Protein kinase C isoforms in muscle cells and their regulation by phorbol ester and calpain.

Objectives were to identify the PKC isoforms in cultured muscle cells, to examine roles of Ca(2+)-dependent proteinases (calpains) in processing of various muscle PKC isozymes and to obtain a mechanistic description of the processing of PKCs by examining the temporal relationships between phorbol ester-dependent translocation of muscle PKCs and calpains between cytosolic and membrane compartments. Using six isoform (alpha, beta, gamma, delta, epsilon, zeta)-specific polyclonal antibodies, PKC alpha, delta and zeta were detected in rat skeletal muscle and in L8 myoblasts and myotubes. PKC alpha and zeta were primarily localized in the cytosolic fraction of L8 myotubes whereas PKC delta was more abundant in the membrane fraction. Phorbol ester (TPA) caused rapid depletion of myotube PKC alpha and PKC alpha and PKC delta isoforms from the cytosolic compartment and rapid appearance of these isoforms in the membrane fraction. However, long-term exposure of myotubes to TPA eventually caused down-regulation of PKCs in the membrane compartment. Down-regulation of PKCs in the membrane fraction was partially blocked by calpain inhibitor II. However, the rapid TPA-dependent cytosolic depletion of PKCs was unaffected by calpain inhibitor. This suggests that calpains may be responsible for membrane-associated down-regulation of PKCs but not for cytosolic depletion. In the final study we assessed the effects of phorbol ester on compartmentation of m-calpain with PKCs in muscle cells. Like the PKCs, TPA caused rapid association of m-calpain with the membrane fraction followed by down-regulation. This demonstrates that phorbol esters cause translocation of both PKCs and calpains to membranes where processing of PKCs may occur via the limited proteolysis exerted by calpains.

MDR-1 gene expression is a minor factor in determining the multidrug resistance phenotype of MCF7/ADR and KB-V1 cells.

The relevance of MDR-1 gene expression to the multidrug resistance phenotype was investigated. Drug-resistant cells, KB-V1 and MCF7/ADR, constantly expressed mRNA of the MDR-1 gene and were more resistant to vinblastine and adriamycin than drug-sensitive cells, KB-3-1 and MCF7. The drug efflux rate of KB-V1 was the same as KB-3-1 although the MDR-1 gene was expressed in only the resistant cell. The higher intracellular drug concentration of KB-3-1 than KB-V1 was due to the large drug influx. In the case of MCF7 and MCF7/ADR, the influx and efflux of the drug had nearly the same pattern and drug efflux was not affected by verapamil. The amount of ATP, cofactor of drug pumping activity of P-glycoprotein, was not changed by the resistance. These observations suggested that drug efflux mediated by MDR-1 gene expression was not a major determining factor of drug resistance in the present cell systems, and that the drug resistance could be derived from the change in drug uptake and other mechanisms.

Effects of dexamethasone on protein degradation and protease gene expression in rat L8 myotube cultures.

We examined the effects of a synthetic glucocorticoid (dexamethasone; Dex) on protoeolysis and on protease messenger RNA (mRNA) concentrations in rat L8 skeletal myotube cultures. Protein degradation was measured as release of radioactive trichloroacetic acid-soluble material from intracellular proteins pre-labelled with [3H]tyrosine. Dex (1 microM) stimulated protein degradation (P < 0.01). This effect was entirely blocked by the glucocorticoid antagonist, RU38486 (mifepristone; P < 0.01). Hence, actions of Dex on muscle protein degradation are mediated via intracellular glucocorticoid receptors. Molecular mechanisms by which glucocorticoids stimulate protein degradation in skeletal muscle are not known. Here, we investigated the regulation of protease (cathepsin B, cathepsin D, proteasome C2 subunit and m-calpain) mRNA concentrations by Dex in cultured L8 muscle cells. Cathepsin B mRNA concentration was enhanced 3.3-fold by Dex. This effect was blocked by RU38486. RU38486 alone did not affect cathepsin B mRNA concentration or mRNAs of other proteases. Concentrations of cathepsin D and m-calpain mRNAs were also increased by Dex. These effects were also abolished by RU38486. Proteasome C2 mRNA was unaffected by Dex and Dex reduced alpha-tubulin mRNA. Thus, glucocorticoids specifically regulate the concentrations of mRNAs encoding some proteases in muscle cells. The regulation of protease mRNA concentration is mediated via interaction between Dex with glucocorticoid receptors and is independent of the actions of Dex on mRNA encoding house-keeping proteins. These changes may underlie glucocorticoid-dependent control of proteolysis in muscle.

