Biomedical subjects

R Bernhardt

Publications and source records attributed to R Bernhardt.

At least 19 recordsLinked to original sources

PubMed1995

Rabbit liver cytochrome P-450 2B5: high-level expression of the full-length protein in Escherichia coli, purification, and catalytic activity.

Rabbit liver cytochrome P-450 2B5 (P-450 2B5) was expressed in Escherichia coli using the D(+)-galactose-inducible expression vector pJL-2, containing the full-length cDNA encoding P-450 2B5. Stimulation by galactose of protein synthesis in the presence of the heme precursor 5-aminolevulinic acid peaked 72 h after addition to the inducer to yield 108 nmol membrane-bound P-450 2B5 per liter of culture medium. The recombinant enzyme was purified to near homogeneity by a two-column procedure involving chromatography on DE-52 cellulose and hydroxylapatite. The hemoprotein was isolated mainly in the low-spin iron configuration and exhibited a reduced CO-difference spectrum with a Soret band at 451 nm. Second-derivative spectral analysis in the middle-UV region revealed that type I binding of 4-nitroanisole to ferric P-450 2B5 abolished absorption bands ascribable to tyrosine residues within the polypeptide chain. Pseudo-first-order rates of NADPH-driven reduction of the pigment were lower when reconstituted with NADPH-cytochrome P-450 reductase than with the mitochondrial adrenodoxin/NADPH-adrenodoxin reductase redox couple. The enzyme was catalytically active toward 4-nitroanisole and androstenedione; metabolic rates were enhanced to different extents by the presence of cytochrome b5. The recombinant hemoprotein did not catalyze bioactivation of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, a potent pulmonary carcinogen. The methods described here should facilitate further studies on the biophysical basis of the complex interactions of P-450 2B5 with its redox partners.

Amino Acid Sequence↗

PubMed1995

Mutational effects on the spectroscopic properties and biological activities of oxidized bovine adrenodoxin, and their structural implications.

Of the aromatic 1H-NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a through-space ring-current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P-450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation. As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P-450(11 beta) (CYP11B1)-dependent and P-450scc (CYP11A1)-dependent substrate conversions were studied. No influence on Vmax values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the Km values were increased, as was the Ks value for binding of CYP11A1 to the [H56T]adrenodoxin. The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary-structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent-exposed region of the molecule.

Adrenodoxin↗

PubMed1995

Conformational stability of bovine holo and apo adrenodoxin--a scanning calorimetric study.

Holo and apo adrenodoxin were studied by differential scanning calorimetry, absorption spectroscopy, limited proteolysis, and size-exclusion chromatography. To determine the conformational stability of adrenodoxin, a method was found that prevents the irreversible destruction of the iron-sulfur center. The approach makes use of a buffer solution that contains sodium sulfide and mercaptoethanol. The thermal transition of adrenodoxin takes place at Ttrs = 46-57 degrees C, depending on the Na2S concentration with a denaturation enthalpy of delta H = 300-380 kJ/mol. From delta H versus Ttrs a heat capacity change was determined as delta Cp = 7.5 +/- 1.2 kJ/mol/K. The apo protein is less stable than the holo protein as judged by the lower denaturation enthalpy (delta H = 93 +/- 14 kJ/mol at Ttrs = 37.4 +/- 3.3 degrees C) and the higher proteolytic susceptibility. The importance of the iron-sulfur cluster for the conformational stability of adrenodoxin and some conditions for refolding of the thermally denatured protein are discussed.

Adrenodoxin↗

PubMed1995

Cloning and stable expression of the human mitochondrial cytochrome P45011B1 cDNA in V79 Chinese hamster cells and their application for testing of potential inhibitors.

