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Biomedical subjects

J Wroblewski

Publications and source records attributed to J Wroblewski.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1985

Effects of calcitonin on elemental distribution and ultrastructure of rat submandibular gland acinar cells.

The effect of calcitonin on rat submandibular gland acinar cells was investigated by X-ray microanalysis and electron microscopy. Calcitonin caused a transient increase of the cellular calcium and magnesium concentration, but did not affect the intracellular concentration of other electrolytes. The relative volume of intracellular mucus increased from 45% in control glands to 72% at 6 h after administration of calcitonin. Calcitonin caused an inhibition of the cellular response of the acinar cells to beta-adrenergic and cholinergic agonists. The changes in elemental composition and ultrastructure of the gland cells are probably due to inhibition of mucus release from the cells.

Animals↗
PubMed1985

Post-mortem storage of tissue for X-ray microanalysis in pathology.

Possible alternatives to rapid freezing in liquid nitrogen of tissue for X-ray microanalysis of electrolytes at the cellular level were investigated. These alternatives might be used in cases where tissue becomes available for examination, e.g., at autopsy, but liquid nitrogen is not immediately available. Rat submandibular gland was used as a test tissue. Freezing of pieces of tissue in a conventional freezer at -80 degrees C or even at -20 degrees C retained the elemental distribution at the cellular level, and also retained the difference between a 'normal' and a 'pathological' (mimicked by an injection of a high dose of isoproterenol) situation. Storage of tissue in a refrigerator, or delaying the autopsy in anticipation of the arrival of liquid nitrogen is not recommended. Significant changes in the cellular ion content occurred if the tissue was left in the animal for 24h post-mortem.

Animals↗
PubMed1984

Intrinsic GABAergic system of adrenal chromaffin cells.

Histochemical and biochemical studies demonstrate that gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (EC 4.1.1.15), and GABA aminotransferase (EC 2.6.1.19) are present in bovine adrenal chromaffin cells. Moreover, [3H]GABA can be taken up and stored by primary cultures of adrenal chromaffin cells. Nicotinic receptor stimulation or KCl depolarization releases the [3H]GABA taken up by these cell cultures. GABA and benzodiazepine recognition sites located in chromaffin cells interact with each other with modalities similar to those described for GABA and benzodiazepine recognition sites located in synaptic membranes prepared from brain tissue. Bicuculline facilitates the release of catecholamine from chromaffin cells induced by nicotinic receptor stimulation but it fails to influence the release of catecholamine evoked by K+ depolarization. Since the GABA-benzodiazepine receptor system appears to modulate nicotinic receptor function, it is suggested that GABA transmission might participate in modulating responsiveness of chromaffin cells to incoming cholinergic stimuli.

4-Aminobutyrate Transaminase↗
PubMed1984

Site of OH radical attack on dihydrouracil and some of its methyl derivatives.

The site of attack of OH radicals on dihydrouracil and five of its methylated derivatives was determined by pulse radiolysis using N,N,N',N'-tetramethylphenylenediamine (TMPD) to detect oxidizing radicals and tetranitromethane (TNM) as well as K3Fe(CN)6 to detect reducing radicals. In the case of dihydrouracil OH radicals abstract preferentially an H atom at C(6) to give the 6-yl radical (greater than or equal to 90 per cent) which at pH approximately 6.5 reduces TNM and K3Fe(CN)6 at almost diffusion-controlled rates. Only a small fraction of OH radicals abstract the H atom at C(5) (less than or equal to 10 per cent). The resulting 5-yl radical oxidizes TMPD to TMPD+ at pH 7-8. With the methylated derivatives of dihydrouracil, OH radicals react less selectively, especially in the case of N(1)-methyl derivatives. This methyl group is activated to a similar degree as the methylene group at C(6). In 1-Medihydrouracil the yield of N(1)-CH2 radicals is about 29 per cent, which has been deduced from the yield of formaldehyde formed after oxidation of this radical by TNM at pH approximately 6.5 and the subsequent hydrolysis. Radicals at the other methyl substituents are generated to a lesser extent (less than or equal to 10 per cent) and are relatively unreactive towards oxidizing agents such as TNM and K3Fe(CN)6 as well as towards the reducing agent TMPD. Although methyl substitution opens new routes for OH attack the preferred site of H abstraction remains C(6) (greater than 60 per cent).

Ferricyanides↗
PubMed1984

X-ray microanalysis of single and cultured cells.

