Biomedical subjects

J Wroblewski

Publications and source records attributed to J Wroblewski.

At least 19 recordsLinked to original sources

PubMed1992

PDGF BB stimulates proliferation and differentiation in cultured chondrocytes from rat rib growth plate.

The role of two isoforms (PDGF AA and PDGF BB) of platelet derived growth factor either alone or in combination with insulin-like growth factor I, on the regulation of proliferation and differentiation of rat rib growth plate chondrocytes was analyzed. PDGF BB increased DNA-synthesis in a dose dependent manner with a half maximal effect at 1 ng/ml. When PDGF BB was combined with IGF-I, an additive effect on DNA-synthesis was observed. PDGF AA and BB alone or combined with IGF-I had no appreciable effects on proteoglycan synthesis. Both homodimers caused an increase in AP-activity, indicating stimulation of cell differentiation. Cultured chondrocytes bound 125I-PDGF AA and 125I-PDGF BB and after stimulation with PDGF expressed c-fos protein. Thus, both homodimers play an important role in chondrocyte differentiation and together with IGF-I interact in the regulation of longitudinal bone growth.

Actins↗

PubMed1992

Very low cadmium concentrations stimulate DNA synthesis and cell growth.

Uptake of cadmium into cultured cells and its effects on cell growth and DNA synthesis are measured over a range of Cd concentrations of seven orders of magnitude. Cd uptake is found to be proportional to the external Cd concentration and to incubation time over a very broad range of concentrations. At least 200 mmol cadmium per kg dry weight of cells can be accumulated in this way, leading to exhaustion of the major intracellular Cd binding sites before cell death. On the other hand, very low cadmium concentrations down to 100 pM stimulate cell growth and DNA synthesis significantly. Stimulation is found in all three mammalian cell types examined: namely L6J1, a rat permanent myoblast cell line, LLC-PK1 porcine renal epithelial cells, and a primary rat chondrocyte culture. Cd acts as a cofactor with serum in L6J1 cultures, but is stimulatory only in serum-free cultures of chondrocytes. Stimulation occurs at Cd concentrations too low to result in a measurable induction of metallothionein. This might implicate the action of response amplifiers in the chain of events leading to Cd-stimulated DNA replication and cell growth.

Animals↗

PubMed1991

Elemental analysis and fine structure of mitochondrial granules in growth plate chondrocytes studied by electron energy loss spectroscopy and energy dispersive X-ray microanalysis.

Electron energy loss spectrometry--EELS, and energy dispersive X-ray microanalysis--XRMA, were used to study the elemental composition of mitochondrial dense granules-mdg. The study was performed on dry cut thin sections (80-200 nm) of freeze-dried and low temperature embedded cartilage. Results obtained by means of XRMA clearly showed high phosphorus and calcium content in the mdg. Using EELS at 100 kV primary voltage we found that small concentrations of elements (i.e. below typically 1% atomic weight) are difficult to analyze and map, this especially in sections thicker than 50-60 nm. Surprisingly, analysis of calcium can be successfully performed on thicker sections though the edge lies above the carbon K edge while this is not possible for the phosphorus edge which is located at lower energies. This is likely due to the edge shapes (sharp for calcium and delayed for phosphorus), and to the more intense contribution of multiple low loss scattering in the background for phosphorus between 100 and 130 eV. By means of EELS elemental mapping a centrally located core was found in numerous mdg. In the calcium map the signal was strongest in the middle of mdg which corresponds to the area of reduced carbon signal. We found that carbon maps might be used for high resolution structural studies of chemically unfixed and anhydrously processed biological tissues. As carbon is the main constituent of Lowicryl resin its distribution is reversed to the distribution of biological tissue in which the proportion of carbon is lower, but is proportional to water content in the specimen in vivo. Use of EELS in combination with electron microscope with accelerating voltages in range of 140-200 kV together with anhydrous techniques of the tissue preparation will provide a new type of information which might lead to better understanding of the etiology and function of small structures in the cell.

