A model of the structure of HOO-Co.bleomycin bound to d(CCAGTACTGG): recognition at the d(GpT) site and implications for double-stranded DNA cleavage.

BACKGROUND: The bleomycins (BLMs) are a family of natural products used clinically as antitumor agents. In the presence of their required cofactors, iron and oxygen, BLMs bind to and mediate single-stranded and double-stranded DNA cleavage. Recently, two dimensional nuclear magnetic resonance (2D NMR) spectroscopic studies and molecular modeling have provided a picture of how the hydroperoxide form of cobalt BLM A2 (HOO-CoBLM), an analog of 'activated' iron BLM (HOO-FeBLM), binds to a d(GpC) motif and of the basis for both sequence specificity and chemical specificity of DNA cleavage. RESULTS: The solution structure of HOO-CoBLM bound to d(CCAGTACTGG) containing a 'hot spot' for double-stranded DNA cleavage at T5 and T15 is reported using constraints from 2D NMR spectroscopy. The mode of binding and basis for sequence specificity and chemical specificity of cleavage is almost identical to that of a d(GpC) motif. This structure has allowed formulation of a structural model for how a single molecule of FeBLM can mediate a double-stranded DNA cleavage event without dissociation from the DNA. CONCLUSIONS: The structural similarity of HOO-CoBLM bound to d(GpT) in d(CCAGTACTGG) compared to a d(GpC) motif suggests a general paradigm for the binding of HOO-CoBLM to DNA and, by analogy, for the binding of the biological significant entity HOO-FeBLM.

Electronic properties of the dissimilatory sulphite reductase from Desulfovibrio vulgaris (Hildenborough): comparative studies of optical spectra and relative reduction potentials for the [Fe4S4]-sirohaem prosthetic centres.

The dissimilatory sulphite reductase (desulfoviridin) from the sulphate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) displays distinct optical and redox characteristics relative to the haem subunit of Escherichia coli assimilatory sulphite reductase. For high-spin pentaco-ordinate desulfoviridin there is minimal change in the absorbance of the oxidized chromophores both after reduction or after addition of exogenous ligands. A ligand-metal charge-transfer band approximately 702 nm is observed in both the oxidized and one-electron-reduced enzyme. E.p.r. spectroscopy has been used to define the relative reduction potentials for sirohaem and [Fe4S4] centres (delta E0 = Es0-Ec0) as a function of sirohaem axial co-ordination. Typically delta E0 lies in a range from -10 to -50 mV. These results show a correlation with the sigma-donor or pi-acceptor properties of the ligand and stand in sharp contrast with estimates for the E. coli enzyme. The electronic properties of the coupled [Fe4S4]-sirohaem redox centre common to both nitrite- and sulphite-reducing enzymes are apparently strongly dependent on the environment generated by protein side chains.

Conformational gating of the dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). Synthesis, characterization, and stopped-flow kinetics studies of 1,5-IAEDANS-labeled desulfoviridin.

The siroheme prosthetic center in the dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris (Hildenborough) readily binds exogenous ligands in the reduced state, but it does not do so in the oxidized state. In contrast, free oxidized siroheme in solution is observed to bind ligands rapidly. This can only be explained by a structural barrier that precludes ligand binding to the enzyme in the oxidized state but is removed after reduction. These observations suggest a redox-linked structural transformation that provides a gating mechanism for enzyme activation. The rate constants defining these structural perturbations, from oxidized-->reduced and reduced-->oxidized states, have been determined by monitoring changes in both the natural emission from desulfoviridin and the emission from a surface-bound fluorophore (1,5-IAEDANS). Consistent results were obtained from these two independent experimental measurements (at 25 degrees C: kox-->red approximately 8 s-1, kred-->ox approximately 0.05 s-1). Activation energies for each transition have been determined from Arrhenius plots (ox-->red: delta G* 16.5 kcal mol-1, delta H* 3.5 kcal mol-1, delta S* -43.8 cal K-1 mol-1; red-->ox: delta G* 19.2 kcal mol-1, delta H* 11.3 kcal mol-1, delta S* -26.6 cal K-1 mol-1). These data are used to further develop a functional model previously proposed for this class of enzyme [Lui, S. M., Soriano, A., & Cowan, J. A. (1993) J. Am. Chem. Soc. 115, 10483; Lui, S. M., Liang, W., Soriano, A., & Cowan, J. A. (1994) J. Am. Chem. Soc. 116, 4531].(ABSTRACT TRUNCATED AT 250 WORDS)

Functional expression and characterization of the assimilatory-type sulfite reductase from Desulfovibrio vulgaris (Hildenborough).

By use of a conjugational transfer method, the gene encoding the assimilatory-type sulfite reductase (ASiR) from Desulfovibrio vulgaris (Hildenborough) has been functionally expressed in Desulfovibrio hosts using the broad-host-range plasmid pDSK519. Production is increased greater than 50-fold relative to natural expression levels. Recombinant enzyme has been characterized by standard biochemical methods, nuclear magnetic resonance, electron paramagnetic resonance, electronic absorption, and activity measurements. It cannot be distinguished from the native enzyme on the basis of spectroscopic characteristics or enzyme activity.