X-ray microanalysis of single or cultured cells is often a useful alternative or complement to the analysis of the corresponding tissue. It also allows the analysis of individual cells in a cell population. Preparation for X-ray microanalysis poses a number of typical problems. Suspensions of single cells can be prepared by either of two pathways: (1) washing - mounting - drying, or (2) centrifugation - freezing or fixation - sectioning. The washing step in the preparation of single or cultured cells presents the most severe problems. Cultured cells are generally grown on a substrate that is compatible with both the analysis and the culture, washed and dried. In some cases, sectioning of cultured cell monolayers has been performed. Special problems in quantitative analysis occur in those cases where the cells are analyzed on a thick substrate, since the substrate contributes to the spectral background.

Animals↗
PubMed1983

Quantitative X-ray microanalysis of semi-thick cryosections.

Methodological aspects of quantitative X-ray microanalysis of semi-thick cryosections (2--6 micrometers) of biological soft tissue were investigated. The preparation of a low background specimen holder is described. Scanning and scanning transmission images of the sections could be obtained, allowing identification and separate analysis of nuclei and cytoplasm. Parallel observations of histochemically stained adjacent sections in the light microscope allowed correlation of the microanalytical data with tissue morphology and histochemistry. Quantitative analysis could be carried out with the help of a standard: a gelatin/glycerol matrix containing mineral salts in known quantities, frozen and sectioned in the same way as the specimen. Mass loss under the electron beam was found to be comparable in specimen and standard. Comparison of various theoretical models for quantitative analysis showed that the 'P/B-method' (determination of the background intensity under the characteristic peak) is the most suitable for semi-thick sections. Factor determining the choice of accelerating voltage were analyzed. The usefulness of this specimen type is illustrated in some biological applications (human oral mucosa, rat salivary gland).

Animals↗
PubMed1983

X-ray microanalysis of cultured chondrocytes.

Methodological aspects of X-ray microanalysis of cultured chondrocytes from rabbit auricular cartilage were investigated. The cells were grown on a Formvar-film covered carbon specimen holder with a hole, which allowed examination in STEM. Several different fluids for washing away the culture medium from the cells were used. The effect of these fluids on the elemental composition of the cells was studied. The composition of cultured chondrocytes was compared with that of auricular chondrocytes in situ. Washing with 0.3 M sucrose gave the most satisfactory results. Subsequently, the specimen holders could be frozen in liquid nitrogen and the cells freeze-dried. The effects of human growth hormone on rabbit chondrocytes were investigated by X-ray microanalysis and transmission electron microscopy. Although no ultrastructural effects of hormone treatment could be observed, marked changes in the elemental concentrations in nucleus and cytoplasm of the chondrocytes were detected.

Animals↗
PubMed1983

Healthy and diseased epithelium of the oral and maxillary sinus mucosa studied by X-ray microanalysis.

Concentrations of Na, Mg, P, S, Cl, K, and Ca in healthy and pathologically transformed epithelial cells of oral cavity and maxillary sinus mucosa in man were measured by X-ray microanalysis. Biopsies from normal mucosa, irradiated and clinically cured squamous cell carcinoma, mucoepidermoid carcinoma from the oral cavity, and polyposis in the maxillary sinus were prepared by rapid freezing and cryosectioning. In normal mucosa, nuclei and cytoplasm of basal, intermediate and apical epithelial cells were analyzed. A slight decrease in the concentration of Na, Mg, P and Cl was found towards the apical cells. The concentration of Na in the nuclei of the different cell layers parallelled that of P. The elemental composition of the pathologically transformed cells differed markedly from that of the cells of healthy oral mucosa (higher Na and Cl). In the irradiated squamous cell carcinoma the concentrations of Mg and K were extremely low.

Aged↗
PubMedDate not supplied

Uptake of radioactive sulphur in the endolymphatic sac. An autoradiographic study.

Autoradiographic uptake of labelled sulphur (S35) in the endolymphatic sac was analysed after performing surgical labyrinthectomy and after systemic administration of glycerol and ethacrynic acid. Accumulation of radioactive substance was observed after surgical labyrinthectomy and associated with a deposition of stainable substance in the endolymphatic sac lumen. Increased activity was noted in the epithelial layer after administration of ethacrynic acid, whereas glycerol did not increase radioactivity in the endolymphatic sac despite the deposition of stainable substance therein. This fact was believed to depend on a too short time interval between the injection and the sacrifice of the animals, as well as a weaker concentration of the sulphated compound within the endolymphatic sac. The results suggest that the endolymphatic sac may secrete sulphur-containing substances presumably associated with a deposition of a stainable substance in the endolymphatic sac.

Animals↗