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Biomedical subjects

F Wohlrab

Publications and source records attributed to F Wohlrab.

At least 37 records · Page 2Linked to original sources

Slight changes in conditions influence the family of non-B-DNA conformations of the herpes simplex virus type 1 DR2 repeats.

The segment inversion site of herpes simplex virus type 1 contains a series of tandem repeats with a purine bias on one strand and high G + C content (DR2 repeats) capable of adopting a non-B-DNA structure under a variety of conditions. Plasmids carrying eight contiguous copies of DR2 sequences undergo a series of supercoil-driven conformational transitions resulting in different extents of relaxation at pH 5.0. These transitions depend on the presence of an appropriate concentration of divalent cations (Mg2+ and Ca2+) which seem to interact specifically with the alternate structure(s). The transitions occurred at approximately the same superhelical density for all lengths of inserts studied. However, the onset of the transition can be shifted to lower negative superhelical densities by increasing NaCl concentrations. This leads to a reduction of the cooperativity of the transition, which takes place over a range of linking isomers under these conditions. Extrapolating from these results, we established physiological conditions where the alternate DNA structure is found at negative superhelical densities as low as -0.035. The existence of non-B-DNA conformations and/or the structural transitions of these sequences located in this region of intense biological activity implies their involvement in the life cycle of the virus.

Base Composition↗

Ducto-insular proliferation of beta-cells after syngeneic islet transplantation into the spleen of streptozotocin-diabetic Lewis rats.

Syngeneic transplantation of cultured and functionally characterized neonatal islets into the spleen of streptozotocin diabetic Lewis rats resulted in long time survival up to 200 d and in plasma glucose levels lower than 9 mmol/L. The daily plasma glucose profile of transplanted rats had shown significantly above that of nondiabetic control rats. Two-hundred d after transplantation, morphologically intact, insulin containing beta-cells were demonstrable in the spleen, thus demonstrating the long-term survival of functioning islet cells. Proliferation of beta-cells was shown in the transplanted islets. In addition, beta-cell clusters were found that derived from pancreatic ductules transplanted together with the isolated islets into the spleen. Mitoses were visible within ductular epithelial cells. The proliferative response of islets after intrasplenic transplantation is probably the result of a long-term stimulation by slightly enhanced plasma glucose values of the transplant acceptors compared to control animals.

Animals↗

Unusual DNA structure in the regulatory region of the human papovavirus JC virus.

The human papovavirus JC virus (JCV) was analyzed for the presence of unusual DNA conformations. Recombinant plasmids containing 60% of the JCV prototype Mad-1 strain DNA were constructed and analyzed with both enzymatic and chemical probes. Fine-mapping studies revealed that the most prominent S1 nuclease-sensitive and bromoacetaldehyde-modified sites were located within the TATA boxes of each 98-base-pair tandem repeat. Further studies revealed that the S1 nuclease-sensitive site in the first TATA box (proximal to the origin) was approximately 50-fold stronger than the site in the second TATA box (distal from the origin). Deletion of the first TATA box drastically reduced the extent of bromoacetaldehyde modification in the second TATA box, whereas deletion of the second TATA box had little or no effect on the reactivity at the first TATA box. Hence, the biological and conformational role of the second TATA box remains unclear. No supercoil-induced relaxation was found, and reactions with the probes were not pH dependent. Also, fragments containing this regulatory region did not appear to be bent, although the A+T-rich segment contained a tract of eight consecutive A's. We conclude that the regulatory region of JCV contains non-B, but right-handed, DNA conformations which account for this behavior.

BK Virus↗

The chemistry and biology of unusual DNA structures adopted by oligopurine.oligopyrimidine sequences.

A family of unusual DNA structures has been discovered in segments with predominantly purines in one strand (pur.pyr sequences). These sequences are overrepresented in eukaryotic DNA and have been mapped near genes and recombination hot spots. When cloned into recombinant plasmids, many pur.pyr sequences are reactive to chemical and enzymic probes that are generally specific for single-stranded DNA. An intramolecular triplex is adopted by mirror repeats of G's and A's. Other non-B DNA structures adopted by similar sequences remain to be fully clarified but may be a family of related conformations. It is likely that these unorthodox structures play an important role in the function of the eukaryotic genome.

Animals↗

[The life and work of Carl Weigert (1845-1904) in Leipzig 1878-1885].

