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Biomedical subjects

C Hartmann

Publications and source records attributed to C Hartmann.

At least 127 records · Page 7Linked to original sources
PubMed1993

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↗
PubMed1993

[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↗
PubMed1993

[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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

[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↗
PubMed1992

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↗
PubMed1991

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)↗
PubMed1991

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↗
PubMed1991

Late onset endophthalmitis associated with intraocular lens: a case of molecularly proved S. epidermidis aetiology.

A case of severe endophthalmitis after cataract extraction followed by posterior chamber lens implantation is reported. Microbiological cultures from a tap of the patient's aqueous humour prior to lens explantation as well as from the explanted lens and aqueous and vitreous humour during operation yielded Staphylococcus epidermidis sensu stricto. Scanning electron microscopy showed massive colonisation of the lens loop by staphylococci. Clonal identity of all isolates was demonstrated by plasmid DNA analysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of extra-cellular products. This is strongly suggestive of the aetiological role of S. epidermidis in this case of late onset endophthalmitis.

Aged↗
PubMed1991

[Damage to the corneal endothelium caused by radial keratotomy].

In our experimental study on 53 rabbits we compared the amount of corneal endothelial damage caused by radial keratotomy (RK) referred to (1) the number of incisions (4, 8, or 16), (2) the postoperative interval (0 h, 48 h) and (3) the direction of the incision [centripetal (cp), centrifugal (cf)]. The endothelial damage was quantified by means of the Janus green photometry technique. Morphological changes were evaluated by scanning electron microscopy (SEM). Depending on the group examined, we found endothelial damage extending over 3-7% of an analysed surface of 64 mm2. One perforation caused endothelial damage of up to 17% of the surface examined. Increasing the number of incisions from 4 to 8 or 16 resulted in a statistically significant increase in the amount of endothelial damage (4.2%, 5.1%, 5.8%; P less than 0.05). At 0 h it was significantly higher than after 48 h (5.5%, 4.6%; P less than 0.05). The direction of the incision had no statistically significant influence in our study (zp: 5.2%, zf: 4.9%). The morphological changes in the rabbit corneal endothelium examined directly after the RK procedure were ruptures in the cell membranes, loss of cells, and posterior corneal protrusions beneath the incisions. After 48 h, we found fewer damaged cells and no denuded Descemet's membranes, but larger polymorphy of the cells and a numerical increase in the microvilli of the cells surrounding the damaged cells. Our results support the crucial argument against RK: the alteration and destabilization of healthy corneal tissue up to the endothelium.

Animals↗
PubMed1991

Biocompatibility of a refractive intracorneal PMMA ring.

Intrastromal ring (ISR) implantation is a promising concept in refractive corneal surgery. In theory, it has two main advantages: eccentric corneal surgery and reversibility after ISR explanation. We present our first experimental data on histobiocompatibility after implantation of a 0.2/1.0 mm vaulted polymethylmethacrylate (PMMA) ISR of 7.5 mm diameter with a specially designed "puzzle lock" using a suction-guided stromal channelling device. Ninety-six rabbit eyes were implanted and enucleated after a 24-h to 8-month follow-up to be period and processed for histology (n = 51), scanning electron microscopy (n = 30) and transmission electron microscopy (n = 9). In this study, the histological data are reported. Two main histopathological phases of stromal reaction were observed: (1) up to 3 months: mild inflammation, mostly mononuclear cell reaction with some macrophages; (2) over 3 months: moderate fibrosis surrounding the ISR filling irregularities of the stromal channel. Mild scarring and rare circumscribed vascularization occurred at the sutured peripheral insertion site of the ISR. Persistent epithelial atrophy without erosion or ulceration was regularly observed over the bulging shoulder of the ISR. There was no significant endothelial surgical trauma due to ISR placement. Major complications occurred in 11 cases (11.5%): stromal abscess starting from the incision (n = 8) and massive neovascularization (n = 3). However, the incidence of complications regressed with improved surgical technique. In conclusion, there is good middle-term tolerance of the PMMA ring with a transparent central cornea.

Animals↗
PubMed1990

Reductive trapping of substrate to methylamine oxidase from Arthrobacter P1.

Methylamine oxidase (EC 1.4.3.6) from Arthrobacter P1 was inactivated by NaCNBH3 in the presence of [14C]benzylamine, leading to the incorporation of 1 mol of radiolabeled substrate/mol of enzyme subunit at complete inactivation. By contrast, no labeling of enzyme was observed using [3H]NaCNBH3 as reductant. These results are analogous to those previously reported for the eukaryotic enzyme, bovine serum plasma amine oxidase [(1987) J. Biol. Chem. 262, 962-965]. The observed pattern of labeling is consistent with the presence of dicarbonyl cofactor at the active site of methylamine oxidase. Further, these studies suggest that our reductive trapping technique, in which the pattern of radiolabeling of an enzyme is compared using C-14 substrate vs tritiated reductant, may serve as a general assay for covalently bound dicarbonyl structures.

Arthrobacter↗