Selective reactivity of rhodanese sulfhydryl groups with 5,5'-dithio-bis(2-nitrobenzoic acid)

The reactivity of the sulfhydryl groups of sulfur-containing and sulfur-free rhodanese (thiosulfate : cyanide sulfurtransferase, EC 2.8.1.1) with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) has been investigated. Only 0.6 sulfhydryl group of the sulfur-containing enzyme reacts with DTNB. After removal of sulfur from persulfide groups a further 0.6 sulfhydryl group (i.e. a total of 1.2) becomes accessible to the reagent. The resulting enzyme-thionitrobenzoate complex shows an absorption spectrum with a shoulder at 325 nm due to bound thionitrobenzoate. Both thiosulfate and cyanide remove thionitrobenzoate from the enzyme restoring its catalytic properties. The modified enzyme is protected from alkylation by iodoacetate until thionitrobenzoate is removed. The existence of a further sulfhydryl group close to the catalytic one is suggested.     

Kinetics of modification of the mitochondrial succinate-ubiquinone reductase by 5,5''-dithiobis-(2-nitro-benzoic acid)

In the present study, we examined the effect of the thromboxane/prostaglandin endoperoxide analogue U46619 on proliferation and hypertrophy in cultured rat vascular smooth muscle cells and the roles of protein kinase C and transforming growth factor-beta (TGF-beta) in the mediation of the hypertrophic response to U46619. Since an increase in basic fibroblast growth factor (bFGF) was previously shown to mediate the hypertrophic response to U46619, we also assessed the relationship between bFGF and TGF-beta in the expression of U46619 actions. U46619 increased [35S]methionine incorporation into protein and protein content of vascular smooth muscle cells but had no effect on cell number. A role for TGF-beta was supported by the following observations: (1) exogenous human TGF-beta 1 increased protein synthesis; (2) antibody to TGF-beta blocked both TGF-beta- and U46619-induced increases in protein content; (3) U46619 increased active and total TGF-beta bioactivities; and (4) the actions of U46619 on protein content and TGF-beta bioactivity were blocked by the thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548. Previous observations had demonstrated a role for bFGF in the expression of U46619 actions on protein synthesis. Results of the present study suggest that TGF-beta and bFGF interact in mediating the protein synthetic response to U46619. First, the concentration of exogenous TGF-beta (10 pmol/L) alone required to produce a protein synthetic response equivalent to that induced by U46619 was much higher than the concentration of endogenous active TGF-beta that accumulated in the media in response to U46619 (0.7 pmol/L). Second, bFGF (20 ng/mL) increased total TGF-beta bioactivity and stimulated protein synthesis. The hyper-trophic response to bFGF was blocked by anti-TGF-beta. The ability of U46619 and bFGF to increase protein synthesis and protein content in vascular smooth muscle cells was associated with TGF-beta-induced suppression of proliferation, as evidenced by the ability of antibody to TGF-beta to enhance U46619- and bFGF-induced increases in [3H]thymidine incorporation into DNA. Results of the present study also supported a role for protein kinase C in the expression of U46619 and bFGF actions. U46619 increased protein kinase C activity in the particulate fraction of vascular smooth muscle cells. Moreover, the protein kinase C inhibitors GF109203X and staurosporine blocked U46619- and bFGF-induced increases in protein synthesis as well as active and total TGF-beta bioactivities. By contrast, the protein kinase C inhibitors did not prevent the increases in protein synthesis induced by exogenous TGF-beta. The results demonstrate that thromboxane/prostaglandin endoperoxide signals increased TGF-beta bioactivity via protein kinase C. Increases in both bFGF and TGF-beta are required for an optimal hypertrophic response to U46619. The hypertrophic response to TGF-beta occurs through a protein kinase C-independent pathway.     

Isoenzymes of creatine kinase in the perinatal period (author's transl)

The serum of new-born children shows a higher catalytic concentration of creatine kinase than adult serum. These values reach normal adult levels within the first 10 days of life. In addition to the MM-, the MB- and BB-isoenzymes of creatine kinase are also found in the serum of women in labour, in cord-serum, and in the serum of newborn children. These findings should be taken into consideration in the analysis of creatine kinase isoenzymes.     

