Coherent dynamics of excitonic wave packets.
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Journal Article↗
Biomedical subjects
C Hartmann
Publications and source records attributed to C Hartmann.
Exciton dynamics in InxGa1-xAs/GaAs quantum-well heterostructures: Competition between capture and thermal emission.
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Journal Article↗
Spectroscopic detection of chemical intermediates in the reaction of para-substituted benzylamines with bovine serum amine oxidase.
Anaerobic, rapid-scanning stopped-flow spectroscopy has been used to investigate the UV-visible absorbance changes (300-540 nm) that occur in the spectrum of bovine serum amine oxidase during reduction by benzylamine, p-hydroxybenzylamine, and p-methoxybenzylamine. The reaction of enzyme with benzylamine generates detectable relaxations at 310, 340, and 480 nm, which are attributed to the production of reduced cofactor (310 and 480 nm) and to an enzyme-substrate Schiff base complex (340 nm). Additional transients have been observed at 440, 425, and 460 nm with p-hydroxybenzylamine, p-methoxybenzylamine, and p-(N,N-dimethylamino)benzylamine, respectively. These relaxations are ascribed to quinonoid species, formed reversibly from Schiff base complexes between oxidized product and reduced cofactor. With the spectral detection of enzyme-product Schiff base complexes, evidence now exists for each of the postulated chemical intermediates along the reaction path of bovine serum amine oxidase [cf. Hartmann, C., & Klinman, J. P. (1991) Biochemistry 30, 4605]. Anaerobic, single-wavelength stopped-flow data, collected in conjunction with rapid-scanning studies for benzylamine and p-hydroxy-benzylamine, provide approximate rate constants for each of the kinetic processes corresponding to enzyme-substrate Schiff base formation, to enzyme reduction, and to the formation and decay of the quinonoid intermediate.
Amine Oxidase (Copper-Containing)↗
A possible role for the 3'-untranslated region in developmental regulation in Trypanosoma brucei.
A series of Trypanosoma brucei transfection vectors was constructed in which transcription of the luciferase gene was driven by the procyclic acidic repetitive protein (procyclin) promoter. The untranslated regions surrounding the luciferase gene were derived from the actin, fructose bisphosphate aldolase, or PARP loci. Trans-splicing of the resulting transcripts occurred as expected, but the site of 3' polyadenylation was upstream of the position anticipated. The nature of the 3'-untranslated region was crucial to the level of expression in bloodstream forms.
Animals↗
Rapid quantitative assays for corneal endothelial cell viability in vitro.
The Janus green photometric technique and the Mosmann's colorimetric MTT-assay to quantify corneal endothelial cytotoxicity were compared. Detergents, studied before by in vitro corneal perfusion, proved to be toxic at the 1% level in the Janus green assay with human corneas and at the 0.4% level in the MTT-assay with bovine endothelial cells. These results correlated well with earlier findings in vitro and in vivo. The use of human corneas reduced the ability of the Janus green photometric technique as a fast screening method because of the heterogeneity and restricted availability of this tissue. With the MTT-assay, large test series could be performed at the same time using cultured cells. Both assays are good tools to screen the cytotoxicity of chemicals and reduce the number of animals needed for endothelial cytotoxicity testing.
Animals↗
[Effect of recombinant human basic fibroblast growth factor (rh-bFGF) on wound healing of the corneal epithelium].
Prior to a possible clinical trial in humans, we studied the wound-healing effect of RH-bFGF in vivo. Healing-rate measurements on 24 deepithelialized rabbit corneas were performed by means of computer-assisted image analysis. A significantly increased healing rate (P = 0.004) was revealed for the 200 ng FGF treated group (rate 1.57 mm2/h) compared to the control group (1.12 mm2/h). To assess the long-term (side) effects of FGF (50 ng topically applied, 2 times daily), 7 rabbits were involved in a model with repeated epithelial scraping of an anterior keratectomy wound (performed with Draeger's rotokeratom to a depth of 0.15 mm). Light and transmission electron micrographs of treated corneas showed an increased fibrogenesis in the anterior stroma with more pronounced activation of keratocytes. No evidence of abnormal neovascularization or inflammation was observed when compared to control corneas. We suggest that topical RH-bFGF promotes corneal wound healing with virtually no detectable adverse reactions. These results seem to be of considerable interest for the possible therapeutic use of rh-bFGF in patients with superficial corneal epithelial lesions.
Animals↗
[Wound healing of the corneal endothelium in vivo after intraocular injection of recombinant human bFGF].
