Diabetes in Patients with ß-thalassemia or other Hemoglobinopathies - Analysis from the DPV Database.

Background: Diabetes mellitus is a common endocrinopathy in patients with thalassemia major, but the occurrence of hemoglobinopathies is rare in Germany and Western Europe. The longitudinal German-Austrian DPV (Diabetes Patienten Verlaufsdokumentation) registry allows a comprehensive characterization of this group of patients. Patients/methods: Patients from the DPV-registry aged<30 years with thalassemia major or other hemoglobinopathies were compared to patients with type 1 diabetes (T1D) and type 2 diabetes (T2D) using the statistical software SAS 9.4. Results: 94 patients (0.13% of patients) with hemoglobinopathies are registered in DPV. 82.4% of 17 patients with thalassemia major, 100% of 12 patients with sickle cell disease (SCD) and >90% of 65 patients with other hemoglobinopathies receive insulin treatment. In the majority of patients with thalassemia major, hemosiderosis is documented. Patients with thalassemia major developed diabetes at a median age of 14.6 [IQR 8.4-18.0] years (9.0 years [5.3-12.5] in T1D; 18.7 years [14.2-25.6] in TD2; both p<0.01). They show high HbA1c/fructosamine levels and frequent hypoglycemia, reflecting poor metabolic control. Conclusion: Diabetes in thalassemia major is probably caused by hemosiderosis due to polytransfusion, while patients with SCD/thalassemia minor are most likely affected by T1D. The high rate of hypoglycemia in patients with ß-thalassemia major may be caused by liver fibrosis and a lack of hepatic glycogen stores.

Normal values for transbulbar sonography and magnetic resonance imaging of the optic nerve sheath diameter (ONSD) in children and adolescents.

PURPOSE: To establish normal values of the optic nerve sheath diameter (ONSD) in children and adolescents for transbulbar sonography and magnetic resonance imaging. MATERIALS AND METHODS: In 99 children and adolescents (age: 5.6 - 18.6 years, mean: 12 years) without neurologic or ophthalmologic disease, measurements of the ONSD with transbulbar sonography were performed. For comparison 59 children and adolescents (age: 5.1 - 17.4 years, mean 12.3 years) with a normal MR examination of the brain had measurements of the ONSD on a T2-weighted thin section sequence of the orbit. Besides establishing modality-related normal values, age dependency, accuracy and reproducibility of measurements were assessed. RESULTS: Overall the mean ONSD was 5.75 ± 0.52 mm for transbulbar sonography and 5.69 ± 0.31 mm for MRI. There was no statistical significance between the 95 % percentiles and age for both transbulbar sonography (p = 0.332) and MRI (p = 0.336). As a parameter for the reproducibility of measurements, the repeatability coefficient (RC) was between 0.34 mm and 0.46 mm. The concordance correlation coefficient (CCC) values revealed a high agreement between readers both for transbulbar sonography (0.868) and MRI (0.796). CONCLUSION: Normal values for ONSD in children and adolescents found in this study are significantly higher than assumed. The values found for transbulbar sonography are confirmed by comparable results for MR measurements. A precise sonographic measurement technique and the consideration of normal values found hereby are essential for correct interpretation of ONSD measurements in children and adolescents.

[Difficult diagnosis in a 17-year-old patient: Type 1 diabetes? Type 2 diabetes? Or "double diabetes"?]. / Erschwerte Differenzialdiagnose bei einem 17-jährigen Patienten. Typ-1-Diabetes? Typ-2-Diabetes? Oder "Doppel-Diabetes"?

