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G Kohler

Publications and source records attributed to G Kohler.

32 records · Page 2Linked to original sources
PubMed1985

Fetal rat chondrocytes sequentially elaborate separate growth- and differentiation-promoting peptides during their development in vitro.

Chondrocytes isolated from the calvaria of rat fetuses proliferate and form cartilage when cultured in a chemically defined, serum-free medium, suggesting that they may elaborate self-regulatory factors. Conditioned media obtained from these chondrocytes stimulated DNA synthesis and proliferation when added to separate cultures of chondrocytes and the closely related osteoblast-like cells, but were not very effective in skin fibroblasts isolated from the same fetuses, as judged by [3H]thymidine incorporation and cell proliferation. Chondrocyte-conditioned medium also promoted chondrocyte differentiation, augmenting 35SO4 incorporation, and the accumulation of type II collagen and cartilage-specific proteoglycan. Stimulation of growth and differentiation appears to be attributable to separate activities, released into the medium sequentially by the cultured chondrocytes during their proliferation and maturation phases. Media obtained from growing chondrocytes stimulated growth, but had little effect on 35SO4 incorporation. Media obtained from mature cultures promoted growth as well as 35SO4 incorporation. The mitogenic and sulfation activities were trypsin inhibitable, but exhibited different solubility characteristics and striking differences in their patterns of elution from gel filtration columns. These results suggest that chondrocytes elaborate autostimulatory peptides, the biological activities of which mirror, at least in part, the developmental stage of the donor cells.

Animals↗
PubMed1984

Adriamycin inhibits PTH-mediated but not PGE2-mediated stimulation of cyclic AMP formation in isolated bone cells.

We have examined the effect of adriamycin, an anthracycline antibiotic which modifies plasma membrane functions, on the cyclic AMP response to PTH and PGE2 in isolated osteoblastlike cells. Adriamycin blunted the increment in bone cell cyclic AMP caused by exposure to PTH. This effect appeared rapidly (within 3 min after bone cells were exposed to adriamycin) and disappeared soon after exposure of adriamycin-treated cells to adriamycin-free incubation medium. Inhibition was evident over the entire time course of PTH action, at low as well as high PTH concentrations, and was one-half maximal at 31 microM adriamycin. It could not be attributed to alterations in cyclic AMP exodus, degradation or interference with the cyclic AMP assay, nor to impaired cell viability. Adriamycin also reduced the stimulatory effect of PTH on adenylate cyclase activity in a crude plasma membrane preparation. By contrast, adriamycin failed to modify the effects of PGE2 on cyclic AMP generation in intact bone cells, and on adenylate cyclase activity in broken cells. Moreover, concentrations of adriamycin that blunted the effect of PTH on adenylate cyclase activity did not inhibit the stimulatory effects of sodium fluoride or of GppNHp. These results suggest that adriamycin selectively alters the interaction between PTH and its receptor or impairs the transmission of information from hormone-receptor complex to adenylate cyclase (or both), perhaps by binding to specific lipid domains in the plasma membrane. Structural analogues of adriamycin, which vary in their lipophilic properties, also varied in their capacity to perturb the cyclic AMP response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1984

Comparison of three methods for detection of prolonged experimental trichinellosis in pigs.

Three methods were employed for the diagnosis of porcine trichinellosis. The pooled sample digestion method and trichinoscopy served as European Community (EC) reference techniques, whereas the reliability of the Enzyme Linked Immunosorbent Assay (ELISA) was tested by 11 laboratories of the European Community and Sweden. Three groups of 6 piglets each were orally inoculated with 50, 150 and 1500 Trichinella spiralis larvae into each animal. Another group of 6 animals served as a non-infected control. Animals were slaughtered and serum and muscle samples were collected at Weeks 4, 12 and 40. The material was mailed under code and examined in all participating laboratories. ELISA proved to be a sensitive technique. ELISA micro assay was the most sensitive procedure. Of the direct techniques the reference pooled sample digestion method was more sensitive than trichinoscopy. It was concluded that both micro and macro ELISA can be used with confidence for the detection of low grade, longstanding experimental T. spiralis infections in swine.

Animals↗
PubMed1983

A high affinity, calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells.

Although acute alterations in Ca2+ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca2+-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca2+-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca2+ of 280 nM, Vmax of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg2+ could replace Ca2+ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg2+ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5'-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca2+-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca2+-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca2+ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.

Animals↗
PubMed1983

Calcitonin response in circulating human lymphocytes.

