Correlation between serum magnesium and blood cyclosporine A concentrations in renal transplant recipients.

BACKGROUND: Low total serum magnesium concentration has been reported in renal transplant recipients on cyclosporine therapy, and this is a risk factor for hypertension and cardiac death. No study is yet available from the Indian subcontinent on the relationship between total serum magnesium and cyclosporine A concentration. METHODS: Blood cyclosporine A and total serum magnesium concentrations were estimated in 31 post renal transplant patients. Sixteen chronic renal failure patients who had not yet received a transplant served as controls. RESULTS: The mean total serum magnesium in post-transplant patients was 0.64+/-0.11 mmol/L (n = 31), which was significantly lower (P <0.001) than in the controls (1.0+/-0.16 mmol/L). An inverse correlation was found between total serum magnesium and blood cyclosporine A concentration (r = -0.532). Systolic and diastolic blood pressures were also higher in the cyclosporine-treated patients. CONCLUSIONS: We conclude that serum magnesium should be monitored regularly in patients receiving cyclosporine A.

Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis.

The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (Em=-368mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the "low-potential" W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25-30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (Em= -335 and -280 mV, respectively). At higher potentials a minor "mid-potential" W(V) species, g= 1.983, 1.956, 1.932, accounting for less than 5 % of the total W, appears with a midpoint potential of -34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major "high-potential" W(V) signal, g= 1.981, 1.956, 1.883, accounting for 30-40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g= 1.981, 1.950, 1.931; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.

Resonance Raman characterization of biotin sulfoxide reductase. Comparing oxomolybdenum enzymes in the ME(2)SO reductase family.

Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sphaeroides biotin sulfoxide reductase expressed in Escherichia coli. On the basis of (18)O/(16)O labeling studies involving water and the alternative substrate dimethyl sulfoxide and the close correspondence to the resonance Raman spectra previously reported for dimethyl sulfoxide reductase (Garton, S. D., Hilton, J., Oku, H., Crouse, B. R., Rajagopalan, K. V., and Johnson, M. K. (1997) J. Am. Chem. Soc. 119, 12906-12916), vibrational modes associated with a terminal oxo ligand and the two molybdopterin dithiolene ligands have been assigned. The results indicate that the enzyme cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) forms with both molybdopterin dithiolene ligands remaining coordinated in both redox states. Direct evidence for an oxygen atom transfer mechanism is provided by (18)O/(16)O labeling studies, which show that the terminal oxo group at the molybdenum center is exchangeable with water during redox cycling and originates from the substrate in substrate-oxidized samples. Biotin sulfoxide reductase is not reduced by biotin or the nonphysiological products, dimethyl sulfide and trimethylamine. However, product-induced changes in the Mo=O stretching frequency provide direct evidence for a product-associated mono-oxo-Mo(VI) catalytic intermediate. The results indicate that biotin sulfoxide reductase is thermodynamically tuned to catalyze the reductase reaction, and a detailed catalytic mechanism is proposed.

Human ferrochelatase: crystallization, characterization of the [2Fe-2S] cluster and determination that the enzyme is a homodimer.

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Previously we have demonstrated that the mammalian enzyme is associated with the inner surface of the inner mitochondrial membrane and contains a nitric oxide sensitive [2Fe-2S] cluster that is coordinated by four Cys residues whose spacing in the primary sequence is unique to animal ferrochelatase. We report here the characterization and crystallization of recombinant human ferrochelatase with an intact [2Fe-2S] cluster. Gel filtration chromatography and dynamic light scattering measurements revealed that the purified recombinant human ferrochelatase in detergent solution is a homodimer. EPR redox titrations of the enzyme yield a midpoint potential of -453+/-10 mV for the [2Fe-2S] cluster. The form of the protein that was crystallized has a single Arg to Leu substitution. This mutation has no detectable effect on enzyme activity but is critical for crystallization. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell constants of a=93.5 A, b=87.7 A, and c=110.2 A. There are two molecules in the asymmetric unit and the crystals diffract to better than 2.0 A resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 A based upon the Bijvoet difference Patterson map.

Probing the heme axial ligation in the CO-sensing CooA protein with magnetic circular dichroism spectroscopy.

The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-)(1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMCD spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA. The spectra are almost identical to each other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.

Anterior encephaloceles: a report of 30 cases.

Thirty cases of anterior encephalocele treated in our centre over an 18-year period (from 1973 to 1990) are presented. At the time of surgery over 60% of the patients were under the age of 2 years, and 40% were aged under 1 year. Only one child was over 10 years of age. Twenty-six patients had the fronto-ethmoidal type of defect, while two each had frontonasal- and naso-orbital-type lesions. Twenty-five children had varying degrees of hypertelorism. Four had an enlarged head and four microcephaly. In 26 patients one-stage repair of the encephalocele and reconstruction of the orbits was undertaken. A ventriculoperitoneal shunt was performed prior to definitive surgery in three patients with gross hydrocephalus. There was no postoperative mortality. Six patients had postoperative CSF rhinorrhoea, three of them requiring a lumboperitoneal shunt. This study highlights the role of one-stage repair of this defect.

Carcinoma of buccal mucosa: incidence of regional lymph node involvement.