UNLABELLED: MEDICAL HISTORY AND CLINICAL FINDINGS: We report on a 17-year-old boy with elevated blood glucose levels, elevated liver enzymes and obesity (BMI 32.3 kg/m2). Clinical examination showed acanthosis nigricans and a vitiligo. The rest of the physical examination was without pathological findings. INVESTIGATIONS: The HbA1c value was 8.6 % (71 mmol/mol), and postprandial C-peptide showed a maximum level of 1.3 nmol/l. The type 1 diabetes-associated autoantibodies against protein tyrosine phosphatase IA-2 and zinc-transporter-8 were positive, while autoantibodies to glutamic acid decarboxylase and insulin were negative. There was no ketonuria. Ultrasound showed steatohepatitis. TREATMENT AND COURSE: Under therapy with metformin up to 2×1 g, blood glucose levels and liver enzymes normalized after a few weeks. After two months, the HbA1c value was 6.0 % (42.1 mmol/mol), and a weight loss of 5 kg was recorded. CONCLUSION: In obese adolescent patients with diabetes, a clear classification right from the beginning is not always possible. Characteristic findings of type 1 and type 2 diabetes may be present simultaneously. In the presented patient, monotherapy with metformin was sufficient in the first year. Close monitoring is essential to detect the transition to insulin dependence in time.

[Acute encephalopathy in salmonella infection]. / Akute Enzephalopathie bei Salmonellenenteritis.

While infections with salmonella typhi may frequently result in encephalopathy, this complication is rare in infections with salmonella enteritidis and has been rarely reported. We report about a 21 months old girl which developed encephalopathy with convulsion, ataxia and cortical blindness following salmonella infection. Moreover, we show a summary of ten previous case reports.

Type 1 diabetes risk assessment: improvement by follow-up measurements in young islet autoantibody-positive relatives.

AIMS/HYPOTHESIS: Combinations of autoantibody characteristics, including antibody number, titre, subclass and epitope have been shown to stratify type 1 diabetes risk in islet autoantibody-positive relatives. The aim of this study was to determine whether autoantibody characteristics change over time, the nature of such changes, and their implications for the development of diabetes. METHODS: Five-hundred and thirteen follow-up samples from 141 islet autoantibody-positive first-degree relatives were tested for islet autoantibody titre, IgG subclass, and GAD and IA-2 antibody epitope. All samples were categorised according to four risk stratification models. Relatives had a median follow-up of 6.8 years and 48 developed diabetes during follow-up. Survival analysis was used to determine the probability of change in risk category and of progression to diabetes. RESULTS: For each stratification model, the majority of relatives (71-81%) remained in the same risk category throughout follow-up. In the remainder, changes occurred both from lower to higher and from higher to lower risk categories. For all four models, relatives aged < 15 years were more likely to change risk category than those aged >15 years (0.001 < p < 0.03). Relatives whose autoantibody status changed from low- to high-risk categories had a higher risk of diabetes than relatives who remained in low-risk categories, and inclusion of autoantibody status during follow-up improved diabetes risk stratification in Cox proportional hazards models (p < 0.001). CONCLUSIONS/INTERPRETATION: Changes in islet autoantibodies are relevant to pathogenesis, and are likely to signal alterations in the disease process. Detection of changes through follow-up measurement will improve diabetes risk stratification, particularly in young individuals.

Interaction of the substrate radical and the 5'-deoxyadenosine-5'-methyl group in vitamin B(12) coenzyme-dependent ethanolamine deaminase.

The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B(12) coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on (2)H(4)-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled (2)H. The strongly coupled (2)H has an effective dipole distance (r(eff)) of 2.2 A, and isotropic coupling constant (A(iso)) of -0.35 MHz. The weakly coupled (2)H has r(eff) = 3.8 A and A(iso) = 0 MHz. The best (2)H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 A and are thus at closest contact. The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 A in the active site of ethanolamine deaminase.

Assessment of the existence of hyper-long axial Co(II)-N bonds in cobinamide B(12) models by using electron paramagnetic resonance spectroscopy.