Coming from Breslau, together with J. Cohnheim (1839-1884), Carl Weigert arrived at the University of Leipzig in 1878. Here, in 1879, he was appointed extraordinary professor at the Department of Pathology. Apart from his growing commitment to autopsy and teaching, due to progressive illness of Cohnheim and the resulting involvement in management of the Department. Weigert studied topical issues of pathology, such as coagulation necrosis and pathogenesis of tuberculosis. His studies into histological staining techniques (principle of elective staining, mordant staining, staining of myelin sheaths) as well as into microtome techniques proved essential to progress in pathology and bacteriology. Weigert left the Leipzig Department of Pathology at the end of March 1885, after the Medical Faculty had failed to appoint him as the successor to Cohnheim. On the 1st of April, 1885, Weigert accepted the position of Director of Senckenberg's Pathological Institute at Frankfurt/Main.

Germany↗

[Spectrophotometric studies on the binding of Victoria Blue 4R to oxidized insulin].

UNLABELLED: The empirical demonstration of insulin by the basic (cationic) dye Victoria Blue 4R (V4R; Ivić 1959) shows the character of a histochemical reaction (Wohlrab et al. 1985), while the chemistry+ of the reaction is not quite understood yet. Aim of this investigation: Will the spectral behaviour of V4R in visible light be influenced by oxidized or non-oxidized insulin? Dependent on the concentration, V4R shows in aqueous solution 2 absorption maxima (lambda = 597 and 558 nm), which represents monomers and dimers of the dye. With increasing dye concentration (200 mumol/l), V4R forms dimers and even higher polymers. Constant V4R concentration (200 mumol/l) results with increasing concentration of oxidized insulin (4 to 16 mumol/l) in a reduction of extinction, while the extinction at lambda = 558 nm (dimers) is more decreased than at lambda = 597 nm (monomers). Non-oxidized insulin has no remarkable influence on the absorption behaviour of V4R. Extinction measurements on V4R stained B-cells of islets of Langerhans after pre-oxidation of the section resulted in a main absorption maximum at lambda = 554 nm. CONCLUSION: Depending on the concentration, the dye V4R is associated with the oxidized insulin in aqueous solution, which is also indicated by the occurrence of an isobestic point in the curve behaviour. This is expressed by the establishment of a concentration-dependent equilibrium between the dye V4R and the oxidized insulin (change in the dissociation and aggregation behaviour respectively of the dye V4R). The determined main absorption maximum (lambda = 554 nm) in the biological material points in the same way to interactions between the stain and oxidized insulin.

Coloring Agents↗

Reaction conditions affect the specificity of bromoacetaldehyde as a probe for DNA cruciforms and B-Z junctions.

The reaction of bromoacetaldehyde (BAA) was investigated further with recombinant plasmids containing tracts of (CG)16, in pRW756, or (CA)32, in pRW777, which adopt left-handed Z-structures under the influence of negative supercoiling. The cruciform structures adopted by the inverted repeat sequences near the replication origins of the pBR322 vectors served as internal controls for the number of unpaired bases. The extent of reaction with the B-Z junctions and the cruciforms was dependent on the reaction and analysis conditions, the method of preparation of BAA, ionic conditions, and the amount of negative supercoiling. In contrast to the previous results of Kang and Wells, B-Z junctions in addition to cruciforms do react with BAA. However, more forcing conditions are required to detect this reaction since B-Z junctions appear to be less reactive than the single stranded loops of cruciforms. The site of reaction with DNA was readily mapped with high precision at the nucleotide level. Also, a simple method is described for determining the concentration of BAA as well as its intrinsic reactivity in a given ionic medium.

Acetaldehyde↗

The segment inversion site of herpes simplex virus type 1 adopts a novel DNA structure.

The 12-base pair (bp) tandem direct repeat sequences (DR2) at the joint region (a sequence) of herpes simplex virus type 1 (strain F) adopt a new type of DNA conformation under the influence of negative supercoiling. The novel conformation is dependent on the number of the DR2 repeats; the 19 mer (228 bp total) and the 14 mer (168 bp) readily form the alternate structure whereas pentamer, trimer, and dimer repeats show somewhat different properties. S1 and P1 nuclease studies reveal that the new conformation has a major structural aberration at its center and conformational periodicities which are not identical on the complementary strands. Also, the effect of salt and pH, the location of reaction with bromo- and chloroacetaldehyde, the type of sequence (direct repeat) involved, and the nature and extent of supercoil-induced relaxations demonstrate that this structure differs from previously recognized conformations including left-handed Z helices, cruciforms, bent DNA, and slipped structures. We propose the existence of a novel conformation, anisomorphic DNA, with different structures on the complementary strands which elicit a structural aberration at the physical center of the tandem sequences. Since the oligopurine X oligopyrimidine sequence may be inherently inflexible, this supercoil-induced structural change and the physical stress on these inserts in recombinant plasmids tend to deform (crack) the DR2 sequences at their centers. Possible roles for anisomorphic DNA in the functions of this segment of intense biological activity are proposed.