The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase

We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibers. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 11 mM glucose as substrate, and supplemented with either 20 mM beta-guanidinopropionic acid (beta-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31 P-NMR, Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, beta-GPA-P, GA-P and GA-P over a similar time course (approximately 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK, Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K(m) and increased Vmax vs control), but none by GA, mGPA or beta-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.     

Pressure oxidation kinetics of orpiment (As2S3) in sulfuric acid

Pressure oxidation kinetics of a massive orpiment (As₂S₃) sample in sulfuric acid solution were systematically investigated. The effects of temperature (170 to 230 °C), mean particle size (48 to 125 μm diameter) and oxygen partial pressure (345 to 1035 kPa) were evaluated. Oxidation rates were found to be affected significantly by changes in temperature and particle size, but were relatively insensitive to changes in oxygen partial pressure. Kinetics appear to be controlled by product layer diffusion over the temperature range of 170 to 210 °C, due to the formation of elemental sulfur on particle surfaces. However, Arrhenius activation energies over this range are large (> 40 kJ/mol) and increase with decreasing temperature, perhaps reflecting the kinetics of sulfur oxidation rather than diffusion. Above 210 °C the rate-controlling step is a surface reaction with an activation energy of approximately 33.8 kJ/mol. The reaction order at 210 °C is approximately 0.2 with respect to oxygen partial pressure.     

Structural characteristics of 2'-O-(2-methoxyethyl)-modified nucleic acids from molecular dynamics simulations

The structure and physical properties of 2'-sugar substituted O -(2-methoxyethyl) (MOE) nucleic acids have been studied using molecular dynamics simulations. Nanosecond simulations on the duplex MOE[CCAACGTTGG]-r[CCAACGUUGG] in aqueous solution have been carried out using the particle mesh Ewald method. Parameters for the simulation have been developed from ab initio calculations on dimethoxyethyl fragments in a manner consistent with the AMBER 4.1 force field database. The simulated duplex is compared with the crystal structure of the self-complementary duplex d[GCGTATMOEACGC]2, which contains a single modification in each strand. Structural details from each sequence have been analyzed to rationalize the stability imparted by substitution with 2'- O -(2-methoxyethyl) side chains. Both duplexes have an A-form structure, as indicated by several parameters, most notably a C3' endo sugar pucker in all residues. The simulated structure maintains a stable A-form geometry throughout the duration of the simulation with an average RMS deviation of 2.0 A from the starting A-form structure. The presence of the 2' substitution appears to lock the sugars in the C3' endo conformation, causing the duplex to adopt a stable A-form geometry. The side chains themselves have a fairly rigid geometry with trans , trans , gauche +/- and trans rotations about the C2'-O2', O2'-CA', CA'-CB' and CB'-OC' bonds respectively.     

The protection of creatine kinase MM sub-bands by EDTA during storage

We examined the effect of a 5 mmol/l concentration of EDTA on the stabilization of the five serum creatine kinase MM isoenzymes, resolved by thin-layer isoelectric focusing. In patient sera, total CK and CK-MB activities were stable during storage of the samples for two months at 4 degrees C even in the absence of EDTA. However, EDTA stabilized the labile MM and MM1 sub-bands, which are the first to appear in the blood after the release from the damaged tissue and its addition to blood samples intended for determining the MM sub-band pattern is recommended. The stabilizing effect of EDTA was emphasized at higher temperatures. EDTA protected the CK-MM pattern in myocardium extracts made in normal serum and incubated at 37 degrees C during 40 h, but was unnecessary when myocardium was homogenized in heat-inactivated serum. It is thought that EDTA could act by inhibiting a heat-labile component of human serum.     

Proficiency testing of creatine kinase and creatine kinase-2: the experience of the Ontario Laboratory Proficiency Testing Program

The Ontario Laboratory Proficiency Testing Program has regularly monitored the analytical performance of total creatine kinase (CK) (approximately 230 participants) and CK isoenzyme-2 (CK-MB) (approximately 160 participants) throughout the entire province. Consistently, a wide dispersion of results has been observed not only between different analyzer systems but also among identical analyzers. Accordingly, the results of the last three proficiency surveys for these analytes were examined statistically to establish both the extent of these variations and the range of values reported for the male upper reference ranges. The results of many of the analyzer systems were significantly different from each other, as were many of the reference ranges. This unsatisfactory situation may only be remedied by the use of reference materials as shown by others. The consequences of these findings also effect the reliability of epidemiological surveys such as the WHO MONICA Project (Circulation 1994;90:583-612), which monitors deaths due to heart disease and includes cardiac enzyme results in its criteria.     