Desulfoviridin, a multimeric-dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). Purification, characterization, kinetics and EPR studies.

Conditions for the rigorous purification of desulfoviridin, the dissimilatory sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) have been established. A final purification by fast protein liquid chromatography yields at least three distinct bands that each exhibit the characteristic absorption spectrum of desulfoviridin. Two of these have been extensively characterized by amino acid analysis, isoelectric focusing, polyacrylamide gel electrophoresis, and formulation of the prosthetic centers. Each contains two pairs of [Fe4S4] and siroheme units. These results stand in marked contrast to recent work claiming significant demetallation of siroheme, excess iron content, and the presence of Fe6S6 clusters. These proposals are critically assessed in light of our results and other published work. Steady-state kinetic parameters have been determined: kcat(SO3(2-) = 0.31 mol SO3(2-).s-1.mol heme-1, Km = 0.06 mM; kcat(NO2-) = 0.038 mol NO2-.s-1.mol heme-1, Km = 0.028 mM; kcat(NH2OH) = 29 mol NH2OH.s-1.mol heme-1, Km = 48 mM. A detailed comparison is made with the Escherichia coli and spinach assimilatory sulfite reductase enzymes and spinach nitrite reductase. Highly purified samples of dissimilatory sulfite reductase display an electron paramagnetic resonance spectrum characteristic of rhombic high spin ferric heme centers, while the fully reduced enzyme shows EPR features typical of [Fe4S4] clusters. The magnetic properties of the prosthetic centers are further characterized by variable temperature experiments and spin quantitation.

A direct spectrophotometric method for monitoring the kinetics of T-cell cytolysis.

A simple and sensitive assay is demonstrated for monitoring the kinetics of T-cell cytolysis by direct spectrophotometric detection of genomic DNA and protein released from target cells. The kinetics of cell lysis has been quantitatively evaluated and shows good agreement with results obtained by use of extracellular fluorescent reporter molecules, or enzyme-coupled assays.

A study on children's condition thalassemia using neutron activation analysis and other techniques.

In this study, 50 thalassemia patients were tested using dual-energy X-ray absorptiometry (DEXA) and in vivo neutron activation analysis (IVNAA) to determine their bone mineral status. Both techniques were suitable for this purpose. Lower age was found to correspond to lower liver iron content and higher bone mineral content in the normal range. Patients undergoing treatment with transfusion had higher bone mineral content. Osteopenic patients had higher hepatic iron content than those with normal bone status. In the case of DEXA, bone mineral content (BMC) divided by height cubed was found to be a better indicator of bone mineral status than the BMD usually given. Liver density as determined by DEXA correlates well with hepatic iron.

The change of penile blood thromboxane B2 and prostacyclin after intracavernous injection of vasoactive drugs for the treatment of arteriogenic impotence.

Penile hypercoagulability during erection may predispose to aging vascular changes and, eventually, arteriogenic impotence. The relationship of penile blood thromboxane B2 and 6-keto-prostaglandin F1 alpha in both psychogenically and arteriogenically impotent patients after intracavernosal treatment with papaverine plus phentolamine or prostaglandin E1 was evaluated. No significant change in penile blood thromboxane B2 was observed with treatment of these vasoactive drugs. On the other hand, penile blood 6-keto-prostaglandin F1 alpha was significantly increased with the injection of 30 mg of papaverine plus 0.5 mg of phentolamine, and of 20 micrograms prostaglandin E1. Furthermore, the prostacyclin-to-thromboxane A2 ratio for the patient who received papaverine plus phentolamine was significantly lower than that for the same individual receiving prostaglandin E1. Our preliminary findings suggest that penile blood prostacyclin may participate in the pathogenesis of arteriogenic impotence and priapism.

Synthesis, cloning and expression of a synthetic gene for high potential iron protein from Chromatium vinosum.

A synthetic gene encoding the peptide sequence for the low molecular weight (M(r) approximately 9600 Da) high-potential iron protein (HiPIP) from the photosynthetic bacterium Chromatium vinosum has been constructed by shotgun ligation of twelve complimentary oligonucleotides varying in size from 42-mers to 48-mers. After cloning the gene into a pET-21d(+) vector, expression of holoprotein in yields of 35 mg/liter of culture was obtained following induction with isopropyl-beta-D-thiogalactoside (IPTG). The recombinant protein was characterized by electronic absorption, 1H NMR, electrochemistry, N-terminal sequencing and amino acid analysis. This is the first example of the expression of a high potential ferredoxin containing a fully constituted [Fe4S4] cluster.

The change of urinary 11-dehydro-thromboxane B2 and 2,3-dinor-6-keto-prostaglandin F1 alpha in arteriogenic impotence.