The concentration of cAMP increases in human mononuclear leukocytes after exposure to salmon calcitonin (SCT). This response is lost when the cells are separated into adherent (monocytic) and nonadherent (lymphocytic) cells, although the appropriate response to prostaglandin E2 remains in both groups. Adherent and nonadherent cells, each cultured alone for 16 h, do not regain the SCT response. Coculturing adherent and nonadherent cells together for 16 h restores the SCT response in the lymphocytes. The addition of a cyclooxygenase inhibitor to this culture system prevents development of the SCT response. The SCT response may be induced in nonadherent cells by culturing them for 16 h in medium previously conditioned by the growth of mixed mononuclear leukocytes.

Calcitonin↗
PubMed1981

Calcium-mediated enhancement of the cyclic AMP response in cultured bone cells.

We have examined the influence of extracellular Ca2+ on cyclic AMP metabolism in an osteoblast-enriched population of bone cells isolated from the calvaria of rat fetuses. The cyclic AMP response to stimulators of cyclic AMP formation (PTH and PGE2), but not basal cyclic AMP levels, increased progressively as the extracellular Ca2+ concentration was raised from 0.2 to 4.0 mM. The response to changes in extracellular Ca2+ were rapid (within 3.5 min), and the level of responsivity that characterized each Ca2+ concentration persisted for at least 6 h when the Ca2+ concentration was kept constant. The effect of Ca2+ spanned the entire time course of PTH action, was not accompanied by altered excretion of cyclic AMP from the cells, and was evident at low as well as at high hormone concentrations. Ca2+ augmented the action of PTH in the presence as well as in the absence of cyclic AMP phosphodiesterase inhibitors, and failed to decrease cyclic AMP phosphodiesterase activity in the short term. Mn2+ and, to a smaller degree, Ba2+ substituted for Ca2+ in promoting the cyclic AMP response to PTH. Verapamil, an inhibitor of Ca2+ penetration, blunted the Ca2+-mediated increments in the cyclic AMP response, and the divalent cation ionophore A23187 enhanced these increments. These results indicate that Ca2+ and other cations are positive effectors of the stimulated cyclic AMP response in isolated bone cells. Accumulation into an as yet unknown cellular compartment may be required for the cation effect. The data are most consistent with enhancement of adenylate cyclase reactivity as the mode of cation action.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Hormonal and nonhormonal desensitization in isolated bone cells.

Prior exposure to PTH markedly decreased the responsiveness of isolated, cultured bone cells to the stimulatory effect of the hormone on cyclic AMP formation. This process of desensitization developed within 30 min, persisted during prolonged incubation of the cells in PTH-free medium, and could not be attributed to enhanced excretion of cyclic AMP from the cells, nor to the extracellular accumulation of an inhibitor of PTH action. Adenylate cyclase activity in a subcellular fraction derived from PTH-treated cells was refractory to PTH and to sodium fluoride. These results indicate that PTH-mediated desensitization reflects, at least in part, impaired cyclic AMP formation. Adenosine and PGE2, known stimulators of bone cell cyclic AMP formation, elicited agonist-specific desensitization, and also desensitized bone cells to the effects of subsequently added PTH. PTH blunted the cellular response to adenosine, but not to PGE2. Modest refractoriness to PTH was evident in cells that had been treated previously with the cyclic AMP phosphodiesterase inhibitors IBMX, theophylline, and Bt2cAMP, whereas treatment with sodium butyrate had no effect. The actions of the inhibitors, like that of PTH, were rapid in onset and long-lasting. Desensitization caused by previous treatment with the phosphodiesterase inhibitors, and with PTH itself, was accompanied by enhanced phosphodiesterase activity in bone cell homogenates. Induction of phosphodiesterase activity may well contribute to desensitization in the bone cell system.

1-Methyl-3-isobutylxanthine↗
PubMed1978

Expression of antibody genes in tissue culture: structural mutants and hybrid cells.

Detailed information on the nature and frequency of somatic mutations has been derived from studies of the clonal diversification of the myeloma MOPC 21 in tissue culture. A screening procedure is described that permitted the isolation of four spontaneous mutations at the gamma1 structural gene locus. These originate from four mutation events. Two seem to be point mutations: a "nonsense" and a "mis-sense." Of the other two, one is a frameshift leading to mistranslation and early termination, the other a large deletion due to perhaps an intrachromosomal translocation or a mitotic recombination. Fusion between myeloma-producing cells has shown that variable and constant region genes cannot be scrambled. Differentiation from stem to plasma cells seems to involve changes in the primary sequence of the DNA. Fusion between myeloma cells and spleen cells from immunized animals is a satisfactory method for the derivation of permanent tissue culture lines producing specific antibody. The hybrids express the myeloma as well as the specific antibody light and heavy chains. By subcloning and selection, one can derive lines that selectively lose individual chains. Lines that no longer express the myeloma components can thus be derived. The use of appropriate defective variants of the myeloma parental line is another way of avoiding the presence of the myeloma components.

