a protective agent for hyperlipidemia, associated with ADR induced nephrotic syndrome.

The radiosensitising effect of gemcitabine and the influence of the rescue agent amifostine in vitro

In this study, the radiosensitising effect of different concentrations of gemcitabine and the combination of gemcitabine/radiotherapy with the rescue agent amifostine was investigated in different human tumour cell lines. The cells were treated with gemcitabine (0–8 nM) for 24 h prior to radiation (0–8 Gy). Amifostine (ami) and alkaline phosphatase (AP) were added 30 min before radiation. Cell survival was determined 7 or 8 days after radiation treatment by the sulforhodamine B (SRB) test. For ECV304 cells, the dose enhancement factor (DEF) varied from 1.39 to 2.98 after treatment with 1–6 nM gemcitabine. FaDu, H292, A549 and CAL-27 seemed to be less sensitive, with DEFs ranging from 1.02 to 2.67. These cells were also less sensitive to the cytotoxic effects of single-agent gemcitabine. Amifostine with AP clearly showed a protective effect in combination with gemcitabine/radiotherapy. In H292 cells, the protection factor (PF) of amifostine after treatment with gemcitabine and radiotherapy varied from 1.64 to 1.86. In ECV304 cells, the PF varied from 2.20 to 2.29. In conclusion, a clear concentration- and cell line-dependent radiosensitising effect of gemcitabine was observed in all cell lines. Amifostine with AP showed protection against the radiosensitising effect of gemcitabine. If the protection in vivo indeed occurs selectively in normal tissues, then amifostine could prevent or strongly minimise the increased toxicity resulting from the radiosensitising effect of the combination of gemcitabine and radiotherapy, without influencing the antitumour effect.

Postoperative Management and Complications in Living-related Liver Transplantation

Living-related liver transplantation is widely accepted as a treatment for patients with end-stage liver disease, with survival rates of up to 80%. Liver transplant recipients are at risk for the same postoperative complications as any patient undergoing a major intraabdominal operation, in addition to several complications specific to this procedure. Maintenance immunosuppression relies principally on administration of tacrolimus and methylprednisolone. Nevertheless, approximately 36% of liver transplant recipients suffer acute rejection in the early posttransplant period and require bolus steroid therapy as a rescue agent. Vascular complications, including hepatic arterial thrombosis and portal vein thrombosis, are additional major problems. When they occur in the immediate postoperative period, they can produce fulminant hepatic necrosis requiring retransplantation, so intensive anticoagulation therapy is needed as prophylaxis against these vascular complications. If thrombosis of the hepatic artery or portal vein is diagnosed early in the postoperative course, emergency thrombectomy with reanastomosis should be attempted. Outflow obstruction by hepatic vein stenosis sometimes causes liver dysfunction, pleural effusion, and hepatosplenomegaly. Percutaneous transhepatic or transjugular approached hepatic vein dilatation is very useful in case of hepatic vein stenosis. Recipients are generally immunocompromised secondary to immunosuppressive therapy and their poor clinical condition and are at high risk for postoperative infection. Infection is a major cause of morbidity and the most common cause of death in liver transplant recipients. Antibiotic, antifungal, and antiviral agents are used empirically, and serologic examinations and bacterial investigations of blood, sputum, stool, urine, and discharge from drains should be performed as well as antibiotic sensitivity tests when necessary. Other complications related to the operation are intraabdominal bleeding, bile leakage, biliary anastomotic stenosis, and intestinal perforation. The postoperative course of liver transplant recipients with these complications depends on making an accurate diagnosis promptly and initiating appropriate management. Postoperative complications of living-related liver transplantation are protean, so it is very important to communicate with professionals in each specialized field to ensure optimal treatment.

Humanized anti-CD154 antibody therapy for the treatment of allograft rejection in nonhuman primates.