In the present study, we investigated the potential role of recombinant human basic fibroblast growth factor (rh-bFGF) on the regeneration of corneal endothelium in the rabbit. For in vivo wound-healing experiments, standardized wounds were created by mechanically scraping the endothelial cells within the boundaries of a central epithelial trephine mark. A single dose of 1.5 micrograms rh-bFGF was injected into the anterior chamber of the right eye immediately after wounding, while the left eyes received the ointment vehicle only (n = 27 rabbits). Functional recovery and wound closure rates were assessed by means of ultrasonic pachymetry, cell counts after endothelial vital staining and direct computer-assisted surface analysis of Janus green-stained endothelial lesions. Measurements were carried out 0, 1, 2, 4, and 7 days after injury. For the time points day 2 and day 4, significant stimulation of endothelial regeneration in rh-bFGF treated eyes was observed with all parameters measured. These results demonstrate that rh-bFGF could prove to be useful in clinical conditions, e.g. to conserve endothelial function after intraocular surgery or as a supplement to corneal intermediate preservation media.
Animals↗
Baculovirus expression and characterization of catalytically active horseradish peroxidase.
Studies of horseradish peroxidase (HRP), a prototypical enzyme, have provided much of the information that is available on the mechanisms and functions of hemoprotein peroxidases. HRP itself is widely used in biotechnological applications. Further progress in defining the structure and function of the enzyme, however, requires its expression in a heterologous system. We report here baculovirus-mediated, high yield expression of a synthetic gene for HRP in Spodoptera frugiperda cell culture. Expression of the soluble, glycosylated protein requires the 5'-leader sequence of the native gene. Recombinant horseradish peroxidase reacts with H2O2 to give compound I, II, and III spectra and a guaiacol oxidation activity, identical to those of the native enzyme. The integrity of the recombinant active site is confirmed by NMR spectroscopy and by catalytic reaction with ethylhydrazine to give a stabilized isoporphyrin that decays exclusively to delta-meso-ethylheme. Furthermore, thioanisoles are oxidized by recombinant and native HRP with the same enantiomeric specificity. HRP expressed in a baculovirus system, despite probable differences in glycosylation, is essentially identical to the native enzyme.
Amino Acid Sequence↗
Synthesis and evaluation of a new series of mechanism-based aromatase inhibitors.
A series of new 4-(alkylthio)-substituted androstenedione analogues was designed as potential suicide inhibitors of aromatase on the basis of mechanistic considerations on the mode of action of the enzyme. Their synthesis and biological evaluation are described. Among the most interesting are the 4-[(difluoromethyl)thio]-, 4-[(fluoromethyl)thio]-, and 4-[(chloromethyl)thio]androstenediones 12, 13, and 14 with respective IC50's of 2.7, 0.8, and 0.94 microM. Compound 12 was a reversible inhibitor of aromatase while compounds 13 and 14 displayed time-dependent kinetics of inhibition with respective KI's and half-times of inactivation of 30 nM and 3.75 min for 13 and 30 nM and 3 min for 14. The inhibition of aromatase by 14 was NADPH-dependent, and was protected by the presence of substrate (0.5-1 microM), while beta-mercaptoethanol (0.5 mM) failed to protect the enzyme from inactivation. Dialysis failed to reactivate aromatase previously inactivated by 14. The mechanistic implications of these findings are discussed.
Androstenedione↗
Nuclear genes control changes in the organization of the mitochondrial genome in tissue cultures derived from immature embryos of wheat.
Although the mitochondrial genomes of the Chinese Spring and Aquila varieties of wheat are normally similar in organization, this is not so in tissue cultures initiated from their immature embryos where the mitochondrial genomes of both are rearranged and in different, characteristic, ways. However, the mitochondrial genomes of tissue cultures of reciprocal F1 crosses between these varieties were almost identical to one another, showing that nuclear genes control the rearrangement processes. These rearrangements are either due to the appearance of new structures or else result from changes in the relative amounts of subgenomic components. The severe reduction in the amount of certain molecular configurations in tissue cultures from reciprocal crosses is probably due to the presence of dominant information in the Aquila nuclear genome. Data obtained from tissue cultures initiated from F2 embryos of the cross Aquila x Chinese Spring suggest that at least two complementary genes are involved in this control. In contrast, the presence of new molecular arrangements appears to be under the control of a dominant allelic form of a Chinese Spring gene or genes. Thus, this study demonstrates that at least two sets of nuclear genes control the reorganization of the mitochondrial genome which occurs when tissue cultures are initiated from the immature embryos of wheat.
Blotting, Southern↗
Recombinant human basic fibroblast growth factor (Rh-bFGF) in three different wound models in rabbits: corneal wound healing effect and pharmacology.