Animals↗
PubMed1974

Comparison of three methods for the detection of Trichinella spiralis infections in pigs by five European laboratories.

Three methods employed in the diagnosis of trichinosis (trichinoscopy, digestion method, and immunofluorescence technique) were compared by laboratories in 5 countries of the European economic community. For this purpose, material from 32 pigs infected with 50, 150, 500, and 1 500 T. spiralis larvae was examined. With none of the three methods was it possible to detect with sufficient reliability a T. spiralis infection in pigs infected with 50 larvae. The digestion method and the immunofluorescence technique yielded more reliable results when the infection dose was 150 larvae or more. With trichinoscopy, reliable results were obtained in pigs infected with 500 and 1 500 larvae. With the digestion method and trichinoscopy, the onset of infections was detectable from 3 weeks post infection, the digestion method being more reliable; the immunofluorescence technique yielded positive results from approximately 4-6 weeks post infection. The immunofluorescence technique is applicable for epidemiological surveys. As a routine diagnostic procedure in the slaughterhouse, trichinoscopy and the digestion method are possible alternatives, the latter being more sensitive.

Animals↗
PubMedDate not supplied

[Epiphyseal dysplasia--symptoms and differential diagnostic aspects].

AIM: Children complaining of hip pain most likely suffer from Perthes' disease. Similar morphological changes are seen in the hereditary diseases of the epiphyseal dysplasias which have a prevalence of about 40 of 100 000 inhabitants. We now show the differentiation between bilateral Perthes' disease, epiphyseal dysplasia and skeletal disorders like mucopolysaccharidosis and pseudoachondroplasia. METHOD: We describe the diseases of multiple epiphyseal dysplasia and spondyloepiphyseal dysplasia on the basis of two case reports. Furthermore, we discuss differential diagnostic aspects, therapeutic options and prognosis. RESULTS: Epiphyseal dysplasias are classified as osteochondral dysplasia. The characteristic feature of these heredopathies is short statue due to skeletal dysplasias at various sites. Multiple epiphyseal dysplasia is caused by an abnormality of enchondral ossification. A distinction is made between the severe Fairbank form, the milder Ribbing form and a mild but rather localised Meyer form. In addition to the femoral head, the spinal bodies can also be affected by the same enchondral ossification disorders and this is defined as spondyloepiphyseal dysplasia. CONCLUSION: The diagnosis of a Perthes' disease can be made from an X-ray of the hip, showing either an advanced necrosis of the femoral head or early signs of it. Typical radiological findings allow the differentiation to other skeletal dysplasias like epiphyseal dysplasia and metabolic disorders.

Adolescent↗
PubMedDate not supplied

Potentially therapeutically relevant differences of antisense oligonucleotide uptake in CD34+ hematopoietic stem cells, leukemic blasts and cancer cells from patients.

Antisense oligonucleotide (ON) technology, e.g. against drug resistance or antiapoptotic factors, may play an important role in future cancer chemotherapy. An unanswered question in this field is the capacity for uptake of antisense molecules in normal and malignant patients' cells. Therefore, we examined the cellular uptake of FITC-labeled phosphorothioate modified ONs in: i) cells from the T-lymphoblastoid cell line CCRF-CEM, ii) CD34+ hematopoietic progenitors from healthy donors, iii) blasts from ALL or AML patients, iv) cells from the ovarian cancer cell line A2780 and v) cancer cells from malignant fluids. The cationic polymer ExGen was taken as a carrier for transfection, while FITC-ON uptake was evaluated by flow cytometry. We found marked differences between these cell types. Cancer cells from the cell line A2780 and from patients showed a distinctly enhanced uptake compared to hematopoietic progenitors and leukemic blasts. Since bone marrow toxicity substantially limits any conventional chemotherapeutic regimen, a better ON uptake in cancer cells compared to hematopoietic precursors might give an advantage for therapeutic approaches using e.g. ON-based chemosensitization.

Antigens, CD34↗