The anti-CD154 antibody hu5C8 prevents acute allograft rejection and prolongs allograft survival after withdrawal of therapy in nonhuman primates. This study describes the use of hu5C8 as a rescue agent for rejection developing after the withdrawal of hu5C8. Twelve rhesus monkeys that had received renal allografts under hu5C8 induction and subsequently rejected were studied. Rescue with hu5C8 was analyzed based on the histological character of the rejection (acute versus chronic) and whether conventional therapy was received at the time of rescue or induction. The diagnosis of rejection and response to therapy was based on allograft function and histology. Four monkeys that had acute rejection associated with conventional immunosuppression and hu5C8 were not reversed by hu5C8 rescue. Four animals with isolated chronic rejection following prolonged rejection-free survival after the withdrawal of hu5C8 did not respond to hu5C8 rescue therapy. Hu5C8 rescue therapy effectively reversed acute rejection occurring in two monkeys after hu5C8 withdrawal. One of two animals with combined acute on chronic rejection responded to hu5C8 rescue therapy. Hu5C8 effectively reverses acute but not chronic allograft rejection and appears to have no synergistic effect with conventional rescue agents.

Rescue of hypogonadotropic hypogonadism-causing and manufactured GnRH receptor mutants by a specific protein-folding template: misrouted proteins as a novel disease etiology and therapeutic target.

In the present study, we demonstrate pharmacological rescue (assessed by ligand binding and restoration of receptor coupling to effector) of five naturally occurring GnRH receptor (GnRHR) mutants (T(32)I, E(90)K, C(200)Y, C(279)Y, and L(266)R), identified from patients with hypogonadotropic hypogonadism, as well as rescue of other defective receptors intentionally manufactured with internal or terminal deletions or substitutions at sites expected to be involved in establishment of tertiary receptor structure. The pharmacological agent used is a small, membrane-permeant molecule, originally designed as an orally active, nonpeptide receptor antagonist, but is believed to function as a folding template, capable of correcting the structural defects caused by the mutations and thereby restoring function. After rescue, this agent can be demonstrably removed. The rescued receptor, now stabilized in the plasma membrane, couples ligand binding to activation of the appropriate effector system. For comparison, low-, intermediate-, or high-affinity peptide antagonists of GnRHR (that do not penetrate the cell) were unable to effect rescue, as was a nonbinding peptidomimetic congener of the rescue agent; this latter effect demonstrates specificity of the rescue agent. Our findings, taken in concert with an earlier study showing rescue of a mutant by modifications to the receptor structure that enhance plasma membrane expression of the GnRHR, suggest that mutant GnRHRs have frequently not lost intrinsic functionality and are subject to rescue by techniques that enhance membrane expression. The present findings demonstrate the efficacy of an approach based on pharmacological rescue and suggest the basis of new approaches for intervention in this and similar diseases.

Bayesian population model of methotrexate to guide dosage adjustments for folate rescue in patients with breast cancer.

Methotrexate (MTX) infusions may induce severe side-effects, and alkaline hydration along with folinic acid rescue is a common way to reduce such toxic risks. The purpose of this study was to develop an adaptive rescue strategy based upon the early detection of patients with impaired MTX elimination. In this study, we propose a simple population-based Bayesian approach for predicting MTX plasma concentration from a limited number of samples, so as to adapt both duration and dosage of the rescue agent to be used next. Ten kinetic profiles obtained after 10 courses of MTX (1.5 g/m2) in seven patients with inflammatory breast cancer were used to establish the population pharmacokinetic parameters (Cl, 8.16 L/h; t1/2, 12.7 h). This population was next involved in the Bayesian estimation of MTX individual pharmacokinetic parameters from only two blood samples (T24 and T36 h), thus allowing one to forecast the elimination of this drug by predicting MTX levels at 48 h. According to the MTX concentrations predicted during the elimination phase, folinic acid rescue was then prolonged in patients likely to be overexposed. The Bayesian estimation presented in this study was an easy and convenient method to efficiently detect patients with impaired MTX elimination in routine clinical practice. This information enabled the introduction of strategies for minimizing the risk of severe drug toxicity.

