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Biomedical subjects

B Zimmermann

Publications and source records attributed to B Zimmermann.

At least 19 recordsLinked to original sources
PubMed1992

Kinetics of beta-glycerophosphate-induced endochondral mineralization in vitro. Calcium accumulation, alkaline phosphatase activity, and effects of levamisole.

Isolated mesenchymal limb bud cells from day-12 mouse embryos grown at high density in organoid culture at the medium/air interphase differentiate into chondrocytes and form cartilage nodules. Upon addition of beta-glycerophosphate (beta-GP), cartilage undergoes endochondral mineralization. This beta-GP-induced mineralization was investigated by measuring the calcium content in the cultures and the activity of alkaline phosphatase (AP) in the cell mass and the medium. Calcium incorporation depended on the amount of beta-GP added. After continuous treatment, mineralization began on day 8 of the culture period and increased linearly until day 15. In long-term cultures, periodical treatment for 6 days caused an increase in mineralization the older the cultures were, but the slope of increase was proportionately less steep. Treatment at the latest period on days 19-24 resulted in a markedly reduced mineralization. After short-term treatment (48 hours), mineralization increased also the older the cultures were and proceeded during further cultivation in beta-GP-free medium. This kinetic behavior indicates a dependency of mineralization on cartilage maturation in this in vitro system. AP activity increased enormously and nearly logarithmically in the cell mass in beta-GP-free medium, whereas beta-GP treatment inhibited this drastic increase. In the medium, considerable activities of AP were also measurable from day 10 onward. It increased in beta-GP-free medium up to day 14, but was diminished after mineralization had been induced. Levamisole inhibited AP activity dose dependently when added directly to the enzyme-containing medium (100% inhibition at 10(-3) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1992

Degeneration of osteoblasts involved in intramembranous ossification of fetal rat calvaria.

Ossification of calvariae from day-21 rat fetuses was reinvestigated by electron microscopy using different fixation techniques (glutaraldehyde/OsO4, tannic acid, ruthenium red, K-pyroantimonate). An osteoid layer with scattered mineral deposits was found at the mineralization front. Directly beyond this layer, a sheet of one to two layers of necrotic and degenerating osteoblasts was present. Above this sheet, normal and healthy cells were seen, formed by six to eight layers of flattened cells, embedded in a collagenous matrix. The osteoblasts on the less mineralizing opposite side of the calcified calvariae and the osteocytes embedded in the calcified calvariae appeared healthy. Closer inspection of the necrotic zone revealed apatite crystals in vesicles which most probably originated from mitochondria of the degenerated cells. Large K-pyroantimonate deposits were found throughout the osteoid and the necrotic zone, whereas only small granules were scattered in the cytoplasm and at the plasma membrane of the healthy cells directly adjacent to the necrotic zone. A concept of intramembranous mineralization is outlined, according to which osteoblasts store enormous amounts of calcium, which are liberated by physiological cell death in the vicinity of the mineralizing front.

Animals↗
PubMed1992

Cryptococcal meningitis in systemic lupus erythematosus.

Two cases of cryptococcal meningitis occurring in patients with systemic lupus erythematosus (SLE) are presented, and 24 additional cases from the literature are reviewed. The insidious onset of this infrequent complication is emphasized. The nonspecific neurological findings associated with this infection are often mistakenly diagnosed as a central nervous system manifestation of SLE. Earlier diagnosis and effective antifungal therapy have improved the prognosis of cryptococcal meningitis in SLE patients in recent years. Strategies for the treatment of patients with this complication are discussed.

Adult↗
PubMed1992

Activation of the fifth component of human complement, C5, without cleavage, by methionine oxidizing agents.