Animals↗

PubMed1990

A low temperature vacuum embedding procedure for X-ray microanalysis of biological specimens at subcellular level.

The validity of freeze-drying and low temperature embedding in Lowicryl resins has been investigated in studies of ion distribution in mouse embryological inner ear, rat rib growth plate, liver and submandibular gland. The morphological preservation of the tissues was adequate for the identification of different intracellular compartments and extracellular structures. It was also possible to analyze extracellular fluids in the vestibular part of the developing inner ear. Compared with thin cryosections, Lowicryl sections are easier to produce and are more stable during analysis. Freeze-dried embedded material can be easily reorientated during cutting and adjacent sections can be used for other purposes such as histochemical and morphological investigations. We found that hydrophilic Lowicryl K11M, which is normally used for immunocytochemical investigations, also can be used for microanalysis. That opens the possibility for combined immunocytochemical and microanalytical studies. However, infiltration and polymerization steps have to be carried out at slightly higher temperatures than when the hydrophobic Lowicryl HM23 is used.

Acrylic Resins↗

PubMed1989

Binding of insulin-like growth factor-I (IGF-I) to primary cultures of chondrocytes from rat rib growth cartilage.

Binding of insulin-like growth factor I (IGF-I) to cultured resting, proliferative and hypertrophic growth plate chondrocytes was investigated. The optimal binding conditions and the extent of degradation of the 125I-IGF-I at 20 degrees C were analyzed in a time-course study. The maximal binding without noticeable degradation was observed after 3 h. The binding of IGF-I to the proliferative cells was 2-fold higher than to the resting and the hypertrophic cells. On the proliferative chondrocytes two classes of receptors with different affinities were found. 125I-IGF-I could be displaced from the proliferative cells by unlabelled IGF-I, IGF-II and insulin, respectively. Half maximal binding was observed at 0.3 nmol/l (= 2.2 micrograms/l) of IGF-I, 4.3 nmol/l (= 32 micrograms/l) of IGF-II and 350 nmol/l (= 2000 micrograms/l) of insulin. No specific binding of human growth hormone (hGH) could be demonstrated. When binding of epidermal growth factor (EGF) to the proliferative cells was assessed, little, but specific binding was observed.

Animals↗

PubMed1989

Effects of IGF-I, rGH, FGF, EGF and NCS on DNA-synthesis, cell proliferation and morphology of chondrocytes isolated from rat rib growth cartilage.

The effects of IGF-I, rGH, FGF, EGF and NCS on DNA-synthesis were analyzed in resting, proliferative and hypertrophic chondrocytes obtained by fractionation. Proliferation and morphology were studied on non-fractionated cells. The highest stimulation of DNA-synthesis was induced by NCS followed by IGF-I at all stages of chondrocyte differentiation. DNA-synthesis was also stimulated by a low concentration of FGF (1 microgram/1) in proliferative and hypertrophic chondrocytes, while FGF in a higher concentration (10 micrograms/1) had no significant mitogenic effect. Cell proliferation was stimulated by both NCS and IGF-I, whereas FGF and EGF only caused morphological changes. Our data indicate that IGF-I is the main serum growth factor regulating growth and proliferation by interacting with chondrocytes at all stages of differentiation.

Animals↗

PubMed1989

Strontium and bromide as tracers in X-ray microanalysis of biological tissue.