Prior to a clinical trial in humans, we studied the effect and pharmacological distribution of recombinant human basic fibroblast growth-factor (Rh-bFGF) in vivo. Healing experiments on de-epithelialized rabbits corneas (n = 24 animals) compared the efficacy of three bFGF doses to controls and revealed a significantly increased healing rate for both 200 ng and 500 ng per application Rh-bFGF treatment groups compared to the control groups. To assess possible side effects of Rh-bFGF (500 ng topically applied for up to 7 days, twice daily), ten rabbits were involved in a model of an anterior keratectomy wound (performed with Draeger's roto-keratome to a depth of 0.15 mm). Light microscopy of thin sections of treated corneas showed an increased fibrogenesis in the anterior stroma with a more pronounced activation of keratocytes. No evidence for abnormal neovascularization or inflammation was observed when compared to control corneas. Ocular penetration and systemic distribution of topically applied labelled 125I FGF was assessed in three models (iodine vapour epithelial burn, anterior keratectomy and penetrating autokeratoplasty) in 24 rabbits. No intraocular penetration of bFGF occurred as shown by direct gamma counting. Macroautoradiography showed a selective labelling of epithelial basement membrane when denuded and intact, as previously described. Evidence for systemic absorption of breakdown products was confirmed by heparin-sepharose chromatography of blood and urine samples. Under these conditions, we suggest that topical Rh-bFGF promotes corneal wound healing without morphological adverse reaction or intraocular and systemic penetration.
Animals↗
RU54115, a tight-binding aromatase inhibitor potentially useful for the treatment of breast cancer.
RU54115 is a new potent inhibitor of aromatase. In vitro, it inhibits the enzyme from human placental microsomes with a Ki of 0.5 nM, which places it among the tightest reported steroidal inhibitors of aromatase. In vivo, it lowers the amount of circulating estradiol in pregnant mare serum gonadotrophin (PMSG)-primed female rats with an ED50 of 0.4 mg/kg when given s.c. and 4 mg/kg when given orally. An oral dose of 25 mg/kg when given once daily to female rats was able to inhibit the growth of DMBA-induced mammary tumors.
9,10-Dimethyl-1,2-benzanthracene↗
Human recombinant bFGF stimulates corneal endothelial wound healing in rabbits.
We have previously shown that bovine, human placenta extracted and recombinant human basic Fibroblast Growth Factor (bFGF) are effective in enhancing corneal epithelial wound healing in vivo. In the present study, we investigated the effect of rh-bFGF on the regeneration of injured rabbit endothelium. A standardized wound was created by scraping of endothelial cells with a special device within the boundaries of a central epithelial trephine mark of 7 mm in diameter. A single dose of 1.5 micrograms rh-bFGF was injected into the anterior chamber immediately after wounding, while control eyes received the vehicle only (n = 27). Functional recovery and wound closure rates were assessed by means of ultrasonic pachymetry, corneal button wet weight, endothelial vital staining as well as direct computer assisted surface analysis of Janus green stained corneal buttons. Measurements were carried out 1, 2, 4, and 7 days after injury. Morphological evaluation and cell counts at D4 and D7 were also performed. Significant stimulation of endothelial regeneration in rh-bFGF treated eyes, was observed with all methodological approaches. These results demonstrate the effectiveness of rh-bFGF in enhancing experimental corneal endothelial wound healing and advocate for a possible clinical application of this growth factor in order to preserve endothelial cell function or to promote healing of this important monolayer in case of disease or injury.
Animals↗
[Transcranial Doppler contrast study--an ideal method for detection of paradoxical cerebral embolism?].
Cerebral paradoxical embolizations can easily be detected by transcranial Doppler investigations using intravenous echo contrast medium. In some cases of TIA or stroke without vascular or cardiac disease, this method will reveal a right-to-left vascular shunting (e.g. by a patent foramen ovale). However, demonstrating a leakage through the atrial septum does not necessarily reveal the source of an embolus or the cause of the stroke. Thus the question of prolonged antithrombotic therapy must be decided in each individual case.
Adult↗
Whole-body cesium 137 activity up to 4 years after the Chernobyl reactor accident in premature newborns, newborns, infants, and children.
Cesium 137 activity was measured after the Chernobyl incident in a whole-body radiation counter (4-pi-scintillation counter) in 85 premature and mature newborns (group 1), 174 infants and young children up to 2 11/12 years (group 2), and 48 children between 3 and 8 years (group 3) from Bonn (Germany) and surroundings. In 1987 the mean level of radioactivity in group 2, at 3.7 Bq/kg body weight corresponding to a mean radiation exposure of 11 muSv/y, was lower than that of group 1 (5.8 Bq/kg, 17 muSv/y) and 3 (9.4 Bq/kg, 28 muSv/y). Up to 1990 the values of all groups revealed a continuous decrease. The latest measurements showed mean values of 0.5 Bq/kg (1.5 muSv/y) in group 1, 0.6 Bq/kg (1.8 muSv/y) in group 2, and 0.8 Bq/kg (2.4 muSv/y) in group 3. A comparison with present cesium 137 values and determinations of the end of the 1950s and beginning of 1960s, both in adults, showed good agreement. The effective dose-equivalent rates amounted to less than 1% of that from natural radiation exposure. These levels should present no teratogenic risks to the population studied and, while there are theoretical mutagenic risks, the dose is so low that no increase in measurable mutagenic effects should be observed.