Kinetic fluorimetric determination of the antineoplastic methotrexate (MTX) in human serum

A kinetic study of the oxidation of methotrexate (MTX) in acidic medium and in the presence of potassium permanganate has been made on the basis of the fluorescence–time curves. A kinetic method for the determination of MTX was developed with a range of application between 0.22 and 3.30 μM. The proposed kinetic method permits us to determine MTX in human serum and to avoid the natural fluorescence of the serum. A detection limit of 0.18 μM was calculated in the presence of ascorbic acid as activator. Only 100 s per sample is necessary for the analysis. The interference of pteridin derivatives and the rescue agent folinic acid (leucovorin) was tested.

Chemical rescue in catalysis by human carbonic anhydrases II and III.

The maximal velocity of catalysis of CO(2) hydration by human carbonic anhydrase II (HCA II) requires proton transfer from zinc-bound water to solution assisted by His 64. The catalytic activity of a site-specific mutant of HCA II in which His 64 is replaced with Ala (H64A HCA II) can be rescued by exogenous proton donors/acceptors, usually derivatives of imidazole and pyridine. X-ray crystallography has identified Trp 5 as a binding site of the rescue agent 4-methylimidazole (4-MI) on H64A HCA II. This binding site overlaps with the "out" position in which His 64 in wild-type HCA II points away from the zinc. Activation by 4-MI as proton donor/acceptor in catalysis was determined in the dehydration direction using (18)O exchange between CO(2) and water and in the hydration direction by stopped-flow spectrophotometry. Replacement of Trp 5 by Ala, Leu, or Phe in H64A HCA II had no significant effect on enhancement by 4-MI of maximal rate constants for proton transfer in catalysis to levels near 10(5) s(-1). This high activity for chemical rescue indicates that the binding site of 4-MI at Trp 5 in H64A HCA II appears to be a nonproductive binding site, although it is possible that a similarly effective pathway for proton transfer exists in the mutants lacking Trp 5. Moreover, the data suggest that the out position of His 64 considered alone is not active in proton transfer in HCA II. In contrast to isozyme II, the replacement of Trp 5 by Ala in HCA III abolished chemical rescue of k(cat) by imidazole but left k(cat)/K(m) for hydration unchanged. This demonstrates that Trp 5 contributes to the predominant productive binding site for imidazole, with a maximal level for the rate constant of proton transfer near 10(4) s(-1). This difference in the susceptibility of CA II and III to chemical rescue may be related to the more sterically constrained and electrostatically positive nature of the active site cavity of CA III compared with CA II. The possibility of nonproductive binding sites for exogenous proton donors offers an explanation for the unusually low value of the intrinsic kinetic barrier obtained by application of Marcus theory to chemical rescue of H64A HCA II.

Substitution reactions of [Pt(dien)Cl]+, [Pt(dien)(GSMe)]2+, cis-[PtCl2(NH3)2] and cis-[Pt(NH3)2(GSMe)2]2+ (GSMe =S-methylglutathione) with some sulfur-bonding chemoprotective agents

