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Biomedical subjects

C Hartmann

Publications and source records attributed to C Hartmann.

At least 91 records · Page 5Linked to original sources
PubMed1994

Mitochondrial DNA variability detected in a single wheat regenerant involves a rare recombination event across a short repeat.

The mitochondrial genome of the selfed progeny of a plant regenerated from long-term somatic tissue culture displays specific structural rearrangements characterized by the appearance of novel restriction fragments. A mitochondrial DNA library was constructed from this selfed progeny in the SalI site of cosmid pHC79 and the novel fragments were subsequently studied. They were shown to arise from reciprocal recombination events involving DNA sequences present in the parental plant. The regions of recombination were sequenced and the nucleotide sequences were aligned with those of the presumptive parental fragments. We characterized an imperfect short repeated DNA sequence, 242 bp long, within which a 7-bp DNA repeat could act as a region of recombination. The use of PCR technology allowed us to show that these fragments were present in both parental plants and tissue cultures as low-abundance sequence arrangements.

Base Sequence↗
PubMed1994

An analysis of the Washington Conference Report on bioanalytical method validation.

The Washington Conference Report on bioanalytical method validation is analysed with respect to the requirements for precision and accuracy. It is shown that if the requirements are interpreted too literally, this could lead to disappointment in practice. A better approach is to separate the total measurement error into its constant (bias) and random (precision) components. To ensure that 95% of all methods fall within the acceptance interval of +/- 15% around the true value, would require, for example, the bias to be < or = 8% and the method precision to be < or = 8% relative standard deviation (RSD; n = 5).

Chemistry Techniques, Analytical↗
PubMed1994

A two-step mode of stripe formation in the Drosophila blastoderm requires interactions among primary pair rule genes.

The stripe pattern of pair rule gene expression along the anterior-posterior axis of the Drosophila blastoderm embryo represents the first sign of periodicity during the process of segmentation. Striped gene expression can be mediated by distinct cis-acting elements that give rise to individual stripe expression domains in direct response to maternal and first zygotic factors. Here we show that the expression of stripes can also be generated by a different, two-step mode which involves regulatory interactions among the primary pair rule genes hairy (h) and runt (run). Expression of h stripes 3 and 4 is directed by a common cis-acting element that results in an initial broad band of gene expression covering three stripe equivalents. Subsequently, this expression domain is split by repression in the forthcoming interstripe region, a process mediated by a separate cis-acting element that responds to run activity. This second mode of pair rule stripe formation may have evolutionary implications.

Animals↗
PubMed1994

Alloimmune neonatal neutropenia resulting from immunization to a high-frequency antigen on the granulocyte Fc gamma receptor III.

BACKGROUND: Alloimmune neonatal neutropenia is mainly caused by NA- or NB1-specific alloantibodies. An antibody in the serum of a Turkish mother who had given birth to an infant with alloimmune neonatal neutropenia showed no NA or NB specificity and was therefore investigated further. STUDY DESIGN AND METHODS: The number of antibody-binding sites was calculated by determination of elutable IgG from granulocytes using a quantitative sandwich enzyme-linked immunosorbent assay. Complement activation was tested by immunofluorescence (C3d) and cytotoxicity tests. The antigen was identified using the antigen-capture assay, monoclonal antibody-specific immobilization of granulocyte antigens, and a modified immunoprecipitation method based upon biotinylation of proteins and visualization by luminescence (luminoimmunoprecipitation). Family study and determination of antigen frequency were done by immunofluorescence and agglutination tests. RESULTS: A noncytotoxic, granulocyte-specific alloantibody that recognized the Fc gamma receptor III, independent of the NA phenotype, was detected, and 242,000 binding sites per cell were calculated. Of granulocytes from 150 randomly selected German blood donors, the alloantibody bound to all. The maternal cells were typed NA1/NA2- and NB1-positive. CONCLUSION: These data reveal the presence of a previously unrecognized, high-frequency epitope on the granulocyte Fc gamma receptor III. Luminoimmunoprecipitation proved to be a simple, nonradioactive technique that was useful in identifying the molecule involved.

Antigens↗
PubMed1994

Hierarchies of RNA-processing signals in a trypanosome surface antigen mRNA precursor.

Nearly all trypanosome mRNAs are synthesized as polycistronic precursors, from which mature mRNAs are excised by trans splicing and polyadenylation. Polyadenylation of a procyclic acidic repetitive protein (PARP, or procyclin) transcript was studied by transient transfection of constructs bearing a chloramphenicol acetyltransferase gene linked to the PARP intergenic region. Polyadenylation usually occurred at A residues, about 100 bases upstream of a trans-splicing acceptor signal. The wild-type polyadenylation site has a cryptic trans-splicing signal about 100 bp downstream: deletion or inversion of this signal results in polyadenylation at multiple sites, upstream of other cryptic trans-splicing signals. The PARP mRNA precursor appears to contain a hierarchy of possible processing signals, the function of cryptic ones being revealed only when the dominant ones are deleted or moved. Correct polyadenylation can be restored by addition of trans-splicing signals from other loci. The results indicate that polyadenylation is coupled to downstream trans splicing but that the products of the trans-splicing reaction are not necessarily functional mRNAs.

Animals↗
PubMed1994

Basic fibroblast growth factor modulates corneal wound healing after excimer laser keratomileusis in rabbits.

We have previously demonstrated beneficial effects of basic fibroblast growth factor (bFGF) on corneal epithelial healing in different in vivo models. In the present study, we investigated the influence on epithelial and stromal healing of topical bFGF after excimer laser keratomileusis in a rabbit model. The corneas of 24 New Zealand white rabbits were deepithelialized (7-mm diameter) and subsequently received identical deep stromal laser ablations (depth, 50 microns; 6 D; diameter, 5 mm). The animals were randomly assigned to one of four treatment groups: PBS (control); bFGF at 500 ng/application; dexamethasone 0.1%, and bFGF+dexamethasone (n = 12 eyes/group). All treatments were given four times daily; with bFGF being given until complete epithelial healing and dexamethasone and PBS, until 3 months postsurgery. Wound surface regression on time was determined by means of computer-assisted image analysis of fluorescein-stained corneas. Corneal opacity was scored using slit-lamp observation and graded following a previously established scoring system. As compared with the other three treatment groups, a highly significant acceleration in the rate of epithelial wound healing was found after bFGF application for 2-3 days only (P < 0.001). A combined therapy (bFGF+steroid) had no effect on the healing rate. As compared with control values, the peak scores for subepithelial haze were significantly lower in eyes that had received bFGF; the regression on time of the haze in the growth factor group paralleled that in eyes receiving continuous steroid treatment. These results demonstrate for the first time the efficiency of a growth factor in modulating the wound-healing response after excimer laser photoablation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

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)↗
PubMed1993

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↗
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↗