Protein control of cobalt-axial nitrogen ligand bond length has been proposed to modulate the reactivity of vitamin B(12) coenzyme during the catalytic cycle of B(12)-dependent enzymes. In particular, hyper-long Co-N bonds may favor homolytic cleavage of the trans-cobalt-carbon bond in the coenzyme. X-ray crystallographic studies point to hyper-long bonds in two B(12) holoenzymes; however, mixed redox and ligand states in the crystals thwart clear conclusions. Since EPR theory predicts an increase in Co(II) hyperfine splitting as donation from the axial N-donor ligand decreases, EPR spectroscopy could clarify the X-ray results. However, the theory is apparently undermined by the similar splitting reported for the 2-picoline (2-pic) and pyridine (py) adducts of Co(II) cobinamide (Co(II)Cbi(+)), adducts thought to have long and normal Co-N axial bond lengths, respectively. Cobinamides, with the B(12) 5,6-dimethylbenzimidazole loop removed, are excellent B(12) models. We studied Co(II)Cbi(+) adducts of unhindered 4-substituted pyridines (4-X-py's) in ethylene glycol to separate orbital size effects from Co-N axial distance effects on these splittings. The linear increase in splitting with the decrease in 4-X-py basicity found is consistent with the theoretically predicted increase in unpaired electron spin density as axial N lone pair donation to Co(II) decreases. No adduct (and hence no hyper-long Co(II)-N axial bond) was formed even by 8 M 2-pic, if the 2-pic was purified by a novel Co(III)-affinity distillation procedure designed to remove trace nitrogenous ligand impurities present in 2-pic distilled in the regular manner. Adducts formed by impurities in 2-pic and other hindered pyridines misled previous investigators into attributing results to adducts with long Co-N bonds. We find that many 2-substituted py's known to form adducts with simple synthetic Co models do not bind Co(II)Cbi(+). Thus, the equatorial corrin ring sterically impedes binding, making Co(II)Cbi(+) a highly selective binding agent for unhindered sp(2) N-donor ligands. Our results resolve the apparent conflict between EPR experiment and theory. The reported Co(II) hyperfine splitting of the enzyme-bound cofactor in five B(12) enzymes is similar to that of the relevant free cofactor. The most reasonable interpretation of this similarity is that the Co-N axial bond of the bound cofactor is not hyper-long in any of the five cases.

Redox state dependence of rotamer distributions in tyrosine and neutral tyrosyl radical.

Redox state-dependent changes in the relative orientation of the phenol side chain and the peptide group in model tyrosine have been characterized using specific 2H isotopic labelling and X-band electron paramagnetic resonance (EPR) spectroscopy. Tyrosyl radicals were generated by UV photolysis of tyrosine trapped in rigid polycrystalline basic-aqueous medium at T < or = 170 K. Ring-2H(4) and beta-2H(2) substitutions on tyrosine were used to enhance the lineshape contributions from beta-hydrogen or ring-hydrogen hyperfine interactions, respectively. The EPR lineshape at 120 K of the trapped ring-2H(4)-tyrosyl radical is altered dramatically after annealing at 235 K. In contrast, the lineshape of the beta-2H(2)-tyrosyl radical is impervious to annealing. The effect of annealing on the lineshape therefore arises from a change in the isotropic hyperfine coupling between unpaired pi-electron spin density at the ring carbon atom C(1) and the beta-hydrogen nuclei, which is caused by rotational relaxation of the ring and peptide group about the C(1)-C(beta) bond. EPR simulations indicate angular distributions of the peptide group (R-) of 0 degrees < or = theta(R) < or = 30 degrees and 0 degrees < or = theta(R)< or = 18 degrees in the rigid and relaxed radical states, respectively. Redox-induced changes in the C(1)-C(beta) rotamer distribution must be accounted for in assessments of stable amino acid side chain equilibrium structures, and may influence catalytic tyrosyl radical/tyrosine function in enzymes.

Identification of dimethylbenzimidazole axial coordination and characterization of (14)N superhyperfine and nuclear quadrupole coupling in Cob(II)alamin bound to ethanolamine deaminase in a catalytically-engaged substrate radical-Cobalt(II) biradical state.