Sulfur-containing compounds are used as rescue agents to protect normal tissue from the toxic side effects of cis-[PtCl2(NH3)2] (cisplatin) without affecting its anti-tumour activity. Reactions of the model complexes [Pt(dien)Cl]+ (1) and [Pt(dien)(GSMe)]2+ (2) (GSMe = S-methylglutathione) with the rescue agents glutathione (GSH), thiourea, thiosulfate, and diethyldithiocarbamate (DDTC) have been studied in a 1.0 mol dm−3 aqueous perchlorate medium at 37 °C and pH 7.30 using stopped-flow and conventional UV/VIS spectrophotometry. The reactions of 1 with 2-mercaptoethanesulfonate (mesna) and 2-mercaptoethylamine (cysteamine), which is the parent compound of 2-(3-aminopropylamino)ethylphosphorothioic acid (WR2721), and of cis-[Pt(NH3)2(GSMe)2]2+ with thiosulfate have also been studied. The reactions between cisplatin and DDTC, thiourea and thiosulfate were investigated in an unbuffered 0.10–0.05 mol dm−3 chloride medium at 37 °C. All reactions follow the rate law: −d[complex]/dt = k2[Pt(II)][Nu] where k2 denotes a second-order rate constant and [Nu] is the total concentration of nucleophile. The rate law indicates that the reactions proceed via a direct nucleophilic substitution pathway. The rescue agents display a relatively narrow range of reactivity towards 1, mesna < Hcysteamine < GSH < thiourea < thiosulfate < DDTC with rate constants k2 of 0.156 ± 0.001, 0.22 ± 0.01, 0.237 ± 0.003, 1.17 ± 0.01, 5.57 ± 0.04, and 8.0 ± 0.1 mol−1 dm3 s−1, respectively. The sequence is consistent with the report that DDTC is the most effective rescue agent discovered so far when administered after cisplatin. The activation parameters ΔH≠ and ΔS≠ have been determined and the products of the reaction of 1 with the rescue agents have been characterised.

Sirolimus for rescue and primary immunosuppression in transplanted children receiving tacrolimus.

The role of sirolimus (SRL) as a rescue agent (n=42) and as a component of primary immunosuppression (n=8) was evaluated in a mixed population of 50 transplanted children receiving tacrolimus (liver: 26, heart: 5, intestinal: 5, liver-intestine: 9, lung: 1, bone marrow: 1, liver-kidney: 1, multivisceral: 1). Rescue indications for tacrolimus (TAC) failure were recurrent acute rejection and acute rejection complicating withdrawal of immunosuppression in posttransplant lymphoproliferative disorder (PTLD). Rescue indications for TAC toxicity were nephrotoxicity, pancreatitis, seizures, hypertrophic cardiomyopathy, and graft-versus-host disease. Mean age at rescue was 11.5 years and mean follow-up was 204 (range 18-800) days. As primary immunosuppression, SRL+TAC prevented early acute rejection in 7/8 children. The indication for rescue resolved in 33/42 children. In children with TAC toxicity, this was associated with decrease in TAC doses by 50%, significant improvements in renal function, and continuing decline in Epstein-Barr virus (EBV) viral load in PTLD patients. Serious adverse events led to discontinuation of SRL in 9/42 rescue patients, 3 of them also experienced acute rejection. Three additional children also experienced acute rejection on SRL therapy (overall incidence 6/50, 12%). Pharmacokinetic analysis in the first week of SRL administration suggested a short half-life (11.8+/-5.5 hr, n=21). SRL and reduced-dose TAC may achieve adequate immunosuppression without compromising renal function or enhancing EBV viremia significantly.

Pentoxifylline Rescue Preserves Lung Function in Isolated Canine Lungs Injured With Phorbol Myristate Acetate