Identification of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid binding sequences in alpha-crystallin

The hydrophobic binding sites in alpha-crystallin were evaluated using fluorescent probes 1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS), 8-anilino-1-naphthalene sulfonate (ANS), and 1-azidonaphthalene 5-sulfonate (1,5-AZNS). The photolysis of bis-ANS-alpha-crystallin complex resulted in incorporation of the probe to both alphaA- and alphaB-subunits. Prior binding of denatured alcohol dehydrogenase to alpha-crystallin significantly decreased the photoincorporation of bis-ANS to alpha-crystallin. Localization of bis-ANS incorporated into alphaA-crystallin resulted in the identification of residues QSLFR and HFSPEDLTVK as the fluorophore binding regions. In alphaB-crystallin, sequences DRFSVNLNVK and VLGDVIEVHGK were found to be the bis-ANS binding regions. Of the bis-ANS binding sequences, HFSPEDLTVK of alphaA-crystallin and DRFSVNLNVK and VLGDVIEVHGK of alphaB-crystallin were earlier identified as part of the sequences involved in their interaction with target proteins during the molecular chaperone-like action. The hydrophobic probe, 1,5-AZNS, also interacted with both subunits of alpha-crystallin. Localization of 1,5-AZNS binding site in alphaB-crystallin lead to the identification of HFSPEEK sequence as the interacting site in this subunit of alpha-crystallin. Glycated alpha-crystallin displayed decreased ANS fluorescence and loss of chaperone-like function, suggesting the involvement of glycation site as well as ANS binding site in chaperone-like activity display.     

Mutation of cis-proline 207 in mitochondrial creatine kinase to alanine leads to increased acid stability

SETTING: Hamad General Hospital, the tertiary health centre for Doha, Qatar. OBJECTIVE: The purpose of the study was to define and correlate the role of radiology with clinical and pathological findings in abdominal tuberculosis. MATERIAL AND METHODS: A total of 59 patients (47 males and 12 females) diagnosed bacteriologically and/ or histologically for abdominal tuberculosis were radiologically assessed. Evaluation was based on the analysis of plain abdominal radiographs, gastro-intestinal contrast studies (barium meal follow through and barium enema), ultrasonography and computed tomography. RESULTS: Plain abdominal radiographs performed in 38 patients were positive in 19 cases (50%). Gastrointestinal contrast examinations were positive in 27 out of 34 cases (80%). Ultrasound examinations were abnormal in 25 out of 31 cases (81%), while computed tomography, performed in 24 patients, revealed abnormal findings in 19 cases (80%). Combined radiographic and imaging procedures revealed peritoneal involvement (ascites) in 16 patients (27%), bowel involvement in 36 (61%), mass lesion in 11 (19%), lymphadenopathy in 13 (22%) and organ involvement in 13 (22%). CONCLUSION: There was no single radiological method that provided all necessary information suggestive of abdominal tuberculosis. Although unequivocal diagnosis of abdominal tuberculosis can only be made by culture and histological findings, combined computed tomography and ultrasound findings were the most important imaging tools in the diagnostic process for abdominal tuberculosis, while contrast studies helped to assess the extent of bowel disease, hence influencing decisions concerning surgery.     

Base sequence-independent distorsions induced by interstrand cross-links in cis-diamminedichloroplatinum (II)-modified DNA

Physico-chemical and immunological studies have been done in order to further characterize the distorsions induced in DNA by the interstrand cross-links formed between the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) and two guanines on the opposite strands of DNA at the d(GC/GC) sites. Bending (45 degrees) and unwinding (79 +/- 4 degrees) were determined from the electrophoretic mobility of multimers of 21- 24-base pairs double-stranded oligonucleotides containing an interstrand cross-link in the central sequence d(TGCT/AGCA). The distorsions induced by the interstrand cross-link in the three 22-base pairs oligonucleotides d(TGCT/AGCA), d(AGCT/AGCT) and d(CGCT/AGCG) were compared by means of gel electrophoresis, circular dichroism, phenanthroline-copper footprinting and antibodies specifically directed against cis-DDP interstrand cross-links. The four different technical approaches indicate that the distorsions are independent of the chemical nature of the base pairs adjacent to the interstrand cross-link. The general conclusion is that the interstrand cross-link induces a bending and in particular an unwinding larger than other platinum adducts and the distorsions are independent of the nature of the bases (purine or pyrimidine) adjacent to the d(GC/GC) site.     