Cobalt(II)-(14)N superhyperfine and (14)N nuclear quadrupole couplings in cryotrapped free and ethanolamine deaminase-bound cob(II)alamin have been characterized in the disordered solid state by using X-band electron spin-echo envelope modulation (ESEEM) spectroscopy. Enzyme-bound cob(II)alamin was cryotrapped after formation by substrate-initiated, thermally activated cleavage of the cobalt-carbon bond of adenosylcobalamin. Free dimethylbenzimidazole axial base-on cob(II)alamin was formed by photolysis of the corresponding adenosylcobalamin and cryotrapped in glycerol-aqueous glass. Three-pulse ESEEM experiments were performed by using microwave pulse excitation at the g( perpendicular) value of Co(II) at magnetic field values of 287.0 and 345.0 mT and over a range of tau values from 227 to 1316 ns. Two common sets of (14)N features are distinguished in the ESEEM spectra. One set is assigned to the remote (N1) nitrogen in the dimethylbenzimidazole alpha-axial ligand by using two independent approaches: (a) comparison of ESEEM from cob(II)alamin with ESEEM from cob(II)inamide-ligand model compounds and (b) from the correspondence between the N1 (14)N nuclear quadrupole parameters derived from ESEEM simulations and those computed by using density functional theory. The second set is assigned to the corrin ring (14)N nuclei. The results identify the coenzyme's on-board dimethylbenzimidazole moiety as the alpha-axial ligand to cob(II)alamin in ethanolamine deaminase in the substrate radical-Co(II) biradical catalytic intermediate state. Thus, Co(II) is a pentacoordinate, alpha-axial liganded complex during turnover. We infer that dimethylbenzimidazole is also the alpha-axial ligand to the intact coenzyme in the resting enzyme. A 14% increase in the isotropic hyperfine coupling of the remote dimethylbenzimidazole (14)N nucleus in enzyme-bound versus free base-on cob(II)alamin shows an enhanced delocalization of unpaired spin density from Co(II) onto the axial ligand, which would contribute to the acceleration of the cobalt-carbon bond cleavage rate in situ.

Variations of p53 in cultured fibroblasts of patients with lung cancer who have a presumed genetic predisposition.

The authors studied cultured skin fibroblasts of 64 patients with lung cancer for constitutive mutations of the p53 tumor suppressor gene by using polymerase chain reaction and single-strand conformation polymorphism covering the entire coding region. The patients were considered to be genetically predisposed because lung cancer had developed in 25 of them before age 46 and because 42 of them had at least one first-degree relative with lung cancer. One mutation was detected at position 235 coding for serine instead of asparagine in the conserved DNA binding domain. The Pro/Pro genotype at codon 72 of p53, considered to harbor an increased risk for lung cancer, could not be detected with increased frequency in this study's patients. From these data, the authors conclude that constitutive variations of the p53 gene do not represent a major genetic determinant for lung cancer.

Tyrosyl radicals in enzyme catalysis: some properties and a focus on photosynthetic water oxidation.

Enzymes that require a redox-active amino acid for catalysis or function have emerged as a distinct class of proteins. For the tyrosine-based radical enzymes, we show that the spin-density distribution in the radical follows an odd alternate pattern that is invariant to within 10% across the class. General properties of the radical enzymes are summarized from which we conclude that their essential role in catalysis is to initiate substrate metabolism by hydrogen-atom abstraction. These ideas are extended to the YZ and YD tyrosines in Photosystem II and a radical-based hydrogen-atom abstraction model for water oxidation is discussed. Differences in rates of oxidation of YZ and YD by the reaction-center chlorophyll, P680+, under various conditions, are considered and rationalized on the basis of changes in reorganization energy induced by the local protein structure and by the presence or absence of the (Mn)4 cluster that binds substrate water.

Sleep deprivation does not induce sister chromatid exchange in humans.

In a preliminary study Bamezai and Kumar (1992) reported that a 24-h period of sleep deprivation may raise sister chromatid exchange (SCE) frequencies up to 193% in peripheral blood lymphocytes. This was reinvestigated to clarify the role of sleep duration as a confounder for SCE, which is a well-established parameter for biomonitoring in occupational medicine. In our study, the SCE baseline and the influence of a 24-h period of sleep deprivation (test period) on SCE were investigated for 20 non-smoking volunteers (10 females and 10 males; 20-29 years of age). There was no significant difference (Pall = 0.094) between the deviations of the two SCE rates of the control period (mean: -0.21 +/- 0.90 SCE) and the differences between SCE rates before and after sleep deprivation (mean: 0.42 +/- 0.94 SCE) of each proband. No significant difference was detected between females and males, and SCE did not correlate with age or sleep duration. Therefore we conclude that the influence of sleep deficit on SCE is in the range of a normal day-to-day variance, and has not to be taken into account when SCE is used for a genotoxic monitoring at the workplaces.

