Alteration of pectic polysaccharides in cell walls, extracellular polysaccharides, and glycan-hydrolytic enzymes of growth-restricted carrot cells under calcium deficiency

Suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells were grown in calcium (Ca²⁺)-deficient and normal liquid media. Cell growth was limited by the Ca²⁺ deficiency. Similar amounts of pectic fractions were extracted from the walls of control and Ca²⁺-deprived cells, but the fractions from the walls of Ca²⁺-deprived cells showed a substantial decrease in galacturonic acid content. However, after 15 days of culture, Ca²⁺-deprived cells released galacturonic acid-rich extracellular polysaccharides at twice the rate of control cells. The polysaccharides consisted of a mixture of several polymers containing predominantly arabinose, galactose and galacturonic acid. Ca²⁺-deprived cells also secreted three times more extracellular proteins, containing many glycan-hydrolytic enzymes, into the medium than did normal cells. SDS-PAGE analysis revealed several distinct changes in the polypeptide pattern in the medium of control and Ca²⁺-deprived cells. Activities of α -galactosidase, β -glucosidase and exo- polygalacturonase increased considerably during Ca²⁺ deficiency, whereas α - l -arabinofuranosidase and β -galactosidase activities were much reduced.     

Role of protein kinase C in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary cells

The effects of staurosporine and K-252a, potent inhibitors of protein kinases, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on catecholamine secretion and protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells were investigated. Staurosporine and K-252a (0.01-10 microM) did not cause large changes in catecholamine secretion evoked by Ca2+ in digitonin-permeabilized cells whereas these compounds strongly prevented TPA-induced enhancement of catecholamine secretion in a concentration-dependent manner. Incubation of digitonin-permeabilized cells with [gamma-32P]ATP resulted in 32Pi incorporation into a large number of proteins, detected as several major bands and darkened background in autoradiograms. Ca2+ and TPA increased phosphorylation of these proteins. Staurosporine and K-252a markedly inhibited Ca(2+)-induced and TPA-induced increases in protein phosphorylation as well as basal (0 Ca2+) protein phosphorylation in digitonin-permeabilized cells. Long term treatment (24 h) of adrenal medullary cells with 1 microM TPA markedly decreased total cellular protein kinase C activity to about 5.3% of control. Pretreatment of the cells with 1 microM TPA strongly inhibited the TPA-induced enhancement of catecholamine secretion whereas it did not cause large changes in total cellular catecholamine amounts, Ca(2+)-induced catecholamine secretion, and cAMP-induced enhancement of catecholamine secretion from digitonin-permeabilized cells. From these results we conclude that protein kinase C plays a modulatory role in catecholamine secretion rather than being essential for initiating catecholamine secretion.     

G6PD Ube, a glucose-6-phosphate dehydrogenase variant found in four unrelated Japanese families.

A total of 6,120 Japanese males were screened for glucose-6-phosphate dehydrogenase deficiency (G6PD). Five cases with the deficiency were discovered. Two of them and an additional two cases have the same variant, G6PD Ube, characterized by moderate enzyme deficiency, fast moving enzyme activity on electrophoresis, high Ki Nadph, utilization of substrate analogues, kinetics, pH optima, and stability. This variant was distinguished for G6PD A- and from other Oriental variants by biochemical parameters. Differences in the frequency and type of the variants between southern Asia and Japan, suggest that the Japanese who have been isolated on islands where malaria is not endemic, may have developed their own variant traits.     

Differences in the photobleaching process between 7-cis- and 11-cis-rhodopsins: a unique interaction change between the chromophore and the protein during the lumi-meta I transition.