Since energy dispersive X-ray microanalysis cannot distinguish between isotopes of the same element, alternative methods have to be used to get information similar to that obtained in experiments with radioactive tracers. In the present study, strontium was used as a tracer for calcium, and bromide as a tracer for chloride. Rats were injected with strontium chloride in vivo, and the uptake of strontium in the acinar cells of the submandibular gland was studied. Eventually a Sr/Ca ratio of 0.3 was reached. In some animals, secretion of mucus had been elicited by stimulation with isoproterenol 4 h prior to injection of strontium chloride. Exchange of calcium for strontium was enhanced by prior injection with isoproterenol. In a second experiment, rats were injected with sodium bromide, and the uptake of bromide by the submandibular acinar cells was followed in time, both in pilocarpine-stimulated and unstimulated glands. Under the experimental conditions, bromide was rapidly taken up by the cells, and the cellular Br/Cl ratio was close to that found in serum. Submandibular glands take up Br somewhat faster than other tissues (liver, heart muscle, skeletal muscle). The uptake of Br in pancreatic acinar cells was studied in vitro. These experiments showed a 1:1 ratio (molar) exchange of Cl for Br.

Animals↗

PubMed1989

X-ray microanalysis of mammalian salivary glands.

X-ray microanalysis was used to study the elemental composition of major salivary glands in rat and man. The elemental composition of the acinar cells is influenced by a variety of factors. Stimulation of fluid secretion by cholinergic, alpha-adrenergic or peptidergic agonists leads to a sustained increase of the Na/K ratio. The elemental composition and ultrastructure of the acinar cells of rat submandibular gland, as well as flow rate and composition of pilocarpinestimulated submandibular saliva are significantly affected by thyroxine, but less by the synthetic glucocorticoid dexamethasone. Hypercalcemia (induced by chronic vitamin D treatment) as well as hypocalcemia (induced by calcitonin) cause accumulation of intracellular mucus in the submandibular acinar cells, with concurrent increase in the cellular calcium concentration, and a significantly decreased flow rate after pilocarpine stimulation. Several animal models have been proposed for the generalized exocrinopathy cystic fibrosis (CF), which may be caused by a defective regulation of chloride transport in epithelial cells. A potential new model is proposed: the chronically furosemide-treated rat, which shows several abnormalities in salivary gland structure and function that parallel the abnormalities found in the human disease. Another experimental model of potential interest in the study of CF is short-term treatment of experimental animals with the adenylate-cyclase inhibitor alloxan.

Animals↗

PubMed1989

Energy dispersive X-ray microanalysis, neutron activation analysis and atomic absorption spectrometry--comparison using biological specimens.

X-ray microanalysis, neutron activation analysis and atomic absorption spectrometry were performed on normal and injured skeletal muscle. X-ray microanalysis of tenotomized rat soleus muscle showed significantly elevated levels of sodium and chlorine and lower potassium compared with normal muscle. Similar ion shifts could be demonstrated by neutron activation analysis and atomic absorption spectrometry. The concentrations of sodium and chlorine obtained by these techniques were somewhat higher and that of potassium lower than the values obtained by X-ray microanalysis. This can probably be attributed to the fact that in atomic absorption spectrometry and in neutron activation analysis the entire muscle biopsy contents are measured while in X-ray microanalysis only the content of muscle cells unaffected by extracellular, non-muscular components are determined. It can be concluded that X-ray microanalysis is a reliable technique to study the elemental content of biological tissue, especially tissue undergoing pathological changes affecting the extracellular spaces. Other types of analysis should be used when elements not detectable by X-ray microanalysis are of interest.

Activation Analysis↗

PubMed1988

Low temperature techniques for X-ray microanalysis in pathology: alternatives to cryoultramicrotomy.

Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozen-hydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation or when data from a large number of samples have to be acquired. Also X-ray microanalysis of cultured or single cells prepared by freeze-drying can be used to rapidly collect information on a large number of cells. Analysis at high resolution has to be carried out on thin sections: Cryosections or sections of freeze-substituted or freeze-dried embedded tissue. For the latter type of specimens, the use of low-temperature embedding methods may have important advantages.

Animals↗

PubMed1988

Effects of IGF-I, EGF, and FGF on proteoglycans synthesized by fractionated chondrocytes of rat rib growth plate.