Determination of low level exposure to volatile aromatic hydrocarbons and genotoxic effects in workers at a styrene plant.

OBJECTIVES: Low exposures to volatile aromatic hydrocarbons and cytogenetic effects in peripheral white blood cells were determined in 25 healthy workers employed in different areas of a styrene production plant in the former German Democratic Republic. The results were compared with 25 healthy unexposed controls (matched for age and sex) employed in the same company. METHODS: The concentrations of aromatic hydrocarbons determined from active air sampling in all areas of the factory (styrene: 73-3540 micrograms/m3 (< 0.01-0.83 ppm); ethylbenzene 365-2340 micrograms/m3 (0.08-0.53 ppm); benzene 73-3540 micrograms/m3 ( < 0.02-1.11 ppm); toluene 54-2960 micrograms/m3 (0.01-0.78 ppm); xylenes 12-94 micrograms/m3 ( < 0.01-0.02 ppm)) were considerably lower than in the pump house ( > 4000 micrograms/m3 styrene, ethylbenzene, benzene, and toluene; > 500 micrograms/m3 xylenes), which was only intermittently occupied for short periods. Passive personal monitoring, biomonitoring of exhaled air and metabolites (mandelic, phenylglyoxylic, trans, trans-muconic, hippuric, o-, m- and p-methylhippuric acids, and phenol) in urine samples collected before and after an eight hour working shift was used to assess individual exposure. Questionnaires and examination of company records showed that the historical exposure was far higher than that measured. Genotoxic monitoring was performed by nuclease P1-enhanced 32P-postlabelling of DNA adducts in peripheral blood monocytes, and DNA single strand breaks, sister chromatid exchange, and micronuclei in lymphocytes. The content of kinetochores in the micronuclei was determined by immunofluorescence with specific antibodies from the serum of CREST patients. RESULTS: No genotoxic effects related to exposure were detected by DNA adducts or DNA single strand breaks and sister chromatid exchange. The only effect related to exposure was an increase in kinetochore positive micronuclei in peripheral lymphocytes; the frequency of total micronuclei in peripheral lymphocytes did not change. Smoking was confirmed by measurement of plasma cotinine, and no confounding effect was found on any of the cytogenetic variables. CONCLUSIONS: Low occupational exposure to styrene, benzene, and ethylbenzene did not induce alterations of genotoxicological variables except kinetochore positive micronuclei. This is the first reported use of the CREST technique for an in vivo study in occupational toxicology, which thus could serve as a valuable and sensitive technique for toxicogenic monitoring.

A hydrogen-atom abstraction model for the function of YZ in photosynthetic oxygen evolution.

Recent magnetic-resonance work on YZ suggests that this species exhibits considerable motional flexibility in its functional site and that its phenol oxygen is not involved in a well-ordered hydrogen-bond interaction (Tang et al., submitted; Tommos et al., in press). Both of these observations are inconsistent with a simple electron-transfer function for this radical in photosynthetic water oxidation. By considering the roles of catalytically active amino acid radicals in other enzymes and recent data on the water-oxidation process in Photosystem II, we rationalize these observations by suggesting that YZ functions to abstract hydrogen atoms from aquo- and hydroxy-bound managanese ions in the (Mn)4 cluster on each S-state transition. The hydrogen-atom abstraction process may occur either by sequential or concerted kinetic pathways. Within this model, the (Mn)4/YZ center forms a single catalytic center that comprises the Oxygen Evolving Complex in Photosystem II.

Determination of DNA single-strand breaks in lymphocytes of smokers and nonsmokers exposed to environmental tobacco smoke using the nick translation assay.