Histopathological findings in 57 surgical specimens of T3 and T4 buccal mucosa cancers are reviewed. The incidence of the well-differentiated tumours (including verrucous carcinoma) was 61%. A moderate degree of lymphocytic infiltration of the tumour was present in 37% of patients. Histologically proven cervical lymph node deposits were present in only 16% of the patients. Amongst the group of patients with clinically enlarged lymph nodes, metastatic disease was histologically demonstrated in 17.5% at level I and 14% at level II of neck nodes. Amongst group of patients with no palpable nodes in the neck, metastatic disease was histologically demonstrated in 11.7% at level I and 9% at level II of neck nodes. These findings contra-indicate an elective neck dissection and indicate the need to confirm histologically the presence of lymph node deposits doing a radical neck dissection in patients with buccal mucosa cancer. This well-differentiated tumour has a much lower tendency to metastasize than cancers in other sites of the oral cavity.

Outcome of patients with pre-existing hepatitis B virus infection following renal transplant.

Twenty four patients who were HBsAg positive prior to renal transplantation were studied with respect to their hepatic and renal outcome. The kidneys in all patients were obtained from living related donors. The standard immunosuppressive therapy consisted of azathioprine and prednisolone. Post-renal transplant follow-up varied from 32-86 months (mean 55 +/- 9). Renal functions, liver functions, HBsAg and HBsAb status was closely monitored during follow-up. One patient developed acute hepatitis four months after transplantation; this resolved in 10 weeks. Four patients showed transient elevations of liver enzymes. The results of liver biopsy in 16 cases revealed, normal histology (8), virus in hepatocytes (5), and acute hepatitis (3). None of the patients had evidence of chronic liver disease. Two patients died due to chronic rejection/chronic renal failure and two patients died due to septicaemia. Five living patients have mild to moderate chronic rejection. It was concluded that pre-existing HBsAg carrier state in transplant patients had no deteriorating effect on the liver. The mortality was related to chronic rejection and septicaemia.

Pancreatic islet transplantation: utility of ductular obstruction and exocrine atrophy model?

Introduction of 'silent' exocrine atrophy (and endocrine 'enrichment') in pancreatic grafts following ductular blockade may have a role in human diabetes by circumventing currently elusive islet isolation/purification protocols. To explore this potential, pancreatic isografts were performed in 12 pairs of inbred Wistar NIN rats. Donor pancreatectomy was performed after distal clamping and canulation of common bile duct and injection of 0.5 ml. polyacrylamide gel (blocked n = 7) or normal saline (un-blocked n = 5) respectively. One to 2 m.m. fragments of the resulting mildly distended pancreases were transplanted in to 2 sites (renal capsule and iliac fossa subcutaneously) of cach recipient. Post-operative biopsies of the transplanted grafts (unilateral nephrectomy and iliac fossa biopsies) revealed macroscopic and microscopic evidence of necrotizing pancreatitis in both the groups at both the sites (histiocytic and giant cell infiltration, fat necrosis and focal calcification with destruction of exocrine and endocrine cells) as early as 1 and 3 weeks. Possible detrimental factors include: volume and pressure of ductal injection, graft sites (confined spaces), post-operative wound infection and bio-compatibility of the material used for ductular blockade.

Functional estrogen receptors as biochemical marker for the detection of hormone-dependent breast cancer.

The translocatable receptors categorized as functional receptors were quantitated in a cross-incubation study of breast cancer nuclei with receptor-rich uterine cytosol. Data demonstrated that tumors that contained cytosolic estrogen receptor (ER) but had translocation defect might not be hormone dependent, whereas tumors with low ER but intact nuclear translocation step will respond to antiestrogen therapy. Cytosolic ER was estimated in 114 primary breast cancer tissues and ten metastatic axillary lymph nodes; 58% of postmenopausal and 54% of premenopausal breast cancer tissues were ER+ with a cutoff value of 10 fmoles and 3 fmoles/mg protein, respectively. Of tumors in the premenopausal and postmenopausal state, 62% and 57%, respectively, were positive for nuclear ER, with a cutoff value of 10 fmoles/100 micrograms DNA. This study suggested that evaluation of functional ER level would reduce the number of false-negative and false-positive tumors.

Monoclonal antibody MAb C-50 in buccal mucosa cancer cells.

Staining patterns of cancerous and normal tissues from 20 cases of proven buccal cancer and ten control individuals respectively to monoclonal antibody MAb C-50 were studied. Nine of 20 patients and 4 of 10 control individuals showed positive immunofluorescence respectively. There was no correlation between clinical stage of disease and MAb C-50 staining nor any significant difference between the ulcerative or exophytic growth groups. One section with T4 lesion of buccal cancer with normal salivary tissue showed positive immunofluorescence of normal salivary cells. In our study the presence of receptor for MAb C-50 on buccal mucosa cancer cells was found to be statistically insignificant.

Significance of transplant perfusion index in the diagnosis of acute rejection in live related renal allograft recipients.

The study was undertaken to assess the usefulness of transplant perfusion index (TPI) in the differential diagnosis of renal allograft dysfunction with special reference to acute rejection. It was observed that the TPI has a sensitivity of 100 per cent and specificity of 98.1 per cent in the diagnosis of acute rejection. It was also observed that the serial values of TPI provided valuable clues and guide in the management of transplant dysfunction and helped in the immediate and long term follow-up of patients with renal allografts.