Purified human C5 was incubated with chloramine T (Cl-T) or N-chloro-succinimide (N-Cl-S) in barbital buffer, pH 7.2. The treatment led to C5 activation: Cl-T- and N-Cl-S-treated C5 acquired a binding site for C6; upon incubation with C6 and subsequent addition of C7, C8 and C9 a membrane attack complex formed which lysed non-sensitized guinea pig red cells (reactive lysis). While the physiological activation of C5 follows its specific cleavage, the resulting fragment C5b representing the activated C5 and expressing the C6 binding site, the treatment with the mentioned chemicals does not lead to fragmentation of the C5 protein. So, functionally, the product of the chemical treatment is C5b-like, but chemically, it comprises the whole protein; no C5a is released. Cl-T and N-Cl-S are known to more or less selectively oxidize methionine residues in proteins, dependent on the conditions. Other sensitive amino acid residues are tryptophan and cysteine. Conditions were chosen for treatment of C5 with Cl-T which exclude attack on tryptophan, and we have ensured that human C5 does not contain free cysteine residues. Further, oxidation of about 60% of the methionine residues of C5 by Cl-T was demonstrated by amino acid analysis. So, all evidence points to methionine residue(s) as the site of attack of Cl-T and probably also of N-Cl-S. The oxidation product of methionine, its sulphoxide, may cause a change in structural conformation of C5 which involves expression of the C6 binding site. Earlier it was found that oxidation of C5 by hydroxyl radicals leads to its activation without cleavage. Since the properties of this C5b-like product resemble those of the product of treatment with Cl-T and N-Cl-S, it is suggested that the formerly found activation of human C5 by hydroxyl radicals is also mediated by oxidation of methionine residue(s) in the C5 protein.

Chloramines↗
PubMed1992

Changes in glycosaminoglycan binding to collagen during desmoid mineralization as revealed by different electron-microscopic staining techniques.

Mineralization at collagen fibrils is regulated by glycosaminoglycans (GAG). Alterations in proteoglycan composition during mineralization as well as inhibition of mineralization by GAGs are well documented. Collagen-GAG interactions during desmoid osteogenesis in fetal rat calvariae were investigated ultrastructurally by means of different fixation techniques. Mineralization was restricted to the collagen of the osteoid at the ectocranial side. Beyond the osteoid, one layer containing degenerated cells was found, followed by sheets of healthy osteoblasts with nonmineralized collagen fibrils. These fibrils were ordered in bundles, but were irregularly arranged in the mineralized osteoid. After fixation in glutaraldehyde-ruthenium red (GA-RR), small RR-positive granules were periodically attached to the fibrils of the nonmineralized collagen. These granules were absent at collagen in the mineralized osteoid. Periodically bound granules (periodicity of 62 nm) could clearly be demonstrated along collagen fibrils by pretreatment with the positively charged protamine sulfate and subsequent fixation in GA-RR in the nonmineralized collagen. In the mineralized osteoid, however, these granules were present, but periodic binding was missing. Heparin pretreatment followed by fixation in GA-RR revealed periodically bound fine strands between collagen fibrils running parallel in the nonmineralized collagen; these threads were absent in the mineralizing osteoid. Restriction of mineralization to osteoid at the mineralization border may be reflected by the observed changes in GAG binding to collagen fibrils within the osteoid of developing fetal calvariae in contrast to binding to collagen in nonmineralized areas.

Animals↗
PubMed1991

Organoid reorganization of human tumors under in vitro conditions.

In the present study we describe a new method to cultivate human tumors, which allows organoid differentiation under in vitro conditions. Diverse tumors of different origin and various histopathology which had been heterotransplanted to athymic mice were dissociated into single cells and seeded at high cell density onto a membrane filter consisting of cellulose nitrate at the gas-medium interface. Within a few days, the tumor cells reorganized and differentiated into organoid structures which exhibited the typical histological characteristics of the original tissues. Due to the formation of organoid aggregates, which was also previously seen with normal fetal cells, this type of culture has been described as 'organoid culture'. In the case of adenocarcinomas of the lung and the colon including the rectum, glandular structures with central lumina, adjacent microvilli, and junctional complexes were formed. Numerous specific intercellular contacts such as desmosomes and tight junctions occurred as well as interdigitations of adjacent cell membranes. In a tumor of the rectum, a typical brush border differentiated at the surface of the reorganized tumor-tissue aggregate. Epidermoid carcinomas of the head and neck developed structures resembling the spinous layer of the epidermis, exhibiting numerous desmosomes and intracytoplasmic bundles of tonofilaments radiating into the desmosomes. Most tumors produced a fragmentary monolayered or multilayered basal lamina of similar morphological appearance as under in vivo conditions. These results illustrate the organoid reorganization and differentiation of human tumor cells under the experimentally rather simple conditions of the organoid culture systems and clearly demonstrate that this in vitro system comes close to the in vivo situation as far as certain differentiation phenomena are concerned.