We hypothesized that pentoxifylline, administered after phorbol myristate acetate (PMA), would diminish the severity of lung injury. Animal research laboratory. Comparative study. Mongrel dogs (n = 33). Baseline measurements were obtained from the isolated blood-perfused dog lung lobes after 1 h of stable perfusion and ventilation. Four different measures of lung compliance were obtained along with WBC and neutrophil counts. Pulmonary vascular resistance (PVR) and capillary filtration coefficient (Kf) were calculated, and the ratio of a normalized maximal enzymatic conversion rate to the Michaelis-Menten constant (Amax/Km) was used to assess perfused capillary surface area. The control lobes (n = 8) were ventilated and perfused for an additional 40 min while the injured lobes (n = 17) received PMA (0.1 microg/mL of perfusate). The pentoxifylline-protected lobes (n = 8) were treated with pentoxifylline (1 mg/mL of perfusate) 10 min after injury with PMA. All measurements were then repeated. The three groups did not differ significantly at baseline. The control lobes remained relatively stable over time. The injured lobes demonstrated marked deterioration in compliance: 8.79 +/- 0.7 to 5.97 +/- 0.59 mL/cm H(2)O (p < 0.05) vs 10.1 +/- 1.0 to 8.07 +/- 0.72 mL/cm H(2)O and 9.6 +/- 1.1 to 9.9 +/- 0.85 mL/cm H(2)O in the control and protected lobes, respectively. Both groups receiving PMA had similar drops in WBC and neutrophil counts, but the pentoxifylline-protected lobes had preservation of all four compliance measures. PVR increased from 37.8 +/- 1.8 to 118.6 +/- 12.7 cm H(2)O/L/min (p < 0.05) in the injured lobes vs 35.4 +/- 0.5 to 36.3 +/- 2.8 cm H(2)O/L/min and 40.4 +/- 0.04 to 46.7 +/- 2.8 cm H(2)O/L/min (p < 0.05) in the control and protected lobes, respectively. Kf increased < 25% in the protected group but more than tripled in the injured group. Amax/Km dropped from 559 +/- 36 to 441 +/- 33 mL/min (p < 0.05) in the injured lobes vs 507 +/- 14 to 490 +/- 17 mL/min and 609 +/- 34 to 616 +/- 37 mL/min in the control and pentoxifylline-protected lobes, respectively. The use of pentoxifylline as a rescue agent prevented the PMA-induced deterioration of lung compliance, vascular integrity, and endothelial metabolic function in this acute lung injury model, despite significant pulmonary neutrophil sequestration.

Tacrolimus versus cyclosporine after lung transplantation: a prospective, open, randomized two-center trial comparing two different immunosuppressive protocols

Background: The need for better immunosuppressive protocols after lung transplantation led us to investigate tacrolimus (Tac) in combination with mycophenolate mofetil (MMF) and steroids or cyclosporine (CsA) in combination with MMF and steroids in a prospective, open, randomized trial after lung transplantation. Methods Between September 1997 and April 1999, 50 lung transplant recipients were randomized to receive either Tac ( n = 26) or CsA ( n = 24) in combination with MMF and steroids. All patients underwent induction therapy with rabbit antithymocyte globulin (ATG) for 3 days. Freedom from acute rejection (AR), patient survival, infection episodes, and side effects were monitored. Results There was no difference in patient demographics between the two groups. Six-month and 1-year survival was similar (84.6% and 73.1% in the Tac group vs 83.3% and 79.2% in the CsA group). Freedom from AR at 6 months and 1 year after lung transplantation was slightly higher in the Tac group (57.7% and 50% vs 45.8% and 33.3%, p = not significant [n.s.]), whereas the number of treated rejection episodes per 100 patient days in the Tac group was significantly lower (0.225 vs 0.426, p < .05). Four patients in the CsA group had to be switched to Tac. Two patients in the CsA group had to be retransplanted. Incidence of infections was similar in both groups with a trend toward more fungal infections in the Tac group ( n = 7 vs n = 1, p = n.s.). Conclusions The combination of Tac and MMF seems to have slightly higher immunosuppressive potential compared with CsA and MMF. The effectiveness of Tac as a rescue agent is not paralleled with undue signs of overimmunosuppression.

The concentration-dependency of the radiosensitising effect of gemcitabine and the influence of the rescue agent amifostine in vitro

The concentration-dependency of the radiosensitising effect of gemcitabine and the influence of the rescue agent amifostine in vitro

Identification of active site residues in E. coli ketopantoate reductase by mutagenesis and chemical rescue.