V79 Chinese hamster cells are being genetically engineered to express human mitochondrial cytochromes P450 as an analytical tool for studying adrenal steroid synthesis. Here, a V79 derived cell line is presented expressing the enzymatically active human cytochrome P45011B1 (CYP11B1) in a stable and constitutive manner. Full length CYP11B1 cDNA was obtained from surgically removed normal adrenal gland by polymerase chain reaction. The cDNA was recombined with a SV40 early promoter containing plasmid for stable integration and expression in V79 cells upon gene transfer. The presence of the human CYP11B1 cDNA in the genome of the transfected cells was confirmed by Southern analysis. CYP11B1 cDNA directed expression was detected by Northern analysis. CYP11B1 dependent hydroxylation of deoxycorticosterone and 11-deoxycortisol was measured by HPLC analysis. Interestingly, the nonsteroidogenic lung fibroblast derived V79 Chinese hamster cell line was able to support human CYP11B1 mediated steroid hydroxylation without simultaneous heterologous expression of the human electron transfer system, adrenodoxin, and adrenodoxin reductase. CYP11B1 inhibitory potency of metyrapone, spironolactone, and the azole derivatives ketoconazole, clotrimazole, miconazole, and fluconazole was measured using the newly established V79MZh11B1 cell line. Thus, beside steroid metabolism studies in general, this cell line may also serve as an in vitro tool for monitoring the interference of drugs with 11 beta-hydroxylase activity.

Adrenal Glands↗

PubMed1992

Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2 Fe-2S/ cluster ligands.

Expression systems for adrenodoxin into the periplasm and the cytoplasm of E. coli have been developed as a prerequisite for site-directed mutagenesis studies. In both systems the /2Fe-2S/ cluster of the protein was correctly assembled, the cytoplasmic one gives, however, a tenfold higher expression level. To determine which of the five cysteines at positions 46, 52, 55, 92, and 95 coordinate the /2Fe-2S/ center, they have been individually mutated into serines. From these mutants, only C95S forms a functionally active holoprotein. Thus, residues 46, 52, 55, and 92 are the cysteines that coordinate the /2Fe-2S/ cluster in adrenodoxin.

Adrenodoxin↗

PubMed1992

Reconstitution of cytochrome P4502B4 (LM2) activity with camphor and linalool monooxygenase electron donors.

The P4502B4 (LM2) monooxygenase was shown to bind the P450cam and P450lin redoxins with respective dissociation constants of ca. 500 and 100 microM. When the redoxin reductases are added, the heterogeneous complexes demethylate benzphetamine with HCHO yields and Vmax similar to results with the homologous microsomal reductase. Implications of evolutionary conserved P450 redox electron donor binding site residues are discussed.

Acyclic Monoterpenes↗

PubMed1992

Risk factor patterns for coronary heart disease in China, Japan and Germany.

The risk factor patterns for coronary heart disease in China, Japan and Germany were studied. 6,025 Germans, 7,580 Japanese and 2,047 Chinese aged 30-59 were investigated following the protocol of the Göttingen Risk, Incidence, and Prevalence Study carried out in West Germany in 1982. It is concluded that in China, the risk factor intervention focuses mainly on smoking and hypertension; smoking also remains the most important risk factor in Japan; while in Germany the major targets are obesity and hyperlipidemia. However, about 38% of the participants from West Germany showed 3 or more risk factors accumulated per person. Thus the multifactorial risk factor reduction might be necessary in Germany.

Adult↗

PubMed1991

Differences in the risk factor patterns for coronary heart disease in China, Japan, and Germany.

In Asia coronary heart disease mortality is almost 10 fold less frequent than in European countries. These findings attract interest to search for different risk factor patterns. From 1982 to 1985 epidemiologic surveys were carried out in China (n = 2047), Japan (n = 7580) and Germany (n = 6052). Healthy, male subjects, aged 30 to 59 years were enrolled. The prevalence rate of hypertension for the Germans was 20% versus 18% for the Japanese, and 11% for the Chinese. About 69% of Chinese, 55% of Japanese, and 37% of Germans were smokers. About 66% of the Germans were overweight (BMI greater than 25), 17% of the Japanese, and 11% of the Chinese. The highest risk group with cholesterol levels of greater than 300 mg/dl included no Chinese subject, 0.1% of the Japanese, but 5% of the Germans. The lipoprotein profiles among the Japanese and the Chinese collectives typically showed antiatherosclerotic characteristics, whereas most Germans exhibited profiles which support development of atherosclerosis. About 36% of the participants from Germany showed 3 or more risk factors accumulated per person (Japan and China 5%). Multifactorial risk factor reduction for Germany is recommended.

Adult↗

PubMed1991

Incidence and mortality rates of myocardial infarction in Chinese workers aged 40-59 in relation to coronary risk factors. Results of a Chinese prospective study (Wuhan Study) in comparison to the Göttingen Risk Incidence and Prevalence Study (GRIPS).