Adenocarcinoma↗
PubMed1991

Patterns of mineralization in vitro.

Various patterns of mineralization are found in the organism during fetal and postnatal development. Different findings and theories have been published in the literature with regard to the mechanisms of mineralization, many of which are controversely discussed. In the present study the different patterns of mineralization observed in the organoid culture system of fetal rat calvarial cells were investigated by electron microscopy. In organoid culture, calvarial cells grow and differentiate at high density, and deposition of osteoid and mineralization of the matrix occur to a very high extent. Different types of mineralization could be observed more or less simultaneously. It was found that hydroxyapatite crystals were formed at collagen fibrils as well as in the interfibrillar space. Mineralization was frequently seen in necrotic cells and cellular remnants as well as in extra- and intracellular vesicles. Addition of bone or dentin matrices or the artificial hydroxyapatite Interpore 200 to the cells caused an increased mineralization in the vicinity and on the surface of the matrices with and without participation of collagen. On previously formed mineralized nodules, an apposition of mineralizing material appeared due to matrix secretion by osteoblasts. It is concluded that initiation of mineralization occurs--at least in vitro--at every nucleation point under appropriate conditions. These mineralization foci enlarge by further apposition as well as by cellular secretion of a mineralizing matrix. Furthermore, cell necroses may liberate mineralizable vesicles. All these patterns of mineralization are the result of different activities of one cell type.

Animals↗
PubMed1991

Use of an artificial oxygen carrier in isolated rat liver perfusion: first demonstration of net glucose uptake at physiological portal glucose concentrations using a hemoglobin-free perfusate.

A defect in isolated perfused rat-liver (IPRL) preparations has been proposed to explain discrepancies between in vivo and in vitro findings regarding hepatic glucose metabolism. The aim of the present study was to investigate whether a preparation of IPRL using a synthetic hemoglobin-free perfusate was capable of net glucose uptake and glycogen deposition at physiological portal substrate concentrations. Livers from fed anaesthetized rats were perfused in a recirculating system using a fluorocarbon emulsion as artificial oxygen carrier. Depending on the prevailing glucose concentration, livers exhibited net glucose uptake or release with a threshold value of 5.5-6.0 mM glucose. Net glucose uptake was associated with net glycogen deposition (+0.23 to +0.59 mumol C6 min-1 g-1). From 5.8 mM (n = 3) and 10.0 mM (n = 8), initial concentration glucose levels fell to 5.3 +/- 0.2 mM after 210 min (n = 3) and 6.3 +/- 0.9 mM after 120 min (n = 8), respectively. This was equivalent to a net glucose uptake of -0.16 and -0.45 mumol min-1 g-1. Anoxia reversibly switched hepatic glucose balance from net uptake (-0.42 mumol min-1 g-1) to release (+0.69 mumol min-1 g-1) followed by net uptake (-0.50 mumol min-1 g-1) after reinstitution of aerobic conditions. We conclude that the composition of perfusion media might play a pivotal role for studies of glucose metabolism in the isolated perfused rat liver. In our experimental model, using a hemoglobin-free synthetic medium, net glucose uptake was readily demonstrated at physiological portal substrate concentrations similar to the in vivo situation.

Aerobiosis↗
PubMed1991

The extracellular matrix in cartilage organoid culture: biochemical, immunomorphological and electron microscopic studies.