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to D-(-)-pantoate in the biosynthesis of pantothenate. The pH dependence of V and V/K for the E. coli enzyme suggests the involvement of a general acid/base in the catalytic mechanism. To identify residues involved in catalysis and substrate binding, we mutated the following six strictly conserved residues to Ala: Lys72, Lys176, Glu210, Glu240, Asp248, and Glu256. Of these, the K176A and E256A mutant enzymes showed 233- and 42-fold decreases in V(max), and 336- and 63-fold increases in the K(m) value of ketopantoate, respectively, while the other mutants exhibited WT kinetic properties. The V(max) for the K176A and E256A mutant enzymes was markedly increased, up to 25% and 75% of the wild-type level, by exogenously added primary amines and formate, respectively. The rescue efficiencies for the K176A and E256A mutant enzymes were dependent on the molecular volume of rescue agents, as anticipated for a finite active site volume. The protonated form of the amine is responsible for recovery of activity, suggesting that Lys176 functions as a general acid in catalysis of ketopantoate reduction. The rescue efficiencies for the K176A mutant by primary amines were independent of the pK(a) value of the rescue agents (Bronsted coefficient, alpha = -0.004 +/-0.008). Insensitivity to acid strength suggests that the chemical reaction is not rate-limiting, consistent with (a) the catalytic efficiency of the wild-type enzyme (k(cat)/K(m) = 2x10(6) M(-1) s(-1) and (b) the small primary deuterium kinetic isotope effects, (D)V = 1.3 and (D)V/K = 1.5, observed for the wild-type enzyme. Larger primary deuterium isotope effects on V and V/K were observed for the K176A mutant ((D)V = 3.0, (D)V/K = 3.7) but decreased nearly to WT values as the concentration of ethylamine was increased. The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding. The double mutant (K176A/E256A) has no detectable ketopantoate reductase activity. These results indicate that Lys176 and Glu256 of the E. coli ketopantoate reductase are active site residues, and we propose specific roles for each in binding ketopantoate and catalysis.

Evaluation of the role of abciximab (Reopro) as a rescue agent during percutaneous coronary interventions: In‐hospital and six‐month outcomes

Abciximab is effective for the prevention of complications when administered prior to percutaneous coronary intervention (PCI). The efficacy and safety of abciximab as an unplanned or rescue agent for complications of PCI is unknown. Rescue versus planned use was compared in 186 consecutive patients. Primary or rescue PCI for acute myocardial infarction (MI) and shock were excluded. Rescue abciximab use was undertaken in 101 patients (54.3%) and planned abciximab was used in 85 (45.7%). The rescue abciximab patients had a lower incidence of previous MI, preprocedural thrombus, multivessel, and vein graft intervention. In-hospital endpoints in the rescue versus planned abciximab patients were death (1.0% vs. 1.2%, P = 1.0), Q-wave MI (2.0% vs. 2.4%, P = 1.0), any MI (14.9% vs. 9.4%, P = 0.3), target vessel revascularization (TVR; 0% vs. 1.2%, P = 1.0), and composite (15.8% vs. 10.6%, P = 0.3). At 6 months, events were death (4.0% vs. 2.3%, P = 0.69), MI (14.9% vs. 9.4%, P = 0.26), TVR (20.8% vs. 4.7%, P = 0.001), and composite (30.7% vs. 15.3%, P = 0.01). In-hospital complications between the rescue and planned abciximab patients of major bleed (1.0% vs. 1.8%, P = NS), stroke (0% vs. 1.8%, P = NS), and thrombocytopenia (3.0% vs. 1.8%, P = NS) were similar. There was a significantly higher procedural time (99.6 min vs. 86.1 min, P = 0.02), contrast volume (278.8 ml vs. 223.5 ml, P = 0.04), and heparin use (8984 u vs. 6003 u, P = 0.0006) in the rescue group. In this nonrandomized comparison, rescue abciximab allowed for the safe discharge from hospital in the majority of patients. However, during a 6-month follow-up, more patients treated with rescue abciximab required TVR with either repeat PCI or CABG. Further studies are warranted to evaluate the overall strategy of rescue abciximab use in PCI. Cathet. Cardiovasc. Intervent. 51:138–144, 2000. © 2000 Wiley-Liss, Inc.