The photochemical and subsequent thermal reactions of 7-cis-rhodopsin prepared from cattle opsin and 7-cis-retinal were investigated by low-temperature spectrophotometry and laser photolysis, and compared with those of 11-cis-rhodopsin prepared from cattle opsin and 11-cis-retinal. Low-temperature experiments revealed that the absorption maxima of batho and lumi intermediates from 7-cis-rhodopsin were at slightly shorter wavelengths than those of 11-cis-rhodopsin while the meta I intermediates of both rhodopsin isomers showed the same absorption maxima. Kinetic experiments of the photobleaching process of 7-cis-rhodopsin using picosecond and nanosecond laser pulses revealed the formation of intermediates corresponding to the batho, lumi, meta I, and meta II intermediates from 11-cis-rhodopsin. An intermediate of 7-cis-rhodopsin corresponding to photorhodopsin (a precursor of bathorhodopsin), however, was not detected. Batho and lumi intermediates from 7-cis-rhodopsin had shorter lifetimes (approximately 40 ns and 300 microseconds) than those of 11-cis-rhodopsin (250 ns and 800 microseconds), but the lifetime of the meta I intermediate from 7-cis-rhodopsin was identical with that from 11-cis-rhodopsin (12 ms). These results indicate that the difference in configuration of the original chromophore between 7-cis- and 11-cis-rhodopsins is a cause of different chromophore-opsin interactions in the batho and lumi stages, while in the meta I stage the difference has disappeared by the relaxation of the protein near the chromophores. A possible interaction change between the 9-methyl group of the chromophore and its neighboring protein during the lumi-meta I transition will be discussed.     

In vitro screening of Fe2+‐chelating effect by a Fenton's reaction–luminol chemiluminescence system

In vitro screening of a Fe²⁺‐chelating effect using a Fenton's reaction–luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton's reaction and luminol was decreased on capturing Fe²⁺ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10‐phenanthroline were examined. EC₅₀ values for these compounds (except 1,10‐phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe²⁺‐chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Inhibitory action of cyclic GMP on secretion, polyphosphoinositide hydrolysis and calcium mobilization in thrombin-stimulated human platelets

The effect of cyclic GMP (cGMP) on human platelet activation was investigated, using its metabolically stable analogue, 8-bromo cGMP (8-bcGMP). Thrombin-induced serotonin secretion was inhibited by pretreatment with 8bcGMP in a dose-dependent manner. Production of inositol trisphosphate (IP3), a Ca2+ releaser was inhibited by 8bcGMP pretreatment of platelets. Preincubation of platelets with 8bcGMP was without effect on the basal level of cytosolic free Ca2+, measured by fluorescent indicator quin2, but suppressed its thrombin-induced enhancement independently of extracellular Ca2+. These results indicate that cGMP may be implicated in phospholipase C activation and Ca2+ mobilization (both influx through the plasma membrane and efflux from internal stores) in thrombin-activated human platelets.     

Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.     

Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.     

Integrin alphavbeta3 regulates microfibril assembly in human periodontal ligament cells

Fibrillin-1 is the major structural component of extracellular microfibrils. However, the mechanism by which extracellular fibrillin-1 assembles into microfibrils is not fully understood. Fibrillin-1 contains the Arg-Gly-Asp (RGD) motif, which may allow binding to RGD-recognizing integrins. We hypothesized that integrin αvβ3 on the cell surface of human periodontal ligament (PDL) fibroblasts may influence fibrillin-1 assembly into cell/matrix layers. We treated PDL fibroblasts with an integrin αvβ3-specific antagonist to examine fibrillin-1 assembly. Western blotting and immunofluorescence analysis showed that treatment with the integrin αvβ3 antagonist at 5 μM clearly abolished fibrillin-1 deposition. These results provide for the first time evidence that integrin αvβ3 regulates extracellular assembly of fibrillin-1, thereby modulating cell-mediated homeostasis of microfibrils.     

Skin-specific caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18

To study the pathophysiological roles of overexpressed caspase-1 (CASP1), originally designated as IL-1 beta-converting enzyme, we generated transgenic mice in which human CASP1 is overexpressed in their keratinocytes. The transgenic mice spontaneously developed recalcitrant dermatitis and skin ulcers, characterized by the presence of massive keratinocyte apoptosis. The skin of the mice contained the active form of human CASP1 and expressed mRNA for caspase-activated DNase, an effector endonuclease responsible for DNA fragmentation. Their skin and sera showed elevated levels of mature IL-18 and IL-1 beta, but not of IFN-gamma. The plasma from these animals induced IFN-gamma production by IL-18-responsive NK cells. Administration of heat-killed Propionibacterium acnes, a potent in vivo type 1 cell inducer, caused IFN-gamma-mediated lethal liver injury in the transgenic mice, which was completely inhibited by treatment with neutralizing anti-IL-18 Ab. These results indicated that in vivo overexpression of CASP1 caused spontaneous apoptotic tissue injury and rendered mice highly susceptible to exogenous type 1 cell-inducing condition in collaboration with endogenously accumulated proinflammatory cytokines.