Limb bud mesenchymal cells obtained from day-12 mouse embryos were grown at high density on a membrane filter (pore size 0.2 micron) at the medium/air interphase. Chondrogenesis in this so-called cartilage organoid culture was monitored quantitatively by immunological estimation of type I and type II collagen and qualitatively by indirect immunofluorescence and electron microscopy in the course of a 36 days culture period. Three stages of cartilage development could be substantiated: 1. Formation of cartilage between days 2 and 7; 2. maturation of cartilage between days 9 and 13; 3. degeneration of cartilage beginning at day 20. Differentiation in cell aggregates and a loose mesenchymal tissue occurred during the first two days of the culture period. Type II collagen synthesis started in cell aggregates two days after plating and after 6 days in culture distinct cartilage nodules had developed which were embedded in loose connective tissue that contained type I collagen. During this period the type II collagen content increased progressively from 2.3 micrograms (day 3) to nearly 40 micrograms (day 7) per mg dry weight, whereas the type I collagen level increased more linearly from 2.7 to 21.3 micrograms/mg dry weight. The second period was characterized by enlargement and fusion of cartilage nodules and a diminished increase in type II collagen content from 45 to 60 micrograms/mg dry weight. Enlargement and fusion occurred by matrix production as well as by transformation of perichondrial cells into chondroblasts. Type I collagen synthesis enhanced from 29 to 54 micrograms/mg. Hypertrophic chondrocytes could be demonstrated ultrastructurally. At the third stage a nearly continuous layer of cartilage on the membrane filter covered by noncartilagenous tissue had developed. To some extent chondrocytes lost their matrix capsule and changed into fibroblast-like cells accompanied by a switch of collagen synthesis from type II to type I collagen. Quantitative studies yielded a constant level of about 60 micrograms/mg type II collagen and a further increase in type I collagen from 77 to 116 micrograms/mg dry weight. This study reveals an in vitro model of a prolonged, but almost identical image of chondrogenesis in vivo prior to endochondral mineralization which may be useful for investigations on cartilage differentiation, maturation and degeneration.

Animals↗
PubMed1991

A mammary-specific promoter directs expression of growth hormone not only to the mammary gland, but also to Bergman glia cells in transgenic mice.

The whey acidic protein (WAP) promoter has been previously used to target the expression of heterologous genes to the mammary glands of transgenic mice. To direct the expression of human GH (hGH) to mouse mammary glands, hGH-coding sequences have been coupled to WAP promoter sequences (WAP-hGH). Female transgenic mice carrying the WAP-hGH constructs show expression of hGH in the mammary gland, demonstrating the functionality of the transgenes. However, when other organs from these transgenic mice were examined, high level expression of hGH was unexpectedly observed in the brains of all male and female mice. Using in situ hybridization or immunohistochemistry, hGH expression from the transgene was seen to occur specifically in Bergman glia cells. In contrast, mice carrying hGH-coding sequences linked to the metallothionein promoter do not express hGH in these cells. Neither the endogenous WAP gene nor at least three other transgenes in which heterologous genes have been placed under the transcriptional control of the WAP promoter are expressed in the brain. Thus, we propose that the combination of the WAP promoter and the hGH structural gene results in a novel tissue specificity in the Bergman glia.

Animals↗
PubMed1991

Metabolism of crystallin fragments in cell-free extracts of bovine lens: effects of ageing and oxygen free-radicals.

1. The ability of cell-free preparations from bovine lens to degrade fragments of alpha-crystallin has been studied. Crystallin fragments, produced by either chemical cleavage with cyanogen bromide or prolonged treatment with H2O2 and Cu2+ to produce hydroxyl radicals, were labelled with 125I and incubated with preparations obtained from lenses from animals of different age. 2. Results showed that the ability of the preparations obtained from the lens cores (the innermost part of the lens composed of enucleated non-dividing cells incapable of protein synthesis) to degrade crystallin fragments decreased with animal age. No such age-related correlation was obtained with preparations obtained from the cortex (the outer region of the lens surrounding the core). 3. The effect of incubation of the various lenticular preparations with H2O2 and Cu2+ on subsequent ability to catabolise crystallin fragments was also examined. Preparations from the oldest lenses were found to be the least resistant to free-radical attack. 4. The relative susceptibility of the crystallins and non-lenticular proteins to H2O2/Cu(2+)-mediated free-radical attack was examined. Not only were the various crystallins (alpha, beta and gamma) far more resistant to cleavage under these conditions, they also protected the non-lenticular proteins from free-radical-mediated attack. The comparative resistance of the crystallins to attack and their ability to protect other proteins appeared to be dependent on their structural integrity as prior denaturation with acid and/or cleavage with cyanogen bromide eliminated these properties. 5. It is suggested that crystallins (which show sequence homology to some heat-shock proteins) possess homeostatic functions which could protect other proteins (e.g. proteases) from certain forms of free-radical-mediated damage; crystallins may therefore be important in ageing in general where aberrant polypeptides accumulate.

Aging↗
PubMed1991

Seronegative symmetric polyarthritis in Sezary syndrome.

We describe a patient with Sezary syndrome and seronegative symmetric polyarthritis. Detailed analysis of the synovial membrane, including T lymphocyte subset delineation, demonstrated that malignant synovial infiltration was the direct cause of arthritis in this patient.