Interaction of nitric oxide with 2-thio-5-nitrobenzoic acid: implications for the determination of free sulfhydryl groups by Ellman's reagent

Nitric oxide (NO) in an aerobic environment, reacts with the sulfhydryl groups of proteins to form nitroso thiols. Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), DTNB, is widely used for the determination of -SH groups. In this procedure, DTNB, a symmetric aryl disulfide, reacts with the free thiol to give a mixed disulfide plus 2-nitro-5-thiobenzoic acid (TNB) which is quantified by its absorbance at 412 nm. We observed that the presence of NO during the determination of SH groups in a reaction system containing glutathione (GSH) or bovine serum albumin (BSA) plus DTNB resulted in an inhibition in the detection of TNB. Addition of NO donors or NO gas after TNB was already formed led to the bleaching of yellow color and loss of absorbance at 412 nm. These interactions did not occur under anaerobic conditions. Decreased formation of TNB therefore appeared to be due not only to destruction of SH groups of BSA or GSH by NO (S-nitrosation) and consequently to lower TNB formation, but also to direct reaction of NO/O2 with TNB. The mechanism(s) of inhibition of accumulation of TNB by NO was evaluated. NO generated by DEA/NO, SNAP, or spermine/NO, as well as gaseous NO or BSA-NO, directly interacted with TNB, followed by decreased absorbance at 412 nm in a concentration- and time-dependent manner. Kinetics of NO/O2 interaction with TNB were dependent on the ability of the NO donors to release NO as the donors with a short half-life bleached the yellow color of TNB faster. The requirement for O2 suggests that nitrogen oxide or higher oxides of NOx are responsible for interaction with TNB. The UV/VIS spectrum of the final product formed during the interaction of NO with TNB was identical to that of DTNB. These results suggest that interaction of NO (NOx) with TNB resulted in the formation of an unstable nitrosothiol, followed by oxidation and dimerization back to the corresponding disulfide, DTNB. Therefore, determination of SH groups in proteins by Ellman's reagent after or in the presence of NO treatment is complicated since the reduced form of DTNB, TNB, can be reoxidized by NO back to DTNB, with subsequent loss of absorbance at 412 nm.     

Colorimetric determination of cystine (disulfide bond) in hair using dithiothreitol

We developed a simple method for the quantification of cystine (disulfide bond) content in hair by measuring the amount of oxidized dithiothreitol (lambda max 283 nm) derived from dithiothreitol (DTT) with cystine. Because the cystine content in hair is almost fixed for each animal species, it can be used as a reliable indicator of hair weight. The absorbance (A280, y) of the supernatant of the reaction mixture correlated well with hair weight (mg, x) (y = 0.10x + 0.06, r = 1.00, n = 10). Within-run and between-day reproducibilities (C.Vs., %) for the assay were 3.1 and 2.8 (n = 5 each), respectively. Hair cystine content (nmol/mg hair, mean +/- S.D.) in normal volunteers was 903 +/- 50.6 (n = 10) by the present method and 755 +/- 24.9 (n = 10) when an amino acid analyzer was used. After assay by our method, the hair sample can be washed, then used repeatedly to assay other analytes. The present method should be useful for assays of analytes present only in small amounts (2-20 mg), without the need for precise weighing of the hair samples.     

The extraction of iron(III) from aqueous acid solutions by di(2-ethylhexyl)phosphoric acid

The extraction of iron(III) from aqueous solutions containing sulphuric, hydrochloric and nitric acids by di(2-ethylhexyl)phosphoric acid (DEHPA) in kerosene has been investigated under different conditions. As a result, it is found that although extraction is dominated by an ion-exchange reaction, the rate of iron(III) extraction from sulphuric acid solutions to reach equilibrium is relatively slow in comparison with that from hydrochloric or nitric acid solutions. In the extraction from aqueous solutions containing hydrochloric or nitric acid, however, the DEHPA combines with iron(III) according to the solvating reaction at higher aqueous acidity. From studies on the rate of the extraction from sulphuric acid solutions, examined under non-equilibrium, it is confirmed that dependencies of extraction rate on hydrogen ion and DEHPA concentrations are in the first and inverse first orders, respectively. The hydrolyzed species is considered to interpret the extraction mechanism in this system.     