The detection of DNA single-strand breaks (SSB) in human mononucleated white blood cells (MWBC) using a modified version of the nick translation assay is presented. This assay allows rapid and sensitive examination of SSB using only 5 ml heparinized blood for an eightfold determination. The assay was standardized by incubation of MBWC in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a known genotoxic agent. In vitro incubation of MWBC with MNNG induced a dose-dependent increase in DNA-SSB at doses between 5 and 500 microM MNNG. The detection limit for the assay was 5 microM MNNG. To assess the suitability of this assay to detect SSB in vivo a controlled study was performed in which volunteer smokers (n = 5), nonsmokers (n = 5) exposed to environmental tobacco smoke (ETS), and nonsmokers controls (n = 5) were compared. The study lasted 4 experimental days, 2 control and 2 exposure days. On control days (days 1 and 3) smokers and nonsmokers sat in an unventilated 45 m3 room for 8 h. On the exposure days (days 2 and 4) each of the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the ETS generated by the smoking volunteers. High exposure to tobacco smoke was confirmed by dosimetry of carboxyhemoglobin (CO-Hb), plasma nicotine and cotinine levels. Blood was drawn before and after each exposure on all 4 experimental days for determination of DNA-SSB in lymphocytes immediately after isolation of blood cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of hydrocarbon tail structure on quinone binding and electron-transfer performance at the QA and QB sites of the photosynthetic reaction center protein.

Binding free energies of 37 functional replacement quinone cofactors with systematically altered hydrocarbon tail structures have been determined for the QA and QB redox catalytic sites of the reaction center protein isolated from Rhodobacter sphaeroides and solubilized in aqueous and in hexane solutions. The first two and part of the third isoprene units of the 10-unit tail of the native ubiquinone-10 cofactor interact with the protein interior at each site. Contributions of the same tail structures to the binding free energies of quinones at the QA and QB sites are comparable, suggesting that the binding domains share common features. Comparison of the affinities of a homologous series of 10 n-alkyl-substituted ubiquinones resolves the binding forces along the length of the tail binding domain and shows that strong steric constraints oppose accommodation of the tail in its extended conformation. Differences in the contributions of identical tail substituents to ubiquinone- and menaquinone-QA site affinities, and tail-induced changes of up to 5-fold in the rates of QA site-mediated electron-transfer reactions, suggest that the tail adjusts the position of the quinone ring. Substitution of ubiquinone with the native 10-unit isoprene tail does not alter the affinity for the sites as determined in hexane solution. However, one- and two-isoprene-substituted quinones bind more tightly than analogs substituted with saturated-alkyl tail substituents. The sites therefore exhibit binding specificity for the native isoprene tail structure. Calculations indicate that the binding specificity arises primarily from a lower integrated torsion potential energy in the bound isoprene tails. The results suggest that the in vivo tail-protein interaction is designed to deter competitive interference of quinone function by amphiphilic species present in the native membrane.

Influence of QA site redox cofactor structure on equilibrium binding, in situ electrochemistry, and electron-transfer performance in the photosynthetic reaction center protein.

The native ubiquinone-10 redox cofactor has been removed from the QA site of the isolated reaction center protein from Rhodobacter sphaeroides and reconstitution attempted with 28 non-quinone molecules in order to identify factors governing cofactor function and the selectivity displayed by the site in the electron transfers that it catalyzes. Equilibrium binding, in situ electrochemistry, and the kinetics of electron transfer to and from the QA site occupant were examined. Four classes of non-quinone molecules are distinguished according to their ability to occupy the QA site and conduct intraprotein electron transfers. The minimal requirements for occupancy of the QA site are at least one ring and a heteroatom hydrogen bond acceptor. Thus, binding at the site is not highly selective. The rates of electron transfers to and from the class of non-quinone molecules (four) that satisfy the criteria for cofactor function at the QA site compare well with rates previously determined from 14 to 295 K for 14 quinone replacements with comparable values of the reaction free energy. This indicates that the rates are relatively insensitive to variations in exotic and quinone cofactor reorganization energy and the vibrational frequencies coupled to the electron transfers, and that the exotic and quinone cofactors are bound in the QA site in comparable positions. It appears that any variation in rate is determined predominantly by the value of the reaction free energy. The QA site protein-cofactor solvation contribution to the in situ electrochemical potential is roughly constant for 12 rigid quinone and 2 exotic cofactors (average value-61 +/- 2 kcal/mol). Favorable electrostatic contributions governing the reaction free energy are therefore also relatively insensitive to cofactor structure. However, flexible molecules appear to encounter in situ steric constraints that lower the electron affinity by destabilizing the reduced cofactor species. This is a strong determinant of whether a molecule, once in the QA site, will function. These findings compare well with those from studies of electron transfers in synthetic systems.