Aged↗
PubMed1990

Relationship between insulin stimulation and endogenous regulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes.

The occurrence of the endogenous regulatory response to high rates of 2-deoxyglucose (2-DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2-DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3-L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3-L1 preadipocytes, insulin-sensitive differentiated 3T3-L1 adipocytes displayed the time-dependent cyclic pattern of 2-DG uptake rates characteristic of the membrane-limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2-DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3-L1 cells into a surface covered by numerous microvilli of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilli into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3-L1 adipocytes with 2 mM 2-DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin-induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3-O-methylglucose (3-OMG) in 3T3-L1 adipocytes. This insulin-induced increase of the 3-OMG distribution space exhibited the same time (t1/2 = 2-2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin-induced stimulation of 3-OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilli from the cell surface. Glucose starvation (18 hours at less than 0.5 mM) induced a conspicuous reduction of the length of microvilli on differentiated 3T3-L1 cells. In this state, the stalks of the microvilli are almost invisible and the enlarged spherical tips of the microvilli (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation-induced shortening of microvilli was accompanied by a threefold increase of the basal 3-OMG transport rate and a greater than twofold increase of the intracellular 3-OMG distribution space as compared to fed cells (10 mM; 18 hours).(ABSTRACT TRUNCATED AT 400 WORDS)

3-O-Methylglucose↗
PubMed1990

Endochondral mineralization in cartilage organoid culture.

In the development of secondary bone, mineralization of the cartilage matrix is the first step in endochondral mineralization. The circumstances of cartilage mineralization are not known. Influences of the periosteal tissue have been mentioned. In order to investigate the role of osteoblastic cells in endochondral mineralization, cartilage organoid cultures were induced to mineralize by the addition of beta-glycerophosphate (beta-GP). In cartilage organoid culture, embryonic mouse limb bud mesenchymal cells were grown at high-density. The cells differentiated into mature chondrocytes and produced hyaline cartilage matrix. When cartilage had formed after 6 days in vitro, 10 mM beta-GP was added. The developed mineralized cartilage was investigated by morphological means. Seven days after the addition of beta-GP, the first mineralized spots were visible mainly in the internodular, noncartilage tissue. After 12 to 14 days, large areas of cartilage were mineralized, and after 21 days, nearly the whole culture had been mineralized. Electron microscopic investigations showed a dramatic alteration of the cartilage matrix followed by a homogeneous mineralization of the cartilage matrix. The chondrocytes in the mineralized area died and faded. Typical rod-like apatite crystals were visible at the border between the mineralized and the unmineralized matrix. This result closely resembles the in vivo situation of cartilage mineralization. Addition of osteoblastic calvarial cells enhanced the mineralization process, as did the addition of conditioned medium of calvarial cell monolayers. Under these treatments, mineralization started after 3 days and reached a maximum after 14 days. On the other hand, addition of mouse skin fibroblast-like cells without a direct contact to the cartilage inhibited cartilage mineralization. These results indicate that osteoblastic cells induce endochondral mineralization, whereas fibroblast-like cells inhibit this mineralization via soluble factors.

Animals↗
PubMed1989

Urate-mediated inflammation in nodal osteoarthritis: clinical and roentgenographic correlations.

We studied 25 patients with crystal-proven gout or roentgenographic evidence of gouty arthritis, or both, in finger joints involved with nodal osteoarthritis (OA). These patients were elderly (mean age 71.4 years), and 72% of them were receiving diuretic therapy. Roentgenographic findings, in addition to features typical of OA, included soft tissue densities (tophi), with or without calcification, large intraarticular erosions, characteristic nonmarginal cortical erosions, and periarticular osteolysis. We conclude that urate crystals mediate episodes of acute inflammation in certain patients with nodal OA. This association is noted primarily in elderly patients of both sexes, especially in those receiving diuretic therapy.

Acute Disease↗
PubMed1989

Septic acromioclavicular arthritis and osteomyelitis in a patient with acquired immunodeficiency syndrome.

We describe a patient with acquired immunodeficiency syndrome who developed Staphylococcus aureus septic arthritis and osteomyelitis of the acromioclavicular joint. The case is unusual because of the rarity of reported bone and joint infections in patients with acquired immunodeficiency syndrome and because of the indolent nature of the infection.

Acquired Immunodeficiency Syndrome↗