Label-free spectroscopic characterization of live liver sinusoidal endothelial cells (LSECs) isolated from the murine liver.

Liver sinusoidal endothelial cells (LSECs) represent a highly specialized and unique type of endothelial cell in terms of their morphology and function. The biochemical and functional characterization of LSECs in vitro is restrained by the rapid change of LSECs' phenotype upon culturing under classical experimental conditions. In this work, we present a novel approach to characterize the biochemical content of murine LSECs, freshly isolated from the liver, with the use of microspectroscopic analysis. For comparison, hepatocytes and Hepatic Stellate Cells (HSCs) were analyzed. Our approach, based on label-free confocal Raman imaging of live cells combined with chemometric analysis, provided insight into the biochemical content of freshly isolated LSECs on a subcellular level. LSECs were featured by a distinct biochemical signature in comparison with other major cell types of the liver. Based on our work we claim that the non-invasive and non-destructive confocal Raman imaging may assist in obtaining chemical information spatially distributed within the cells that characterize the phenotype of primary LSECs as well as other types of liver cells. Furthermore, our approach provides a unique insight into LSECs' morphology and chemical composition that may help to understand their functions.

Electromagnetic fields with frequencies of 5, 60 and 120 Hz affect the cell cycle and viability of human fibroblast BJ in vitro.

The impact of electromagnetic field (EMF) on humans has been described in numerous studies, but many questions are still unanswered. The aim of the experiment described in this study was to evaluate the effect of EMF on the viability of human fibroblast BJ in vitro and the percentage of cells in different phases of the cell cycle (G1/G0, S, G2/M) after 2 hours of exposure to sinusoidal continuous and pulsed EMFs with frequency of 5 Hz, 60 Hz and 120 Hz at a magnetic induction of 2,5 mT. The viability of BJ cells exposed to an EMF was estimated immediately after completion of exposure and after 24 hours. Metabolic activity of cells was assessed by MTT assay and compared to a control culture not exposed to EMFs. Cell cycle analysis was performed by BrdU incorporation. The analysis of the viability demonstrated significant differences in field efficiency, depending on its nature. Exposure of cells to pulse EMFs resulted in a decrease in their viability for each of the analyzed frequencies. Reduced viability was maintained for a further 24 hours after the end of exposure of cells to pulsed EMF. In the case of continuous field, reduced BJ cell viability was observed only at the highest applied frequency - 120Hz, and this effect maintained for the next 24 hours. Although there was no significant effect on cell viability (metabolic activity) of cells immediately after exposure to continuous EMF with a frequency of 5Hz, a significant increase was observed after 24 hours of incubation.

Vibrational spectroscopy-based quantification of liver steatosis.

The liver plays a central role in lipid metabolism, and abnormal lipid accumulation in the liver is a key feature of Non-Alcoholic Fatty Liver Disease. In experimental studies, quantification of liver steatosis is commonly based on lipids staining or biochemical analysis. Here, we present a spectroscopic approach for quantitative analysis of the lipid content in the freeze-dried liver. The method is based on vibrational spectroscopy (Raman and infrared) measurements applied for Partial Least Squares (PLS) regression modeling. The obtained PLS models show a good correlation of the spectroscopic data with the reference histological evaluation of steatosis based on Oil Red O (ORO)-stained images of liver cross sections. Vibrational spectroscopy with PLS-based modeling described here represents a useful approach for the fast assessment of the liver steatosis in a small sample of freeze-dried liver tissue. In conclusion, our work demonstrates the easy-to-use method that can be applied in laboratory routine as a beneficial alternative to the established ORO staining.

Hydroxycinnamoyl derivatives and secoiridoid glycoside derivatives from Syringa vulgaris flowers and their effects on the pro-inflammatory responses of human neutrophils.