Acetaldehyde↗

Enzymatic probes for left-handed Z-DNA.

In conclusion, one of the aspects of the DNA polymorphism observed is the formation of Z-DNA under a variety of conditions. Left-handed DNA stretches not only represent alternate structures, but also exert long-range effects due to their influence on superhelical properties on an entire supercoiled DNA as first shown six years ago [49, 50]. The examples given in this review emphasize the site-specificity of enzymes due to structural features rather than sequence itself. In this fashion, the reversible transition from B to Z DNA could modulate site-specific events on many levels of biological regulation. Considering all of the enzymes studied to date (S1, mung bean, BAL31, P1 nucleases, Hha I, BssH II, MHha I, BamH I, EcoR I, RNA polymerase, recl, recA, DNA glycosylase, O6-methylguanine-DNA methyltransferase), only the recl (and possibly the recA) protein seems to recognize and utilize left-handed DNA. A large number of questions related to the biology of Z-DNA are unanswered including: what is the DNA structure (B or Z or other) which is in physical contact with proteins; is Z-DNA recognized by proteins or are junctions the important features; do proteins revert the Z structure to B or to some other right-handed conformation; what other cofactors (perhaps chiral in nature) may be involved; what are the alternate forms of left-handed DNA; does left-handed DNA exist in vivo; what is the biological role(s) of left-handed DNA? The future of this field of investigation will be exciting indeed.

Computer Simulation↗

Morphometric parameters of Müller (glial) cells dependent on their topographic localization in the nonmyelinated part of the rabbit retina. A consideration of functional aspects of radial glia.

Morphometric parameters of Müller cells were evaluated by light microscopy both in whole retinae and in enzymatically isolated cells from adult pigmented rabbits. In spite of the marked decrease in cell densities from visual streak to far periphery, a constant glia-neuron ratio of about 1:15 was found in all regions. The volume of individual Müller cells was found to increase strongly when the cells become shorter, i.e. when the retinal centre was compared to the retinal periphery. The contribution of Müller cell volume to the total retinal volume, however, was shown to be constant at about 6%. Long Müller cells have a thin vitreal process and a small vitreal endfoot surface. The consequences of this rule for the proposed function of Müller cells in retinal K+ clearance are discussed with respect to general features of radial glia. It is suggested that foetal radial glial cells too long to perform sufficient K+ clearance are destined to be transformed into 'adult' multipolar glia by mitotic cell division.

Animals↗

Effects of alpha-aminoadipic acid on the glutamate-isolated P III of the rabbit electroretinogram.

alpha-Aminoadipic acid was intravitreally applied to adult rabbits. After 5 h, the retinae of these animals were examined by electroretinography and histochemistry. The retinal Müller cells were extremely swollen, and the electroretinographic slow P III was extinguished. The mass receptor potential was somewhat diminished. The results are consistent with the opinion that the slow P III is the reaction of the Müller cells to the changed external potassium ion concentration caused by the activity of the photoreceptors.

2-Aminoadipic Acid↗

The effect of 2'-fluoro-2'-deoxycytidine on herpes virus growth.

The effect of 2'-fluoro-2'-deoxycytidine (dCfl) on the growth of certain viruses of the herpes type was investigated. It is shown that the compound has considerable anti-viral activity against HSV-I, HSV-II, pseudorabies virus and equine abortion virus. It has an effect comparable to that of araC and is more efficient than br5dC, but less so than acyclovir. Experiments with thymidine kinase-negative strains of HSV-I indicated that dCfl was phosphorylated by the viral kinase, and its Km appears to be low and close to that of thymidine. Density gradient centrifugation enabled us to show that dCfl was incorporated into cellular and viral DNA and RNA. The cytotoxic activity of dCfl appears to be about 10-times smaller than that of araC. Removal of the nucleoside analog, washing and replacement with deoxycytidine reversed this effect, indicating rather a cytostatic than cytotoxic effect.