Some 2045 male Chinese industrial workers aged 40-59 years living in the city of Wuhan in the People's Republic of China were examined for coronary risk factors in the year 1983. The investigation included a patient history, clinical examination, and ECG and laboratory tests, with special attention to serum lipids. After 5 years, a follow-up investigation of the study group was carried out. The results were compared to the similarly designed German GRIPS project. In comparison to the German population, significantly lower levels for total-, LDL-, and VLDL-cholesterol, apolipoprotein B, triglycerides, uric acid, body mass index, and diastolic blood pressure were found in China. The percentage of smokers, however, was remarkably higher in China than in the Federal Republic of Germany. During the 5 year observation period in the Chinese sample, four subjects suffered from sudden death and four from nonfatal myocardial infarction; in the German study group three times as many fatal myocardial infarction and cases of sudden death and 7.5 times as many nonfatal myocardial infarctions were recorded. Nonfatal coronary heart disease and peripheral vascular disease were also observed less often in China. The incidence of cerebrovascular diseases was 1.5 times higher in China than in Germany. Whereas in Germany, total-, and LDL-cholesterol values were the major distinguishing parameters between infarction and reference groups, in China these values have thus far had no significant influence on the level of risk. Instead in the Chinese incidence group, significantly higher levels for blood pressure, body mass index, uric acid, and the ratio LDL/HDL-cholesterol were found.

Adult↗

PubMed1990

Socioeconomic differences in cardiovascular risk factors in China.

Socioeconomic conditions are important in explaining variation in cardiovascular morbidity in advanced societies. To analyse whether cardiovascular risk factors vary according to socioeconomic status in a developing country, and more specifically, in an urban area of China, we compared data from structured interviews and cardiovascular screenings in a group of 1169 male workers (45-65 years). These men participated in the five-year follow-up of a prospective investigation started in 1982/83 in several plants in Wuhan. Unhealthy behaviours such as heavy cigarette smoking and alcohol consumption were significantly more prevalent among men with low educational attainment and with jobs characterized by heavy physical workload, noise and heat. Men paid according to a new, highly demanding salary system exhibited significant increases in total cholesterol and systolic blood pressure from first to second screening. In conclusion, our findings show significant socioeconomic variation in cardiovascular risk factors among middle-aged male workers in China.

Aged↗

PubMed1989

Two forms of omega-hydroxylase toward prostaglandin A and laurate. cDNA cloning and their expression.

We have isolated cDNA clones for two forms of P-450s, P-450ka-1 and P-450ka-2, from a rabbit kidney cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase (Matsubara, S., Yamamoto, S., Sogawa, K., Yokotani, N., Fujii-Kuriyama, Y., Haniu, M., Shively, J.E., Gotoh, O., Kusunose, E., and Kusunose, M. (1987) J. Biol. Chem. 262, 13366-13371), as a hybridization probe. The cDNAs for P-450ka-1 and P-450ka-2 encode polypeptides of 510 and 511 amino acids, respectively, with sequence similarity of 85% and 87% to P-450p-2. The two deduced primary structures have 87% identity. RNA blot analysis demonstrated that the mRNAs for P-450ka-1 and P-450ka-2 formed single bands at approximately 3.0- and 2.6-kilobase positions, respectively. The mRNA for P-450ka-1 was expressed only in the liver and kidney and was increased remarkably in these tissues by the administration of clofibrate. In contrast, the mRNA for P-450ka-2 was expressed constitutively in the liver, kidney, and small intestine, but its transcription was enhanced only in the liver by clofibrate treatment. Thus, in spite of their high sequence similarity, these P-450 species have different modes of regulatory expression. Comparison of the nucleotide sequences among P-450ka-1, P-450ka-2, and P-450p-2 shows about 90% overall sequence similarity in any pair of the three sequences. Nucleotide replacements are not evenly distributed, but are rather biased. There is a region of approximately 500 base pairs of exceptionally high homology among the three sequences. These results indicate that the gene conversion event occurred during the evolutionary process of these genes.

Amino Acid Sequence↗

PubMed1989

Distance between lysine 384 and heme of cytochrome P-450 LM2 (P-450 IIB4) studied by fluorescence energy transfer measurements.

The distance between FITC-modified lysine 384 of cytochrome P-450 LM2 and the active site, heme, was estimated by fluorescence energy transfer measurements. To avoid differential labelling of P-450 LM2 for protection of the alpha-amino group from FITC modification, deconvolution of measured fluorescence decay curves using a double exponential model was performed. A value of 2.7 nm was obtained for the distance FITC (lysine 384) - heme. This distance is too large to account for a direct electron tunneling from prosthetic group to prosthetic group at this interaction site between reductase and P-450 LM2.

