Biomedical subjects

J Wroblewski

Publications and source records attributed to J Wroblewski.

At least 37 records · Page 2Linked to original sources

PubMed1995

Low concentrations of mercury induce changes in ion composition of cultured myoblasts.

The effects of low concentrations (1 pM, 1 nM, 1 microM) of mercuric chloride on ion distribution in cultured myoblasts were analysed by energy dispersive X-ray microanalysis. An increase in intracellular sodium concentration was observed five minutes after addition of HgCl2 to the culture medium. This increase was dose dependent and accompanied by a transient decrease in potassium concentration. Exposure to 1 nM and 1 microM HgCl2 led to a two-fold increase in the cytoplasmic chlorine concentration. The higher HgCl2 concentration (1 microM) induced morphological alterations in the form of cell membrane blebs, perforations and shrinkage or flattening of the myoblasts. It was concluded that even low concentrations of mercuric chloride cause elemental and morphological changes in cultured myoblasts, which may reflect effects of the metal on membrane permeability.

Animals↗

PubMed1995

Expression of the intermediate filament nestin during rodent tooth development.

The developing tooth represents a suitable model for understanding the molecular mechanisms involved in induction, morphogenesis and differentiation of organs. It is conceivable that the developmental changes could be reflected in the distribution of different cytoskeletal components and in this report we analyze the expression of the intermediate filament nestin during rodent tooth development at the protein and mRNA levels (by immuno light and electron microscopy, and by in situ hybridization). Nestin is expressed at all stages of tooth development, but the expression levels increase after birth in both ectodermal and ectomesenchymal derivatives. The shift in nestin distribution, from the proliferating dental lamina to the dental mesenchyme, indicates that nestin may be involved in inductive phenomena. At early stages of mineralization, nestin is seen within the apical parts of the presecretory ameloblasts. Nestin is also expressed in odontoblasts, both during odontogenesis and after tooth eruption. The increase in nestin expression from early to late developmental stages and sustained expression in a differentiated cell type contrasts with previously observed patterns of nestin expression during nerve and muscle development. This suggests that nestin could be used as a specific marker for the odontoblast.

Animals↗

PubMed1994

Distribution of C-CAM in developing oral tissues.

C-CAM is a cell surface glycoprotein that is involved in cell adhesion and may play a role in histogenesis and organogenesis. It is a member of the carcinoembryonic antigen (CEA) gene family, which is a subfamily of the immunoglobulin gene superfamily. We have analyzed the expression of C-CAM during normal and disturbed craniofacial development in the mouse by immunohistochemistry and in situ hybridization. Developmental disturbances were induced by retinoic acid (RA) treatment of pregnant mice. Normal and malformed fetuses were examined on days 14, 15, 16, 17 and 18 of gestation. The expression of C-CAM was detected first at day 16. With age, the signal became gradually stronger. C-CAM was detected in the epithelia of both ectodermal and mesodermal origin, including oral and respiratory epithelia, epithelia of the developing vessels, glands and their ducts. In the RA-treated fetuses, the expression of C-CAM was higher in the epithelium of the oral cavity than in that of the nasal cavity, with a distinct borderline between differentiating nasal and oral epithelium of the palatal shelves. However, the submucosal nasal glands and ducts showed higher expression than oral glands in both normal and RA-treated mice. The expression of C-CAM did not differ significantly between control and RA-treated animals. The presence of C-CAM in all proliferating craniofacial epithelia indicates that this molecule may play an important role in development.

Adenosine Triphosphatases↗

PubMed1994

Acute zonal occult outer retinopathy (AZOOR) associated with multifocal choroidopathy.

Acute zonal occult outer retinopathy (AZOOR) may be precipitated by various retinal disorders and is characterised by rapid loss of visual field which cannot be explained by the ophthalmoscopic changes consequent upon the initiating disease. The electroretinogram is abnormal, indicating that the field loss is due to retinal dysfunction. The phenomenon was first recognised in the multiple evanescent white dot syndrome (MEWDS) as the enlarged blind spot syndrome. It was subsequently described with multifocal inner choroidopathy and acute macular neuropathy (AMN). We have identified 7 patients who presented with widespread visual loss associated with multifocal inner choroidopathy in whom functional loss was documented with electroretinography and automated visual field testing. All patients were young, myopic, and otherwise healthy women. Initial photopsia was noted by 4 patients. Fundus findings included scattered small partially pigmented yellowish lesions resembling those in multifocal inner choroidopathy or pseudo presumed ocular histoplasmosis syndrome, disc swelling, vitritis, and secondary choroidal neovascularisation. Two patients had bilateral involvement. All patients had an enlargement of the blind spot, and widespread visual field loss which was not explained by fundus changes. All had an abnormal electroretinogram suggesting widespread retinal disease. In AZOOR retinal dysfunction occurs without corresponding visible retinal lesions. This disorder appears to be precipitated by several conditions, although the causal relationship between the initiating event and the widespread functional loss is unknown.

Acute Disease↗

PubMed1994

X-ray microanalysis of endocrine, exocrine and intestinal cells and organs in culture: technical and physiological aspects.