Limitation of Hu-PBL-scid mouse model in direct application to immunotoxicity assessment.

Hu-PBL-scid mice were directly introduced to the methods of immunotoxicity assessments. Human IgG and IgM was detected 1 week after transplantation. Cyclosporin A (CsA) and cyclophosphamide (CP), which were injected i.p. 4 weeks after transplantation, decreased the serum concentration of IgM after 2-4 days of treatment but not that of IgG. Lymphocyte proliferation induced by various mitogens and primary T-dependent antibody responses to sheep red blood cells could not be measured by using splenocytes of hu-PBL-scid mice. These results were correlated with the fact that human cells were not detected in the spleen, thymus, or blood of hu-PBL-scid mouse but were detected in lymph nodes of the intestine, which were observed by flow cytometric and immunohistochemical examinations. The present results suggest using hu-PBL-scid mice in routine immunotoxicity investigations: lymph nodes of intestines could be used as the lymphocyte source. In addition, the determination of serum Ig concentration might be used as a experimental item.

Efficient fixation procedure of human leukemia cells in sulforhodamine B cytotoxicity assay.

The fixation procedures in sulforhodamine B (SRB) assay for human leukemia cells were modified to produce more reliable results. It was found that the concentration of the fixative agent, trichloroacetic acid (TCA), was critical in the selective fixation of cellular protein. While a TCA solution of 80% fixed both cells and serum proteins, a 50% solution fixed only cells with a very low interference of the serum proteins. Accordingly, we selected 50% TCA as a fixative agent which made the final absorbance of the SRB assay to be exactly matched to the cell density with a small deviation and a low background. Besides the change of TCA concentration, a precentifugation of microplate just before fixation also improved the previous assay procedures in the two points of view. The 2-h standing step was simply substituted for only 1 min of centrifugation. Both the rapid and slow application of TCA solution in fixation produced the same extents of fixation. In an actual application, these two kinds of modifications in the previous SRB assay procedure were also proved to be effective in the determination of cytotoxicities of doxorubicin by using human leukemias.

Comparison of glucose-consumption and thymidine-incorporation endpoints in histocultured human superficial bladder tumors.

Glucose consumption and tritiated thymidine incorporation were monitored for 54 days in histocultured human superficial transitional cell carcinoma of the bladder, indicating that long-term histoculture of human transitional cell carcinoma of the bladder is feasible. The glucose-consumption rate was relatively similar among the histocultured specimens and depended on the size of the explant in histoculture. Tritiated thymidine incorporation, on the other hand, varied greatly between the histocultured specimens. In addition, [3H]thymidine incorporation varied in single histocultures between cells in the original explant and those invading the gel. From these results, we conclude that the glucose consumption rate in histocultured human superficial bladder tumor is more reliable and sensitive than thymidine incorporation. The glucose-consumption endpoint is simple to measure and nondestructive, allowing multiple measurements on a single culture, unlike the thymidine incorporation endpoint.

Whole body slotted tube resonator (STR) for proton NMR imaging at 2.0 Tesla.

Design principles of a whole body Slotted Tube Resonator (STR) RF coil are given and fabrication details of the RF coil for a 2.0 Tesla NMR imaging system are presented. Experimental study proved efficient operation of the coil at 2.0 Tesla field and indicated potential for the extension of the technique to the even higher fields.

Effect of extracellular cations on the chemotherapeutic efficacy of anticancer drugs.

Cancer development and the efficiency of chemotherapy relies on the patients calcium-related pathological status such as hyper- or hypocalcemia. In the present study, we investigated the effect of extracellular cations such as calcium and magnesium on the therapeutic efficacy of antitumor drugs. The analytic parameters used were cellular drug uptake/excretion and the chemosensitivity of the human breast cancer cell lines, MCF7 and MCF7/ADR. Both calcium and magnesium ions decreased the membrane permeability of cancer cells, which was determined by cell size analysis. These divalent ions also lowered the drug uptake and the cytoplasmic levels of rhodamine 123 and adriamycin, suggesting that they might interfere with the diffusion of these drugs by modifying the physical properties of the cytoplasmic membrane. The acute cytotoxicity of adriamycin after a short period of incubation correlated with changes in its cytoplasmic level. Our results indicate that these extracellular cations might play an important role in the therapeutic activities of anticancer drugs in cancer patients. These results also provide insight a new aspect of chemotherapy, because they suggest that the therapeutic doses of anti-cancer drugs should be modified in cancer-bearing patients presenting with abnormal blood calcium levels.

Cytotoxicity of urushiols isolated from sap of Korean lacquer tree (Rhus vernicifera Stokes).