Animals↗

PubMed1989

Axonal pathfinding during the regeneration of the goldfish optic pathway.

Retinal ganglion cells in fish and amphibians regenerate their axons after transection of the optic nerve. Fiber tracing studies during the third month of regeneration show that the axons have reestablished a basically normal fiber order in the two brachia of the optic tract; axons originating in the ventral hemiretina are concentrated in the dorsal brachium, axons from the dorsal hemiretina in the ventral brachium. Attardi and Sperry (Exp. Neurol. 7:46-64, 1963) first suggested that the reestablishment of the fiber order reflects path-finding by the regenerating axons. Recently, however, Becker and Cook (Development 101:323-337, 1987) have claimed that the fiber order observed at later stages of regeneration is due to secondary axonal rearrangements and that the initial brachial choice is random. In order to evaluate whether regenerating axons are capable of navigating in the optic tract and brachia and on the tectum, the present study examined the pathway choices and the morphology of regenerating axons en route to their tectal targets in goldfish. Subsets of axons were labeled at various time intervals (2 to 30 days) following an optic nerve crush, by intraretinal application of the lipophilic fluorescent tracer 1,1-dioctadecyl-3-3-3'-3'-tetramethylcarbocyanine (DiI). After a survival time of 18 to 72 hours (to allow for diffusion of DiI along the axons), the experimental animals were perfused with fixative and their right and left optic pathways (nerve, tract, and tectum) were dissected free and separated at the chiasm. Fluorescently labeled axons were traced in whole-mounted pathways. Pathway choices were examined at the brachial bifurcation where axons from ventral and dorsal hemiretinae normally segregate. DiI was found to label axons reliably up to their growth cones, even at the earliest stages of regrowth. The pathway choices of the axons were nonrandom. The majority of the ventral axons reached the appropriate, dorsal hemitectum through the appropriate dorsal brachium of the tract. Dorsal axons reached the ventral hemitectum mainly through the ventral brachium. This suggests the presence of specific guidance cues, accessible to the regenerating axons. Differences in the complexity of the growth cones of the regenerating axons (simple in the nerve and tectal fiber layer, complex in the tract and the synaptic layer of the tectum) provide further evidence for specific interactions between the regenerating axons and their substrates along the pathway. These results argue that regenerating retinal axons in fish are capable of axonal path-finding.

Animals↗

PubMed1988

Regenerated optic fibers in goldfish reestablish a crude sectoral order in the visual pathway.

The goldfish optic pathway is regenerated after an optic nerve crush. We have examined the axonal topography of the regenerated pathway by labeling, with horseradish peroxidase (HRP), axons originating from retinal sectors or annuli. The positions of the labeled axons in the cross section of the pathway were compared to the normal and related to the factors that may influence axonal pathfinding. The positions of retinal axons in the cross section of the normal pathway are predictable from the retinal addresses of the ganglion cells described by the polar coordinates r (the distance from the optic disc) and theta (the sectoral or clockface position). The two coordinates map orthogonally onto the cross section of the pathway; r varies monotonically along one axis; theta varies along a perpendicular axis. The normal r-order, present in the nonregenerated stump of the experimental nerve, was severely degraded and perhaps lost entirely in the regenerated optic nerve, tract, and brachia. Sectoral order was also lost as the axons passed the crush site, but it was reestablished, albeit crudely, in the regenerated tract and brachia where axons tended to occupy positions appropriate to their dorsal, ventral, nasal, and temporal retinal origins. The exit sequence of the regenerated axons from the stratum opticum into the tectal neuropil was normal: temporal first, nasal last. These results suggest that the regenerating fibers followed some theta-specific cue located in the nonaxonal environment. It seems likely that the original axons probably followed the same cue. In contrast, the absence of r-order suggests that there is no r-specific cue for the regenerates to follow. It seems likely that the original r-order was a consequence of nonspecific influences--the orderly spatiotemporal growth of the retina and the existence of a permissive region for axonal growth.

Animals↗

PubMed1988

Axons added to the regenerated visual pathway of goldfish establish a normal fiber topography along the age-axis.