Animals↗

Low dose streptozotocin induced diabetes in mice. Metabolic, light microscopical, histochemical, immunofluorescence microscopical, electron microscopical and morphometrical findings.

The experimental animal model of human insulin-dependent diabetes mellitus (IDDM, type I diabetes), which was for the first time described by Like and Rossini (1976) for Charles River CD-1 mice and produced by the application of multiple subdiabetogenic streptozotocin (SZ) doses, has been reproduced in the mouse strain C57 Bl/KsJ which has been bred over several generations at the Central Institute for Diabetes Karlsburg (since 1975). Male mice were given subdiabetogenic intraperitoneal injections of SZ (40 mg/kg b.w.) on five days running.--The simultaneously performed metabolic, light and electron microscopical, histochemical, fluorescence microscopical, and morphometric examinations show that the small doses of SZ lead to a metabolic disturbance in the islets of Langerhans already on the third day of the experiment (after the first two SZ injections) which is associated with a high-degree reduction or an interruption of the insulin production. Subsequently, on the 8th day (three days after the last SZ injection) up to the 20th day an insulitis occurs which is characterized by a target cell reaction of lymphocytes against the beta cells and leads to the lysis of the majority of the beta cells. In the course and after the insulitis, a persisting insulin deficiency diabetes develops with lacking signs of an attempt to replace the perished beta cells. For the time being, the nature of this target beta cell destruction by lymphocytes remains unclear. According to the enzyme-histochemical and electron microscopical results, the involved lymphocytes are natural killer cells (NK cells) rather than T cells. The release of the target cell reaction is obviously effected by the initial metabolic disturbances in the beta cells intervening in the insulin synthesis. Virus bodies do not play any role in this process. The importance of this animal model to human insulin-dependent diabetes mellitus is discussed.

Animals↗

Morphologic-histochemical characterization of inflammatory cells in insulitis induced by multiple subdiabetogenic doses of streptozotocin in C57B1/KsJ mice.

Insulitis can be induced in C57B1/KsJ mice by injection of subdiabetogenic doses of streptozotocin (SZ, 40 mg/kg body weight) on five consecutive days. Subsequent to the resulting insulitis, a persisting diabetes syndrome develops. This experimental diabetes is regarded as a model of insulin-dependent diabetes mellitus in man (IDDM, type I). Insulitis is considered as a cellular immune reaction against the beta cells in the islets of Langerhans. This paper describes an ultrastructural study showing that lymphoid cells destroy the outer membranes of beta cells. No histochemical indications of the presence of T lymphocytes were found. Some of the lymphocytes are characterized as natural killer cells (NK cells) according to their histochemical and ultrastructural features.

Animals↗

Simultaneous biochemical and morphological investigations on the effect of leukocytes from type I diabetics on isolated rat islets.

The first morphological and biochemical findings on the effect of leukocytes from a newly diagnosed type I diabetic patient on isolated rat islets are presented. Leukocytes from venous blood were co-incubated with isolated neonatal rat islets for 22 h. According to the biochemical and electron-microscopical findings, beta cells localized in the periphery of the islets and a few single beta cells show lytic alterations (single cell necroses). The electron micrographs suggest that the beta cells were lysed after contact with lymphocytes or lymphoid cells from the diabetic patient (target cell reaction). The simultaneous biochemical and morphological investigations reveal that after beta cell lysis, insulin released in a granular state can be phagocytosed by granulocytes and thus escape the estimation of insulin concentration in the medium.

Animals↗

[Histochemical determination of sorbitol dehydrogenase in the rat kidney. Indicator histochemical, immunohistochemical and microelectrophoretic studies].

By comparative indicator- and immunohistochemical technique we have investigated the distribution of sorbitol dehydrogenase (SoDH) in rat kidney. In this connection we have tested some parameters which influenced the histochemical demonstration of SoDH activity by tetrazolium salt indicator technique (e.g. prefixation of tissue sections, elution behaviour of the enzyme, influence of different dialysis membranes, composition of incubating medium). According to these studies the enzyme was demonstrated in native cryostate sections by membrane incubating technique. For the immunohistochemical demonstration of SoDH specific antibodies against this enzyme were prepared in rabbits and used for indirect immunofluorescence method and for unlabelled antibody enzyme (PAP) technique. A good agreement of sorbitol dehydrogenase localization in rat kidney was observed with all the tested methods, but also some discrepancies exist in connection with the glomerular localization of the enzyme.

Animals↗