Accidents↗
Structure-function studies of substrate oxidation by bovine serum amine oxidase: relationship to cofactor structure and mechanism.
The chemical mechanism of substrate oxidation, catalyzed by bovine serum amine oxidase, has been explored by a detailed investigation of structure-reactivity correlations. Past mechanistic studies, involving the reductive trapping of substrate to cofactor [Hartmann, C., & Klinman, J. P. (1987) J. Biol. Chem. 262, 962], implied the intermediacy of a substrate imine complex in the catalytic redox mechanism. These studies led to the proposal of a transamination mechanism for substrate oxidation, analogous to pyridoxal phosphate dependent enzymes. In pyridoxal phosphate catalyzed reactions, the transamination process involves the transient formation of a resonance-stabilized carbanion intermediate. Although evidence has been presented describing the participation of an active site base in bovine serum amine oxidase catalysis [Farnum, M. F., Palcic, M. M., & Klinman, J. P. (1986) Biochemistry 25, 1898], the nature of the intermediate derived from C-H bond cleavage has not been directly addressed. To examine this question, a structure-reactivity study was performed using a series of para-substituted benzylamines. Having prior knowledge of the intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic rate constants from steady-state data [Palcic, M. M., & Klinman, J. P. (1983) Biochemistry 22, 5957]. Deuterium isotope effects on kcat and kcat/Km parameters were determined for all substrates, allowing for the calculation of rate constants for C-H bond cleavage (k3) and substrate dissociation constants (Kd). Pre-steady-state constants obtained for p-acetylbenzylamine, p-(trifluoromethyl)benzylamine, and unsubstituted benzylamine exhibited excellent agreement with values calculated from steady-state isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Amine Oxidase (Copper-Containing)↗
A pharmacokinetic model describing the removal of circulating radiolabeled antibody by extracorporeal immunoadsorption.
Extracorporeal immunoadsorption is a new technique for removal of circulating radiolabeled antibody from the peripheral blood (1) to reduce background activity for improved tumor imaging, and (2) to reduce whole-body and marrow toxicity when high doses of radiolabeled antibodies are used for antitumor therapy. A pharmacokinetic model was developed to describe plasma disappearance of 111In-KC-4G3 prior to, during, and after immunoadsorption in humans. The model is developed based on a two-compartment open model, and during immunoadsorption a third compartment is added for removed radioactivity by the immunoadsorption column. Goodness-of-fit statistics indicate a good fit of the model to the data. The resulting pharmacokinetic parameters for a selected patient are V1 = 2.64 L, VSS = 3.64 L, t 1/2 alpha = 3.77 hr, and t 1/2 beta = 48.5 hr. The immunoadsorption clearance (CLIA = 19.3 ml/min) was 21-fold greater than the patient's plasma clearance (CL10 = 0.899 ml/min), indicating a very effective immunoadsorption process. The model predicts an increase in plasma radioactivity upon termination of immunoadsorption, probably due to redistribution of radioactivity from the extravascular compartment to the plasma in response to the rapid decline in plasma radioactivity during immunoadsorption. Two series of simulations were performed to examine the influence of onset time and duration of immunoadsorption. In series one the onset time was varied and in series two immunoadsorption duration was varied. In series one, the predicted radioactivity amounts adsorbed by the immunoadsorption column ranged from 75% of the injected dose (4-hr onset) to 52% of the injected dose (24-hr onset). In series two, immunoadsorbed radioactivity ranged from 32% (2-hr duration) to 64% of the injected dose (12-hr duration). When instituted as early as 4 hr, the predictions suggest that earlier immunoadsorption onset improves the effectiveness of radioactivity removal, relating to higher early circulation concentrations, and longer immunoadsorption periods remove more radioactivity, but also result in larger predicted radioactivity redistribution form tissue to plasma. To employ the immunoadsorption procedure for tumor imaging and therapy optimally, the data and our predictions indicate that a compromise must be made that will balance immunoadsorption onset and duration against tumor radioactivity uptake and subsequent radioactivity redistribution from tissues back to plasma. Together with biologic considerations providing sufficient antigen-antibody interaction and dependent on the utilized radioisotope, these data support the utility of extracorporeal immunoadsorption during the radioimmunodetection of cancer and for future therapeutic applications.
Adsorption↗