The effects of insulin-like growth factor (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF), or 10% newborn calf serum (NCS) on the amount and structure of the proteoglycans synthesized by fractionated chondrocytes from rat growth plate were investigated. Proliferative cells (fraction II) or resting cells (fraction III) synthesized more proteoglycans than hypertrophic cells (fraction I). Addition of IGF-I to the cultures increased proteoglycan synthesis more than addition of EGF or FGF. EGF and FGF induced synthesis of proteoglycans of smaller molecular size with a lower proportion of aggregates. The size of the constituent glycosaminoglycan chains did not differ between control and growth factor-treated cells. The present study demonstrates that proteoglycan structure and synthesis are modified by growth factors to different extents, depending on the maturation stage of the target cells.

Animals↗

PubMed1988

X-ray microanalysis of cartilage and chondrocytes.

The elemental content of differentiating chondrocytes in situ and in vitro was studied by X-ray microanalysis. The in situ studies were carried out on semi-thick cryosections and on thin sections of freeze-dried low-temperature vacuum-embedded rat rib growth plate. Resting, proliferative, and hypertrophic cells were analyzed separately. The in vitro studies were carried out on cultured cell fractions isolated from rat rib growth plate. The cell fractions were relatively homogeneous and contained resting - early proliferative, proliferative or hypertrophic cells, respectively. Data from cells in vitro are generally not comparable to data obtained from cells in situ. Chondrocytes in situ contain relatively high levels of Na, which is probably due to: (1) high Na levels in the cartilage matrix formed intracellularly, and (2) low supply of oxygen and nutrients. High Na levels in (some of) the proliferative cells may also be related to mitotic activity. The chondrocyte matrix contains relatively high levels of K. The data in the present study are compared to those of previous studies on the elemental content of chondrocytes.

Animals↗

PubMed1988

Elemental microanalysis of biological specimens.

Although X-ray microanalysis in the electron microscope is the most common method for microanalysis of biological specimens, other methods of elemental microanalysis (electron energy loss spectroscopy, scanning Auger microanalysis, and proton, ion, and laser microprobe analysis) may provide important complementary information and help overcome some of the limitations of electron probe X-ray microanalysis. Despite differences in physical principles and instrumentation, the various microanalytical methods have much in common with regard to specimen preparation, quantitative analysis, and interpretation of analytical data. A common approach to microanalytical problems in the biological sciences, irrespective of the analytical techniques used, seems therefore indicated.

Electron Probe Microanalysis↗

PubMed1987

Elemental changes associated with chondrocyte differentiation in rat rib growth plate.

Quantitative X-ray microanalysis was under-taken to follow the elemental changes that occur in the process of chondrocyte differentiation. For analysis at the cellular level, semi-thick freeze-dried cryosections of rat rib growth plate cartilage were used. For evaluation of the elemental concentrations at the subcellular level, thin sections of freeze-dried and low temperature vacuum embedded cartilage were analyzed. Levels of Na, P, S, Cl, K, and Ca were determined in the cells and extracellular matrix in different zones of the cartilage--resting, proliferative, and hypertrophic. Proliferative cells had a sodium concentration that was twice that of resting cells, suggesting that Na may play an important role in the regulation of DNA- and protein-synthesis in chondrocytes, A concomitant rise in Na and S concentration occurred between resting zone and proliferative zone cartilage matrix. The high concentrations of Na and K in the matrix are probably due to the high amount of sulfate in proteoglycans which may bind these cations.

Animals↗

PubMed1987

Subcellular location of IGF-I in chondrocytes from rat rib growth plate.