In the present study methods for preparation of cultured cells and organ cultures for analytical electron microscopy are investigated. These methods allow qualitative and quantitative analysis of mobile ions in combination with biochemical or morphological studies. Cultured cells can be easily prepared for analytical microscopy and therefore use of in vitro systems for X-ray microanalysis has increased over the last few years. Two major, anhydrous preparation techniques, by which loss or redistribution of ions is minimized, were used: (1) Cells were cryosectioned and analysis carried out on freeze-dried sections obtained from frozen cell monolayers, pelleted cells or organ cultures. (2)Cells cultured on supports compatible with elemental analysis were frozen after removal of experimental media by rinsing, freeze-dried and analyzed. The first technique was applied to the studies of the elemental content of isolated Langerhans islets and thyroid follicles cultured in collagen gel. The second was used in studies of the ionic changes in enterocytes. Data obtained from organotypic cell cultures and cultures of single cells were compared with analytical data obtained from sections of corresponding tissues, where isolation, culturing and steps in processing such as removal of culture or experimental medium were omitted. It was shown that often culture systems fully acceptable to physiologists have an elemental composition different from that of tissue in situ and can not be regarded as fully normal tissue.

Animals↗

PubMed1993

Mutations in the human retinal degeneration slow (RDS) gene can cause either retinitis pigmentosa or macular dystrophy.

Mutations in the RDS gene, which encodes the photoreceptor glycoprotein peripherin, have been sought in families with autosomal dominant retinal dystrophies. A cysteine deletion at codon 118/119 is associated with retinitis pigmentosa in one. Three families with similar macular dystrophy have mutations at codon 172, arginine being substituted by tryptophan in two and by glutamine in one. A stop sequence at codon 258 exists in a family with adult vitelliform macular dystrophy. These findings demonstrate that both retinitis pigmentosa and macular dystrophies are caused by mutations in RDS and that the functional significance of certain amino-acids in peripherin-RDS may be different in cones and rods.

Adult↗

PubMed1993

Growth regulation of LLC-PK1 cells: lack of effect of Na(+)-loading.

To gain more information about the growth regulation of renal epithelial cells, we examined the growth stimulatory effect of serum and intracellular sodium in the renal epithelial cell line, LLC-PK1. In subconfluent LLC-PK1 cells serum-starved for 5 days and exposed to [3H]thymidine for 24 h, 22.9% of the cells synthesized DNA. Stimulation with 10% foetal calf serum (FCS) caused an almost three-fold increase in the fraction of labelled nuclei (62.2%). Serum-starved LLC-PK1 cells exposed to 10% FCS responded with an increased abundance of c-jun transcripts. The maximal expression of the c-jun transcripts occurred at 60 min and declined 120 min after serum stimulation. It has been suggested that an increase in Na+ influx plays a role in the growth regulation of renal epithelial cells. This prompted us to study the effect of intracellular Na+ loading on the growth response of LLC-PK1 cells. Serum-starved LLC-PK1 cells were incubated in a low K+ medium or exposed to Nystatin. Incubation in a low K+ medium or with Nystatin resulted in a marked increase in intracellular Na after only 5 min. A low K+ medium did not significantly influence the intracellular pH. No effect was observed on DNA synthesis or the abundance of c-jun transcripts in LLC-PK1 cells. Nor did Na+ loading enhance the growth stimulatory effect of serum. The results suggest that an increase in intracellular sodium does not directly regulate the growth of renal epithelial cells.

Animals↗

PubMed1993

Regulation of growth-plate chondrocytes by insulin-like growth-factor I and basic fibroblast growth factor.

A study was performed in order to investigate the possible functional roles of insulin-like growth-factor I (IGFI) and basic fibroblast growth factor (bFGF) in the regulation of mitotic and metabolic activity of growth-plate chondrocytes. Chondrocytes from the distal radial growth plates of calves and the costal physeal cartilage of rats were exposed to these factors, individually and in combination, in primary monolayer culture, to assess their effects. The data showed that bFGF had both a greater potency and a greater efficacy as a mitogen for bovine growth-plate chondrocytes than did IGF-I. The maximum incorporation of 3H-thymidine by bFGF was 8.3 times that in serum-free (control) cultures; the maximum stimulation of incorporation by IGF-I was 2.5 times that in the control medium. In contrast, IGF-I stimulated a maximum incorporation of 35S-sulfate into glycosaminoglycans that was 2.6 times that in the IGF-I serum-free control cultures, while bFGF had no effect or was mildly inhibitory. When used together, these two factors acted synergistically. Incorporation of 3H-thymidine was more than two times greater than the sum of the effects of the growth factors when used alone and 20.5 times greater than that of the growth factor-free control cultures. Physeal chondrocytes from six-day-old rats were mitotically more responsive to bFGF than to IGF-I, but they were more responsive to IGF-I when they had been derived from twenty-eight-day-old rats. Interaction between bFGF and factors in the serum enhanced the mitotic activity of the rat chondrocytes, but bFGF did not interact with IGF-I under the same experimental conditions. In the presence of bFGF, there was a reduction in the stimulation by IGF-I of incorporation of 35S-sulfate and a decrease in the percentage of chondrocytes containing alkaline phosphatase. These growth factors also influenced cellular morphology in culture. In the presence of IGF-I or serum, the rat chondrocytes manifested the polygonal morphology typical of chondrocytes in culture, while bFGF promoted a more elongated spindle shape. Removal of bFGF and replacement by IGF-I restored the polygonal morphology, indicating that this transition is reversible.

Alkaline Phosphatase↗

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↗