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Biomedical subjects

C Hartmann

Publications and source records attributed to C Hartmann.

At least 55 records · Page 3Linked to original sources
PubMed1995

Corneal wound healing modulation using basic fibroblast growth factor after excimer laser photorefractive keratectomy.

Photorefractive keratectomy with the 193-nm excimer laser is one of the most promising innovations in refractive surgery. However, routine clinical application is hindered by two main obstacles, i.e., postoperative subepithelial opacity and possible regression of the refractive result. Because we have previously demonstrated beneficial effects of basic fibroblast growth factor (bFGF) on corneal epithelial healing in different in vivo models, we investigated the influence on both epithelial and stromal healing of topical bFGF after excimer laser keratomileusis in a rabbit model. After 7-mm circular deepithelialization, the eyes of 24 New Zealand White rabbits received identical deep stromal laser ablations (depth 50 microns, 6 D, diameter 5 mm) and were randomly assigned to one of four treatment groups [control (phosphate-buffered saline, PBS), bFGF 10 micrograms/application, dexamethasone 0.1%, bFGF + dexamethasone, n = 12 eyes/group]. All treatments were administered four times daily--bFGF until complete epithelial healing, 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 observed biomicroscopically and graded using a previously established scoring system. After bFGF application for 2-3 days only, a highly significant acceleration in epithelial wound healing speed was found compared with the rate of the other three treatment groups (p < 0.001). A combined therapy (bFGF + steroid) had no effect on the healing rate. Compared with control values, mean scores for subepithelial haze in the other groups were found to be nearly 50% lower during the first 2 postoperative months.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Altered immunohistochemical expression of small proteoglycans in the tumor tissue and stroma of basal cell carcinoma.

Small proteoglycans have been shown to act as receptors for matrix molecules or growth factors and to influence the attachment and the migration of cells. We therefore report here on the immunocytochemical expression of three small proteoglycans, i.e., decorin, biglycan, and the recently described PG-100, in normal human skin and in basal cell carcinoma. In normal human skin, staining for decorin revealed expression throughout the dermis with an increased signal in the papillary dermis, whereas no expression was observed in the epidermis. Biglycan and PG-100 were mainly detected in the epidermis, with biglycan being expressed only in suprabasal layers. In addition, biglycan could be detected in a narrow zone below the basement membrane. In tissue specimens obtained from 12 basal cell carcinomas, the expression of biglycan and PG-100 was absent or strongly down-regulated in the tumor tissue. Tumor cells thus displayed a staining pattern similar to that found on the basal cells of normal human skin. In the stroma surrounding the tumor, however, the expression of biglycan and to a lesser degree decorin was increased when compared with normal human dermis. The increased deposition appears to be due to an increased synthesis of these molecules, as total RNA extracted from basal cell carcinoma tissue revealed an induction of biglycan and decorin mRNA. This study indicates that the expression of proteoglycans in basal cell carcinoma tumor cells and in tumor stroma is altered from that in normal skin.

Animals↗
PubMed1994

Gene expression mediated by bacteriophage T3 and T7 RNA polymerases in transgenic trypanosomes.

Messenger RNAs of higher eukaryotes share a functionally essential 5' monomethyl CAP structure generated during a reaction that is linked exclusively to RNA polymerase II transcription. In unicellular parasites belonging to the Kinetoplastida, however, mRNAs acquire their 5' CAP through a trans-splicing reaction which effectively uncouples pol II transcription and capping. Consequently functional mRNAs can be produced by endogenous RNA polymerase I. Here we demonstrate the extension of this flexibility to heterologous bacteriophage polymerases. Transgenic Trypanosoma brucei cell lines stably expressing functional, nuclearly localized T3 or T7 RNA polymerase were established and assayed using reporter plasmids bearing the corresponding phage promoters. In these cell lines the levels of phage promoter-driven gene expression ranges from one half to greater than 5 times that mediated by endogenous pol I. Analysis of 5' ends of transcripts synthesized by the T7 polymerase revealed that they are trans-spliced. Thus the usual eukaryotic link between mRNA production and pol II transcription can be by-passed by the introduced phage polymerases, thereby significantly expanding the critically small panel of promoters currently available for exploitation in reverse genetic approaches in T. brucei.

Animals↗
PubMed1994

Relaxing incisions with compression sutures for control of astigmatism after penetrating keratoplasty.

BACKGROUND: Ten per cent of patients with persisting postoperative astigmatism following penetrating keratoplasty (PK) require surgical re-intervention, despite an otherwise "successful" transplant. Relaxing incisions (RIs) in combination with compression sutures seem to be the preferable procedure. However, poor predictability and lack of long-term experience complicate the issue. Here we report the 2-year follow-up results of 25 patients with high PK astigmatism treated by means of RIs and compression sutures. METHODS: Commonly, free-handed RIs were placed at the graft-host interface and 10-0 nylon compression sutures were placed perpendicular to the incisions. PK sutures had been removed no less than 4 months prior to refractive surgery. RESULTS: Nineteen eyes regained a functional vision of > or = 0.4. The net decrease in astigmatism was 6.1 +/- 4.3 D (47 +/- 21%). The mean vector-corrected change in astigmatism was 13.1 +/- 5.7 D. Cylinder axis variation was reasonably low, with a correlation of attempted versus achieved axis of r = 0.85. Within the first 3 months after operation the induced astigmatism regressed by, on average, 5.5 +/- 4.3 D, making intraoperative overcorrection necessary. As an inevitable side effect, refractive procedures resulted in a myopic shift (4.7 +/- 6.9 D) in spherical equivalence. CONCLUSION: RIs and compression sutures are very useful in reducing postkeratoplasty astigmatism if correction of extremely high cylinder (> 10 D) is not intended. However, predictability still remains unsatisfactory and more than one operation may be required.

Adult↗
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↗