Cytotoxicities of four urushiols, congeners isolated from the sap of Korean lacquer tree (Rhus vernicifera Stokes), to 29 human cancer cell lines originated from 9 organs were evaluated. Their values of 50% growth inhibition were below 4 microg/ml, and showed cell line specific cytotoxicity. The present result is the first report on the cytotoxicity of urushiols suggesting that they would have an anticancer activity to human cancer cells.

Comparative studies of adriamycin and 28-deacetyl sendanin on in vitro growth inhibition of human cancer cell lines.

The limonoid compound (28-deacetyl sendanin) isolated from the fruit of Melia toosendan SIEB. et ZUCC. was evaluated on anticancer activity. According to a standard in vitro cytotoxicity assay, eight human cancer cell lines and SRB assay were introduced for present evaluation. As a positive standard, adriamycin was tested in parallel. The cell lines were originated from six different organs. In view of dose-response profiles to 28-deacetyl sendanin, the most sensitive cells were SF-539 and PC-3 which were derived from CNS and prostate, respectively. In contrast, all the cell lines responded similarly to adriamycin to give rise to nearly identical dose-response profiles. By comparison of Gl50 between 28-deacetyl sendanin and adriamycin, six cell lines were more sensitive to 28-deacetyl sendanin and two were more resistant. As a result, 28-deacetyl sendanin had more sensitive and selective inhibitory effects on in vitro growth of human cancer cell lines in a comparison with adriamycin.

[Diagnosis and management of obstetric acute disseminated intravascular coagulation].

Twenty-seven in-patients with obstetric DIC in our hospital from Jan. 1971 to Dec. 1990 were analysed retrospectively. The incidence was 0.12% in the first decade and 0.02%, in the second, showing a difference of significance between them. The most common predisposing factors included amniotic fluid embolism, abruptio placenta and hemorrhagic shock. Bleeding from multi-organs in various extent and coagulation disorders occurred in all those 27 cases. [Besides anti-shock treatment, heparin was employed together with fibrinogen in 4 postpartum and 1 antepartum DIC patients, fibrinogen alone in 8 cases, and hysterectomy in 11 cases. 17 patients were saved and 9 died. It is important that early diagnosis and much attention paid to clinical characteristics together with serial laboratory tests. Key management should include prompt treatment and eradication of predisposing factors. Quick decision spite of to terminate the pregnancy and even hysterectomy should be done in some risks.

X-ray reflectivity study on the structure and phase stability of mixed phospholipid multilayers.

Vertically oriented multilayers composed of two saturated phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), were deposited on silicon. X-ray reflectivity was used to investigate the structures of the variously mixed phospholipid multilayers as a function of composition. Then, the phase stability was investigated at various annealing temperatures under humid conditions. The results indicated that the lipid spacing of the mixed phospholipid multilayers varied systematically as a function of the DPPC/DPPS ratio and that no macroscopic phase separation occurred during the annealing process under both dry and humid conditions.

Retinitis pigmentosa GTPase regulator (RPGRr)-interacting protein is stably associated with the photoreceptor ciliary axoneme and anchors RPGR to the connecting cilium.

Retinitis pigmentosa (RP) is a blinding retinal disease in which the photoreceptor cells degenerate. Mutations in the gene for retinitis pigmentosa GTPase regulator (RPGR) are a frequent cause of RP. The function of RPGR is not well understood, but it is thought to be a putative guanine nucleotide exchange factor for an unknown G protein. Ablation of the RPGR gene in mice suggested a role in maintaining the polarized distribution of opsin across the cilia. To investigate its function, we used a protein interaction screen to identify candidate proteins that may interact physiologically with RPGR. One such protein, designated RPGR-interacting protein (RPGRIP), is expressed specifically in rod and cone photoreceptors. It consists of an N-terminal region predicted to form coiled coil structures linked to a C-terminal tail that binds RPGR. In vivo, both proteins co-localize in the photoreceptor connecting cilia. RPGRIP is stably associated with the ciliary axoneme independent of RPGR and is resistant to extraction under conditions that partially solubilized other cytoskeletal components. When over-expressed in heterologous cell lines, RPGRIP appears in insoluble punctate and filamentous structures. These data suggest that RPGRIP is a structural component of the ciliary axoneme, and one of its functions is to anchor RPGR within the cilium. RPGRIP is the only protein known to localize specifically in the photoreceptor connecting cilium. As such, it is a candidate gene for human photoreceptor disease. The tissue-specific expression of RPGRIP explains why mutations in the ubiquitously expressed RPGR confer a photoreceptor-specific phenotype.

Protein kinase C-epsilon is the likely mediator of mucin exocytosis in human colonic cell lines.

The phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces mucin secretion in the colonic tumor cell line T84 in a Ca(2+)-independent manner. To determine whether a specific protein kinase C (PKC) isoform is involved in colonic cells, we compared PMA-dependent mucin secretion by three human colonic tumor cell lines (T84, HT-29/A1, and LS 180) with the expression of PKC isoforms alpha, beta, delta, epsilon, and zeta, previously identified in human colon (L. A. Davidson, Y. H. Jiang, J. D. Derr, H. Aukema, J. R. Lupton, and R. S. Chapkin. Arch. Biochem. Biophys. 312:547-553, 1994). In each cell line PMA (10(-7) M) caused mucin secretion within 30 min. PMA-dependent mucin secretion was three to four times greater from HT-29/A1 and T84 cells than from LS 180 cells. All three-cell lines contained mRNA for PKC-alpha, PKC-epsilon, and PKC-zeta but not PKC-beta or -delta. Each cell line also expressed PKC-alpha, -epsilon, and -zeta protein. PKC-epsilon expression (mRNA and protein) was three to four times greater in HT-29/A1 and T84 cells than in LS 180 cells, correlating with PMA-responsive mucin secretion, whereas all cell lines contained similar levels of PKC-alpha mRNA and protein. When cells were stimulated by PMA, only PKC-epsilon was translocated from cytosol to membrane fractions early enough to stimulate mucin secretion. Because PKC-epsilon is also a Ca(2+)-independent isoform, it is likely to mediate mucin exocytosis in colonic cells.

Portal and superior mesenteric venous gas with retroperitoneal abscess--CT diagnosis (case report).

We present a case of portal and superior mesenteric venous gas in a 31-year-old diabetic woman with a left-sided retroperitoneal abscess. Five years prior to admission, patient was diagnosed with diabetes mellitus and developed emphysematous pyelonephritis, requiring nephrectomy on the left side. A CT examination showed air distributed throughout the portal venous system and superior mesenteric vein.

Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-epsilon.

Treatment of HT-29 cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces MUC2 expression. To investigate the role of PKC in regulating mucin genes in intestinal cells, we examined the regulation of MUC1, MUC2, MUC5AC, MUC5B, and MUC6 expression in two human mucin-producing colonic cell lines, T84 and HT29/A1. T84 and HT29/A1 cells (at 80-90% confluency) were exposed to 100 nM PMA for 0, 3, and 6 h. Twofold or greater increases in mRNA levels for MUC2 and MUC5AC were observed in both cell lines during this time period, whereas the levels of MUC1, MUC5B, and MUC6 mRNAs were only marginally affected. These results indicated that PKC differentially regulates mucin gene expression and that it may be responsible for altered mucin expression. Our previous results suggested that the Ca(2+)-independent PKC-epsilon isoform appeared to mediate PMA-regulated mucin exocytosis in these cell lines. To determine if PKC-epsilon was also involved in MUC2/MUC5AC gene induction, HT29/A1 cells were stably transfected with either a wild-type PKC-epsilon or a dominant-negative ATP-binding mutant of PKC-epsilon (PKC-epsilon K437R). Overexpression of the dominant-negative PKC-epsilon K437R blocked induction of both mucin genes, whereas PMA-induced mucin gene expression was not prevented by overexpression of wild-type PKC-epsilon. PMA-dependent MUC2 mucin secretion was also blocked in cells overexpressing the dominant-negative PKC-epsilon K437R. On the basis of these observations, PKC-epsilon appears to mediate the expression of two major gastrointestinal mucins in response to PMA as well as PMA-regulated mucin exocytosis.

A retinitis pigmentosa GTPase regulator (RPGR)-deficient mouse model for X-linked retinitis pigmentosa (RP3).

The X-linked RP3 locus codes for retinitis pigmentosa GTPase regulator (RPGR), a protein of unknown function with sequence homology to the guanine nucleotide exchange factor for Ran GTPase. We created an RPGR-deficient murine model by gene knockout. In the mutant mice, cone photoreceptors exhibit ectopic localization of cone opsins in the cell body and synapses and rod photoreceptors have a reduced level of rhodopsin. Subsequently, both cone and rod photoreceptors degenerate. RPGR was found normally localized to the connecting cilia of rod and cone photoreceptors. These data point to a role for RPGR in maintaining the polarized protein distribution across the connecting cilium by facilitating directional transport or restricting redistribution. The function of RPGR is essential for the long-term maintenance of photoreceptor viability.

A new internal ribosomal entry site 5' boundary is required for poliovirus translation initiation in a mouse system.

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.