Throughout a goldfish's life, new generations of ganglion cells are added on the retinal margin and their axons extend centrally to occupy predictable positions in the retinotectal pathway, adjacent to their predecessors and subjacent to the pia. The stacking of successive generations of axons defines the age-axis of the pathway. This study examined whether an ordered array of predecessor axons is a prerequisite for the patterned growth of new axons. One optic nerve was crushed intraorbitally and the fish was injected with 3H-thymidine to label the proliferating cells on the retinal margin. The ring of 3H-thymidine-labeled cells separated retina that was present at the time of nerve crush (inside the ring) from new retina added afterward (outside). After a period of 14-16 months postcrush, both tectal lobes received two punctate applications of horseradish peroxidase (HRP), one in the central and the other in peripheral tectum, to retrogradely label contralateral retinal ganglion cell bodies and their axons. The pattern of HRP labeling from the control tectum confirmed earlier work: axons on the central tectum had somata in the central retina, and axons on the peripheral tectum had somata in the peripheral retina. The labeled cells and axons were both in predictable patterns. The somata that were backfilled from applications to the center of the experimental tectum lay inside the radioactive ring and had therefore regenerated their axons. The patterns of their labeled axons in the optic pathway and of their somata in the retina were typical of the regenerated condition as described in earlier studies. The somata backfilled from the periphery of the experimental tectum were outside the radioactive ring and had been added after the optic nerve crush. The patterns of their labeled axons and somata were comparable to the normal pattern. These observations indicate that new axons do not depend on an ordered array of predecessors to reestablish normal order along the age-axis of the pathway.

Aging↗

PubMed1988

Electrostatic interactions between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase.

At pH 8.2 and a 100-fold molar excess of the amino group specific label 2-methoxy-5-nitrotropone (MNT) over protein 2 mol MNT/mol P-450 LM2 were bound, which caused a 50% decrease in the overall activity due to a decreased electron transfer rate from reductase to the hemoprotein. However, different from FITC modification, which produces the same effects, the label is not selectively bound to the alpha-amino group and to lysine 384, but reacts with lysines in positions 49, 100, 139, 144, 251, 384 and 433. The decrease in the overall activity and reduction rate thereby correlates with a relative increase in the modification of lysines 139, 144, 251 and 384. Thus, besides lysine 384 the epsilon-amino groups of lysines 139, 144 and 251 are further candidates for participation in the interaction with reductase. This finding supports our model of charge-pair contacts between P-450 and reductase, where amino groups of P-450 LM2 form salt bridges to carboxylic groups of reductase. The decrease of reductase supported P-450 reduction velocity in microsomes at high salt concentration (I greater than 222 mM) indicates the dominant electrostatic character of P-450/reductase interaction. Based on these results and data from the literature a model of membrane topography of P-450 LM2 has been proposed. Extension of the charge pair interaction model to interaction mechanisms of other P-450 isoenzymes and forms with their respective electron donors is discussed.

Amino Acid Sequence↗

PubMed1988

A simple determination of the sideness of the NH2-terminus in the membrane bound cytochrome P-450 LM2.

A simple and fast procedure for determining the sideness of the NH2-terminus in membrane proteins using FITC as label has been developed and applied to microsomal cytochrome P-450 LM2. The main advantages of FITC, which was shown not to penetrate lipid membranes, as compared to other labels are that it is fluorescent and that it can be used for manual microsequencing of proteins. By use of FITC it was directly shown that the NH2-terminus of P-450 LM2 is localized at the cytoplasmic side of the endoplasmic reticulum membrane. Based on this result the validity of recently published models of the membrane topology and the membrane insertion of P-450 are discussed.

Animals↗

PubMed1987

Modification of carboxyl groups on NADPH-cytochrome P-450 reductase involved in binding of cytochromes c and P-450 LM2.

Carboxyl groups of NADPH-cytochrome P-450 reductase have been modified with the water-soluble carbodiimide EDC. Although there is no significant loss in DCPIP reduction the activity with cytochrome c and cytochrome P-450 LM2 as electron acceptors was inhibited by about 60 and 85%, respectively (1 h incubation time, 20 mM EDC). The inactivation by EDC was nearly completely prevented in the presence of cytochrome P-450 LM2, but not by bovine serum albumin. These results and crosslinking studies suggest that carboxyl groups of NADPH-cytochrome P-450 reductase are involved in charge-pair interactions to cytochrome c and to at least two amino groups of cytochrome P-450 LM2.

Animals↗