Thirteen new compounds including caffeoyl-glucaric and p-coumaroyl-altraric acid derivatives, one monoterpenoid glucoside, four secoiridoid glycosides, and three hydroxycinnamoyl phenylpropanoid glycosides esterified with an oleoside 11-methyl ester along with fifteen known compounds were isolated from flowers of Syringa vulgaris L. (Oleaceae). Their structures were elucidated by high-resolution spectroscopic methods. The tested compounds were able to decrease the production of reactive oxygen species. Moreover, oleoechinacoside (13), demethylhydroxyoleonuezhenide (14), demethyloleonuezhenide (15), syringaoleoacteoside (25) and oleoacteoside (26) at the concentration of 50µM, moderately suppressed the LPS-stimulated release of pro-inflammatory chemokine IL-8 and TNF-α from human neutrophils. Moreover, oleonuezhenide (12), oleoside 11-methyl ester (16) and oleoacteoside (26) at the concentration of 50µM were able to induce the surface expression of interleukin 10 receptor, which is suppressed by the incubation of monocyte/macrophage cells with LPS.

Interaction of calcyclin and its cyanogen bromide fragments with annexin II and glyceraldehyde 3-phosphate dehydrogenase.

The structural properties of calcyclin protein are quite well characterized but its function remains obscure. To help elucidate the biological role of calcyclin we have performed the in vitro studies of the Ca(2+)-dependent interaction of Ehrlich ascites tumor cells calcyclin and its cyanogen bromide fragments with two potential calcyclin targets: annexin II and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The binding of annexin II, evidenced by the reaction with 125I-calcyclin, was found to be very weak and occurred only for intact calcyclin. On the other hand the interaction between calcyclin and GAPDH was of high affinity and could be assigned to the N-terminal region of calcyclin. Intact calcyclin and its N-terminal fragment bound to GAPDH in the gel overlay and affinity chromatography assay. When examined in the presence of a crosslinking agent the interaction resulted in the formation of 46K covalent adduct between calcyclin monomer and GAPDH subunit. Fluorescence of 5-iodoacetamido-fluorescein-labelled calcyclin was efficiently quenched by GAPDH in the presence of Ca2+. Titration experiments revealed the stoichiometry of one calcyclin monomer binding to each of GAPDH subunits with a binding constant of 10(8) M-1. The results of this work suggest that the binding between calcyclin and GAPDH may have bearing on calcyclin function.

Calcyclin is a calcium and zinc binding protein.

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.

Tissue specific distribution of calcyclin--10.5 kDa Ca2+-binding protein.

Expression of calcyclin in different cell lines and mouse tissues was determined with polyclonal antibodies raised against calcyclin from Ehrlich ascites tumour (EAT) cells. The protein was detected in mouse skeletal and cardiac muscle, in lung, kidney and spleen, and was especially enriched in mouse smooth muscle as well as in rat fibroblasts. No positive immunological reaction was detected in mouse brain, liver and intestine and some tumourigenic cell lines. The level of calcyclin mRNA found in different cells and tissues corresponded well to the calcyclin level estimated by immunoblotting. The calcyclin-like protein was purified from mouse stomach and appeared to be very similar to the EAT protein.

Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons.

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0. 34 ng/microg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein. (J Histochem Cytochem 48:1195-1202)

Validity of questionnaire criteria in mass screening for the diagnosis of peptic ulcer.

Epidemiological studies of peptic ulceration in the stomach and duodenum based on a standard questionnaire were carried out among the employees of a sulphur mine. The results obtained in the preliminary cross-sectional study were checked against a sample of 180 people who had undergone radiological examination of the alimentary tract. The validity of the questionnaire was then assessed by comparison of these two sets of results. The study shows that a more revealing screening test may be based on a wide range of questions rather than on a smaller set of specific questions.

Calcyclin--Ca(2+)-binding protein homologous to glial S-100 beta is present in neurones.

The aim of this work was to confirm the presence of calcylin in brain and to identify calcyclin positive cells. Calcyclin was identified in brain soluble proteins that were bound to phenyl-sepharose in a calcium dependent fashion. Specific antibodies against calcylin were found to stain subsets of brain neurones such as neurones in Ammons horn of hippocampus, granule cells in cerebellum, and neurones in brain stem. Glial cells which contain large amounts of S-100 beta protein were calcyclin negative. These results indicate that calcyclin is present in neurones, but not in glial cells.