Experimental resolution of the free energies of aqueous solvation contributions to ligand-protein binding: quinone-QA site interactions in the photosynthetic reaction center protein.

Equilibrium binding free energies of 14 benzo-, naphtho-, and anthraquinone cofactors have been determined at the QA redox catalytic site of the purified photosynthetic reaction center protein from Rhodobacter sphaeroides solubilized in water (delta G degrees B,w), in hexane solution containing 30 mM water (delta G degrees B,hh), and after partial dehydration (delta G degrees B,dh) with magnesium sulfate. Our aim is to resolve the contributions of aqueous bulk phase solvation and protein hydration contributions to binding in order to characterize in detail the direct interactions between the ligands and protein at the QA site. This is accomplished by comparing the differences between delta G degrees B,w and delta G degrees B,hh (or delta G degrees B,dh) with the water to hexane solvent transfer free energies of the quinones (delta G degrees tr,Q). Values of delta G degrees tr,Q are determined separately in binary solution and range from 0.65 to -5.69 kcal/mol (1 cal = 4.184 J). The results are interpreted in terms of a thermodynamic cycle that links the species involved in the binding and solvent transfer equilibria. Values of delta G degrees B,hh -delta G degrees B,w are linearly correlated with -delta G degrees tr,Q (slope, 0.78 +/- 0.04; ordinate intercept, -0.13 +/- 0.12 kcal/mol). The deviation of the experimental slopes from the predicted value of unity is attributed in part to a systematic decrease of quinone thermodynamic activity in the aqueous binding medium relative to the aqueous phase in the binary partitioning solvent system. The difference between the quinone-QA site binding free energies measured in hydrated hexane and water is therefore related only to the difference in bulk phase quinone solvation, as given by 0.78 delta G degrees tr,Q. The linear relation obtained using delta G degrees B,dh -delta G degrees B,w has the same slope, but the intercept is decreased to -1.48 +/- 0.19 kcal/mol, indicating that quinone binding strengths in the hexane system are uniformly enhanced after partial dehydration. This suggests that the quinones encounter a common opposition to interaction with the site in the hydrated, relative to the partially dehydrated, state. The further utility of the method to directly assess ligand-site binding free energies is demonstrated with examples that address the contributions of molecular size and dipolar or hydrogen bond interactions to the binding of quinones at the QA site.

Detection of DNA single-strand breaks in lymphocytes of smokers.

In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five nonsmokers, not exposed to cigarette smoke. During the 4 days of the study, nonsmoking periods were strictly controlled. On the first day the ten subjects were sham exposed. On the second day the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the environmental tobacco smoke. After another day of sham exposure the smoke exposure was repeated under the same conditions. Blood was drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cells and after 4 h incubation at 37 degrees C, using a modified assay based on the nick translation reaction. Base levels of unscheduled DNA synthesis (UDS) and UDS levels were determined after 1 h incubation with methyl methanesulfonate. Duplicate analysis using the same method was performed in a second laboratory after transportation of blood samples at 0 degree C on a train from Munich to Hamburg. Tobacco smoke exposure of the subjects increased COHb and plasma cotinine levels. SSBs could be detected in all probands with some interindividual day-to-day and morning-to-evening variations. In four of five active smokers, SSB increases were found after smoking. In nonsmokers exposed to tobacco smoke no exposure-related variation in SSB levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Nature of biological electron transfer.

Powerful first-order analysis of intraprotein electron transfer is developed from electron-transfer measurements both in biological and in chemical systems. A variation of 20 A in the distance between donors and acceptors in protein changes the electron-transfer rate by 10(12)-fold. Protein presents a uniform electronic barrier to electron tunnelling and a uniform nuclear characteristic frequency, properties similar to an organic glass. Selection of distance, free energy and reorganization energy are sufficient to define rate and directional specificity of biological electron transfer, meeting physiological requirements in diverse systems.