Role of Arginine 277 in (S)-Mandelate Dehydrogenase from Pseudomonas putida in Substrate Binding and Transition State Stabilization

(S)-Mandelate dehydrogenase from Pseudomonas putida is an FMN-dependent alpha-hydroxy acid dehydrogenase. Structural studies of two homologous enzymes, glycolate oxidase and flavocytochrome b(2), indicated that a conserved arginine residue (R277 in MDH) interacts with the product carboxylate group [Lindqvist, Y., Branden, C.-I., Mathews, F. S., and Lederer, F. (1991) J. Biol. Chem. 266, 3198-3207]. The catalytic role of R277 was investigated by site-specific mutagenesis together with chemical rescue experiments. The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. The k(cat) for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its K(m) value was 40-fold lower; in contrast, k(cat)s for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and K(m) values were 5-15-fold lower compared to R277K. The K(d)s for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. The k(cat) for R277G could be enhanced by the addition of exogenous guanidines or imidazoles; the maximum rescued k(cat) was approximately 70% of the wt-MDH value. Only reagents that were positively charged and could function as hydrogen bond donors were effective rescue agents. Our results indicate that R277 plays a major role in transition state stabilization through its positive charge-consistent with a mechanism involving a carbanion intermediate. The positive charge has a relatively small contribution toward substrate binding. R277 also forms a specific hydrogen bond with both the substrate and the transition state; this interaction contributes significantly to the low K(m) for (S)-mandelate.

Effect of transgenic GDNF expression on gentamicin-induced cochlear and vestibular toxicity.

Gentamicin administration often results in cochlear and/or vestibular hair cell loss and hearing and balance impairment. It has been demonstrated that adenovirus-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) can protect cochlear hair cells against ototoxic injury. In this study, we evaluated the protective effects of adenovirus-mediated overexpression of GDNF against gentamicin ototoxicity. An adenovirus vector expressing the human GDNF gene (Ad.GDNF) was administered into the scala vestibuli as a rescue agent at the same time as gentamicin, or as a protective agent, 7 days before gentamicin administration. Animals in the Rescue group displayed hearing thresholds that were significantly better than those measured in the Gentamicin or Ad.LacZ/Gentamicin groups. In the Protection group, Ad.GDNF afforded significant preservation of utricular hair cells. The data demonstrated protection of the inner ear structure, and rescue of the inner ear structure and function against ototoxic insults. These experiments suggest that inner ear gene therapy may be developed as a clinical tool for protecting the ear against environmentally induced insults.

Integrative Aspects of Zinc Transporters

Cells maintain zinc concentrations with relatively narrow limits. Nevertheless, physiologically relevant changes in free Zn(II) pools or changes in Zn bound to specific ligands or within vesicles may occur without a major change in total cellular zinc concentrations. The task of maintaining such levels rests in part with zinc transporter proteins. The genes for some putative zinc transporters have recently been cloned. As of this time, most have not been directly shown to transport zinc in functional studies, albeit evidence is strong that they have such a function. Zinc transporter (ZnT)-1 was identified as a rescue agent for cells maintained in very high extracellular zinc conditions; therefore, ZnT-1 has been suggested to function as an exporter. ZnT-1 is expressed in a variety of tissues, including intestine, kidney and liver. Intestinal expression is regional, being much greater in duodenum and jejunum and in villus versus crypt cells. Immunolocalization places ZnT-1 at the basolateral membrane of intestinal enterocytes and epithelial cells of the distal renal tubules. Regulation of ZnT-1 mRNA and ZnT-1 protein does not change markedly with changes in dietary zinc level except when a large single oral zinc supplement is provided. ZnT-1 is induced by transient ischemia of the forebrain. ZnT-2 and ZnT-3 may function in tissue-specific vesicular zinc transport. ZnT-4 is believed to be abundant in mammary gland and may be associated with zinc secretion into milk. A mutation of the ZnT-4 gene may account for the lethal milk (lm) syndrome. The putative zinc transporters identified thus far appear to have characteristics commensurate with functions in integrative zinc acquisition and homeostasis.