Thromboxane A2 is a potent vasoconstrictor and a stimulus of platelet aggregation, which may contribute to hypercoagulability. The prostacyclin, prostaglandin I2, has exactly the opposite effect. Measurement of the major urinary metabolites, 11-dehydro-thromboxane B2 and 2,3-dinor-6-keto-prostaglandin F1 alpha (prostaglandin F1 alpha) by radioimmunoassay can accurately reflect in vivo the biosynthesis of thromboxane A2 and prostaglandin I2, respectively. Group 1 consisted of 60 patients less than 50 years old. The mean urinary 11-dehydro-thromboxane B2 level of 3 patients with arteriogenic impotence was significantly greater than that of the 57 control volunteers: 2.66 +/- 0.65 versus 1.74 +/- 0.56 (plus or minus standard deviation) ng./mg. creatinine (p = 0.008). The prostaglandin F1 alpha levels for the patients and controls were 32.74 +/- 8.45 and 37.58 +/- 16.55 ng./mg. creatinine, respectively, which was not significantly different (p greater than 0.05). Group 2 consisted of 96 patients 50 years old or older. The 11-dehydro-thromboxane B2 concentration in the urine was 1.83 +/- 0.58, 2.54 +/- 1.12 and 1.91 +/- 0.73 ng./mg. creatinine in the 47 normal control volunteers, 20 patients with arteriogenic impotence and 29 with arteriogenic impotence plus intracavernous injection of 20 micrograms prostaglandin E1, respectively. The arteriogenic impotence group showed the significantly highest level among the 3 groups (p = 0.0025). Also, the urinary prostaglandin F1 alpha levels in these patients were 45.71 +/- 36.3, 57.71 +/- 35.53 and 59.30 +/- 45.08 ng./mg. creatinine, respectively, which was not significantly different (p greater than 0.05). For the 13 patients with arteriogenic impotence (group 3) we compared the urinary 11-dehydro-thromboxane B2 and prostaglandin F1 alpha levels before and after intracavernous injection of prostaglandin E1 by using a paired t test. The results showed that the change in 11-dehydro-thromboxane B2 levels was 2.78 +/- 1.09 versus 1.99 +/- 0.75 ng./mg. creatinine, which was significantly different (p = 0.005), whereas that for prostaglandin F1 alpha was 62.30 +/- 40.41 versus 58.86 +/- 44.26 ng./mg. creatinine, with no significant difference (p greater than 0.05). Our findings suggest that urinary 11-dehydro-thromboxane B2 may have an important role in the diagnosis and treatment of arteriogenic impotence.

Renal protein synthesis in diabetes mellitus: effects of insulin and insulin-like growth factor I.

Is increased synthesis of proteins responsible for the hypertrophy of kidney cells in diabetes mellitus? Does the lack of insulin, and/or the effect of insulin-like growth factor I (IGFI) on renal tubule protein synthesis play a role in diabetic renal hypertrophy? To answer these questions, we determined the rates of 3H-valine incorporation into tubule proteins and the valine-tRNA specific activity, in the presence or absence of insulin and/or IGFI, in proximal tubule suspension isolated from kidneys of streptozotocin diabetic and control rats. The rate of protein synthesis increased, while the stimulatory effects of insulin and IGFI on tubule protein synthesis were reduced, early (96 hours) after induction of experimental diabetes. Thus, hypertrophy of the kidneys in experimental diabetes mellitus is associated with increases in protein synthesis, rather than with decreases in protein degradation. Factor(s) other than the lack of insulin, or the effects of IGFI, must be responsible for the high rate of protein synthesis present in the hypertrophying tubules of diabetic rats.

Studies of cadmium uptake in bone and its environmental distribution.

Rat experiments indicate that oral ingestion of cadmium through drinking water leads to an accumulation of cadmium in bone, in addition to liver and kidney. After five weeks of cadmium intake in drinking water (50 to 100 mg/L), the bone cadmium levels increased in proportion to the intake concentration. Bone and kidney histology showed no signs of bone or kidney damage up to 5 wk of cadmium ingestion. Cadmium accumulation in bone was a primary phenomenon and not secondary to renal failure. In addition, cadmium levels have been estimated in a variety of sources, e.g., foodstuff, fertilizer, and sewage sludge, using neutron and proton activation analyses and atomic absorption spectrophotometry. Cadmium levels of Canadian foods are in the range of 0.002-0.07 mg/kg, and soils are in the range of 0.55 to 1.72 mg/kg. Fertilizers contain cadmium from 0.3 to 1.25 mg/kg, whereas sewage sludge contains up to 122 mg/kg.

Treatment of unstable fractures of the distal radius by external fixation.

The Roger Anderson external fixator was used in the treatment of unstable fractures of the distal radius in 52 patients, and the results evaluated after a follow-up averaging 58 months. The indications for its use were failure to maintain adequate closed reduction using plaster, and instability of the fracture as determined by the initial radiographs. Our radiological criteria for instability included dorsal angulation of more than 20 degrees, fractures involving the joint, radial shortening of more than 10 mm, and severe dorsal comminution. Using the Lucas modification of the Sarmiento demerit point-rating system, we found that 46 patients (89%) had good or excellent results and six (11%) were classified as fair. There were no poor results. Seven patients (14%) developed complications. None of these affected the long-term results except in one elderly woman where the pins loosened and had to be removed.