Is creatine kinase isoenzyme CK-MB a diagnostic tool for perioperative myocardial infarctions? (author's transl)

There is still controversy of the validity of elevated CK-MB serum activity in the diagnosis of perioperative myocardial infarction after open heart surgery. CK-MB activity was investigated using myocardial and skeletal muscle biopsies and in sera postoperatively in 192 patients. In biopsies CK-MB fraction of total myocardial CPK was 37%, the total-CPK activity of human skeletal muscles still shows a 5% fraction of CK-MB. There has to be more than 8% CK-MB fraction of total CPK-serum-activity to take this as evidence of myocardial damage. 3 h postoperatively enzymatic-immunologic CK-MB test is no longer interfered by enzymes derived from hemolyzed erythrocytes. In patients without signs of myocardial lesions postoperatively mean CK-MB-activity is 11 to 27 U/1 depending on the operative procedure performed. Activity levels exceeding 50 U/1 are almost evident of myocardial infarction. Elevated CK-MB-serum activity is a sensitive parameter for myocardial lesions overestimating an event of infarction. It is a helpful tool diagnosing perioperative myocardial infarction.     

Kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions

The toxicity of NO3- and NO2- to mammals has been widely publicized. However, the kinetic mechanism of inhibition of human muscle creatine kinase by NO3- and NO2- has not been explored. The kinetic theory of the substrate reaction during the modification of enzyme activity previously described by Tsou (Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381-436) has been applied to a study of the kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions (NO3- and NO2-). The kinetic equation of the substrate reaction was derived from theoretical analysis and experimental data, then simplified. The microscopic rate constants for the reaction of the inhibitors with the enzyme were obtained from the simplified equation for the substrate reaction in the presence of the inhibitors. The results show that the apparent forward rate constant A is dependent on ATP concentration, indicating competition between the inhibitor (NO3- or NO2-) and ATP. The results also suggest that binding of creatine-MgADP and the anion with the enzyme is very tight, since their binding constants are much higher than those for normal substrates.     

Cardiac markers in the early hours of acute myocardial infarction: clinical performance of creatine kinase, creatine kinase MB isoenzyme (activity and mass concentration), creatine kinase MM and MB subform ratios, myoglobin and cardiac troponin T

We compared early markers of acute myocardial infarction (AMI) in the first 6 h from the onset of symptoms in 133 non-traumatized patients arriving at the emergency department with chest pain suggestive of AMI. Clinical performance parameters were calculated on the basis of 45 patients with AMI and 88 patients with a non-AMI diagnosis. At admission and in the first 0-3 h after the onset of chest pain the creatine kinase-MB (CK-MB) subform ratio was the most sensitive test at a comparable specificity level of 0.95. In the time interval of 3-5 h, myoglobin, the CK-MB mass concentration and the CK-MB subform ratio were associated with the greatest areas under receiver operating characteristic (ROC) curves, but differences between these tests were small and non-significant. At 6 h from the onset of pain, differences in clinical performance between the same three tests were even smaller whether or not samples drawn after the start of thrombolytic treatment were included in the test comparison. For confirmation of AMI at 6 h after onset of pain, CK-MB (activity and mass concentration) demonstrated the highest positive likelihood ratio, and for exclusion of AMI at 6 h the CK-MB subform ratio was associated with the highest negative likelihood ratio. However, differences between the CK-MB subform ratio, CK-MB mass concentration and myoglobin were not significant as estimated by the substantial overlap between the confidence intervals of the likelihood ratios and the ROC areas at 6 h. Cardiac troponin T (cTnT) demonstrated an ROC area equal to the CK-MB isoform ratio and myoglobin at 6 h. However, the likelihood ratio for ruling out AMI was lower, mostly due to the elevated cTnT in unstable coronary disease not defined as AMI. We conclude that the CK-MB subform ratio, CK-MB mass concentration and myoglobin do not demonstrate any significant differences in clinical performance for ruling in or ruling out acute myocardial infarction at 6 h after the onset of chest pain.