Ultrathin cryosections of segments of growth plates from rat rib and isolated, gelatin-embedded chondrocytes from the same source were used to demonstrate the ultrastructural location of IGF-I in the different layers of the growth plate. Sections were incubated with a polyclonal rabbit antiserum to free IGF-I and colloidal gold coated with goat anti-rabbit IgGs. The gold label was present in all chondrocytes, localized almost exclusively to the cytoplasm. Particularly the cells in the proliferating zone displayed considerable amounts of gold label. In these cells, gold label was predominantly present over the rough endoplasmic reticulum and membrane bound vesicles, with only negligible gold label over the nuclei and cartilage matrix. The distribution of gold label in the chondrocytes from the proliferating zone, especially in the rough endoplasmic reticulum, strongly suggests that IGF-I can be synthesized by these cells. However, further studies are needed to verify whether there is a local synthesis of IGF-I in the chondrocytes and whether it is under control of growth hormone.

Animals↗

PubMed1987

Low temperature techniques in biomedical microanalysis.

Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozen-hydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation, or when many data from a large number of samples have to be acquired. Quantitative analysis is then usually carried out with the peak-to-local background method: by taking the background in the same energy range as the characteristic peak, an intrinsic correction for absorption of X-rays within the sample is made. X-ray microanalysis of cultured cells prepared by freeze-drying can also be carried out in this way. Analysis at high resolution has to be carried out on thin sections: cryosections, or sections of freeze-substituted or freeze-dried embedded tissue. For the latter type of specimens, the use of low-temperature embedding methods may have important advantages. Quantitative analysis of thin sections is carried out by the established continuum method.

Animals↗

PubMed1987

Cell fractions from rat rib growth cartilage. Morphological and X-ray microanalytical investigation.

For in vitro studies of differentiation and proliferation of chondrocytes, a need arose to prepare enriched cell fractions for analysis of the effects of different growth promoting factors. Therefore chondrocytes were isolated from rat rib growth cartilage in young rats by collagenase digestion and subsequently three cell fractions were prepared by centrifugation in a step gradient of Percoll. In a previous paper, matrix molecules synthesized by each fraction were characterized biochemically. In the present study, ultrastructure and elemental content of the fractionated chondrocytes were analyzed by scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray microanalysis. DNA-synthetic activity was measured by means of autoradiography of 3H-thymidine incorporating cells. Both morphology and elemental concentrations differed between fractions and were characteristic for each fraction. The cell fraction with the lowest density consisted of large, polygonal cells that became flattened in culture. The reduced synthetic activity and markedly lowered K content of these cells suggest that they originate from the hypertrophic zone of the growth plate. The cells in the fraction with the highest density were rather homogeneous in size and shape, and had a well developed rough endoplasmic reticulum and Golgi complex, characteristic of proliferating and resting chondrocytes. Concentrations of P and K were also significantly higher in this fraction. The fraction with intermediate density contained an admixture of cells with a predominance of proliferating cells with high DNA-synthetic activity.

Animals↗

PubMed1986

Cell fractions from rat rib growth cartilage. Biochemical characterization of matrix molecules.

In an attempt to isolate and characterize the putative target cells for growth hormone, chondrocytes were isolated from rat rib growth cartilage and fractionated by centrifugation in a discontinuous Percoll gradient. This resulted in three cell fractions with differing properties. The fraction with the lowest density consisted mainly of large, lipid-containing cells which became flattened in subsequent culture. The cells in this fraction were fair collagen producers but synthesized only minor amounts of proteoglycans and apparently no proteoglycan aggregates. These cells probably originate in the hypertrophy zone of the growth plate. The fraction with highest density, on the other hand, consisted of small cells which upon cell culture became polygonal and surrounded with refractile matrix. They synthesized less collagen, but more proteoglycans than the low-density fraction. The proportion of proteoglycan aggregates was rather low (10-20%) but otherwise the proteoglycans were similar to those synthesized by other chondrocytes. This cell fraction was tentatively identified as cells from the upper part of the growth plate. Finally, the middle fraction was heterogeneous, consisting of cells of varying shape. This fraction synthesized large amounts of both collagen and proteoglycans. In all three cell fractions, type II collagen predominated. There were also minor amounts of (1a, 2a, 3a) collagen, and, in the two denser fractions, of type I collagen.

Animals↗