Calcyclin-like protein from Ehrlich ascites tumour cells. Ca2+ and Zn2+ binding, distribution and target protein.

The calcyclin-like protein from Ehrlich ascites tumour cells is a 10.5 kDa heat stable protein, which binds two Ca2+ ions each with different affinity. Upon Ca2+ binding, the protein changes its conformation exposing hydrophobic regions. In this conformation it is able to interact with fluphenazine and with a 36 kDa protein immunologically similar to mammalian calpactin. Calcyclin-like protein binds Zn2+ and forms dimers like other members of the S-100 protein family. The calcyclin-like protein is present in several mouse tissues such as stomach, skeletal muscle, heart, spleen, lung and kidney, but seems to be absent from brain, intestine and liver as well as from some tumorigenic cells lines.

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon.

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound 45Ca2+ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca2+ concentration. Crosslinking experiments with BS3 showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg(2+)-ATPase activity of myosin in the presence of Ca2+.

[Fatty acids in confectionery products]. / Kwasy tluszczowe w produktach cukierniczych.

The content of fat and fatty acids in 144 different confectionery products purchased on the market in Warsaw region during 1997-1999 have been investigated. In examined confectionery products considerable variability of both fat and fatty acids content have been found. The content of fat varied from 6.6% (coconut cookies) up to 40% (chocolate wafers). Saturated fatty acids were present in both cis and trans form. Especially trans fatty acids reach (above 50%) were fats extracted from nut wafers, coconuts wafers.

[The fat and fatty acids content in selected sea fish]. / Zawartosc tluszczu i sklad kwasów tluszczowych w wybranych rybach morskich.

The fat content as well as fatty acids in baltic herring, mackerel and salmon from Norway has been determined. The fat content was 7%, 11.6% and 13.2% respectively. Both fat extracted from mackerel and salmon contained much more of polyunsaturated fatty acids in comparison to that extracted from herring. PUFA content in herring fat was 15% whereas mackerel and salmon 29% and 25% respectively. PUFA represented mostly of omega-3 family.

[Fatty acids in chocolate and chocolate products]. / Kwasy tluszczowe w czekoladach i wyrobach czekoladowych.

Fat content as well as fatty acids composition in 46 chocolate both stuffed and hard type as well as 14 chocolate related products from the market in Warsaw area in the years of 1997-1999 has been analysed. As the result of above investigations the considerable difference in fat content (from 6.6 to 40.0%) as well as fatty acids groups has been detected, in particular in stuffed chocolates and chocolate products.

S100A6 and its extracellular targets in Wharton's jelly of healthy and preeclamptic patients.

INTRODUCTION: In this work we compared the level, localization and binding partners of a calcium binding protein, S100A6, in extracellular matrix of Wharton's jelly of healthy and preeclamptic patients. METHODS: Studies were performed on the umbilical cords taken from 10 newborns delivered by healthy and 10 newborns delivered by preeclamptic mothers. To characterize S100A6 in Wharton's jelly immunoblotting and immunohistochemistry were applied. For identification of S100A6 targets pull down assays and mass spectrometry were performed. Direct interaction of S100A6 with its targets was checked by ELISA while co-localization of these proteins was analyzed by immunofluorescence staining. RESULTS: We have found that the level of S100A6 in Wharton's jelly is higher in patients with preeclampsia than in healthy ones and that post-translational modifications of S100A6 in preeclamptic tissue are different than those of S100A6 in control. We have identified several proteins that might interact with S100A6, among them are lumican and PRELP, found in Wharton's jelly of healthy and preeclamptic patients, and IGFBP-1 identified, as an S100A6 target, only in preeclamptic tissue. We have shown that the interactions between S100A6 and these proteins are direct and that IGF-1 competes with S100A6 for binding to IGFBP-1. CONCLUSION: In Wharton's jelly of preeclamptic tissue S100A6 is up-regulated and binds to different targets than in control. This suggests involvement of S100A6 in development of preeclampsia.