Generation of B220low B cells and production of autoantibodies in mice with experimental amyloidosis: association of primordial T cells with this phenomenon.

To investigate the immunological state in amyloidosis, mice were twice intraperitoneally injected (2-week interval) with casein emulsified in complete Freund's adjuvant. Two weeks after the treatment, amyloid deposits were detected in the spleen and other organs of these mice. The number of lymphocytes yielded by the liver and spleen increased significantly. The most affected lymphocyte subset was found to be B cells, namely, the total number of B cells increased and unusual B220low B cells were newly generated in the liver and spleen. In other words, not only normal B220high B cells but also unusual B220low B cells were detected in these organs of mice with amyloidosis. In parallel with this phenomenon, autoantibodies against denatured DNA were detected in sera. Since such autoantibodies are known to accompany the functional activation of NKT cells, NKT cell-deficient mice were used for the induction of amyloidosis. Such mice showed less formation of amyloidosis and lower levels of autoantibodies in sera. Athymic nude mice were NKT cell-deficient but NK1.1- TCRint cells were present. These athymic mice showed an intermediate induction of amyloidosis. The cytokine profile seen in mice with amyloidosis was the Th0 type, showing simultaneous production of IL-4 and IFNgamma. These results suggest that the generation of B220low B cells and the production of autoantibodies in aid of primordial T cells may be major immunological mechanisms in amyloidosis mice.

Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts.

PURPOSE: We compared the ability of adenoviral-mediated wild-type p53 RPR/INGN201(Ad5/CMV/p53) to radiosensitize non-small cell lung carcinoma (NSCLC) and normal lung fibroblast cells. MATERIALS AND METHODS: NSCLC cell lines (A549 and H322) and human lung fibroblast cells (MRC-9 and CCD-16) were used in this study. Radiosensitivity was determined by clonogenic assay and tumor growth delay. Expression of p53, Bax, and p21WAF1 protein were evaluated by immunoblot. A FITC conjugate of annexin V was used for flow cytometric detection of apoptosis. RESULTS: Clonogenic and apoptotic assays indicated that Ad5/CMV/p53 enhanced the radiosensitivity of both NSCLC cell lines. On the other hand, the two normal human fibroblast cell lines appeared to be resistant to the cytotoxic effects of Ad5/CMV/p53 and were not radiosensitized compared to the NSCLC cells. According to immunoblot analysis, Bax expression was increased in the NSCLC cells treated with the combination therapy; Bax expression, however, was unchanged in normal cells. In in vivo studies, tumor growth suppression was enhanced by this combination strategy in xenograft tumors growing in nude mice compared to Ad5/CMV/p53 or radiation therapy when used alone. CONCLUSIONS: Our data indicate that therapy using Ad5/CMV/p53 and irradiation in combination is more effective than either treatment when used alone on NSCLC cells, is not limited to cells with defective endogenous p53, and does not enhance the radiosensitivity of normal cells.

Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53-dependent manner.

We examined the influence of adenovirus-mediated wild-type p16INK4a (Ad/p16) expression on the radiation sensitivity of NSCLC cell lines, all of which lacked constitutive p16INK4a but each of which varied in p53 status: A549 (-p16INK4a/ +pRb/wt-p53), H322 (-p16INK4a/ +pRb/mt-p53), and H1299 (-p16INK4a/ +pRb/deleted-p53). The in vitro clonogenic survival results indicate that Ad/p16 enhanced the radiosensitivity of A549 but not H322 or H1299. Further analysis indicated that the apoptosis induced by combination therapy using Ad/p16 plus irradiation was dependent on the endogenous p53 status of the cancer cells. We performed Western blotting to analyse the p53 protein expression of A549 cells treated with either Ad/p16 or Ad/Luc. Endogenous p53 protein levels were higher in A549 cells transfected with Ad/p16 than in those transfected with Ad/Luc. Furthermore, when wt-p53 protein expression was restored in H1299 using Ad/ p53, Ad/p16 stabilized p53 protein expression and radiosensitized the cells. These results suggest that Ad/ p16-induced stabilization of p53 protein may play an important role in Ad/p16 mediated radiosensitization by enhancing or restoring apoptosis properties. Thus, Ad/ p16 plus radiation in combination may be a useful gene therapy strategy for tumors that have wt-p53 but nonfunctional p16INK4a.

Immunohistochemical expression of vascular endothelial growth factor can predict response to 5-fluorouracil and cisplatin in patients with gastric adenocarcinoma.

The combination of 5-fluorouracil (5-FU) and cisplatin is used most commonly for gastric carcinoma. Recent studies have indicated that vascular endothelial growth factor (VEGF) is related to drug delivery through angiogenesis and vascular permeability. In this study, we evaluated the efficacy and toxicity of continuous infusion of 5-FU and low dose cisplatin infusion as first-line treatment in patients with unresectable gastric adenocarcinoma. We also examined the relationship between chemotherapy response and immunohistochemical expression of VEGF in the biopsy samples of gastric primary. All 30 patients enrolled in this study were assessable for response, adverse reactions, and VEGF expression. The regimen consisted of 5-FU (350 mg/m2/day every day by continuous venous infusion) and low dose cisplatin (7 mg/m2/day by drip infusion over 1 h on days 1-5 every week). This treatment was repeated weekly for 3 consecutive weeks. Four weeks after the second cycle, mesurable lesions were estimated for response. An overall response rate was 46.7% (14/30). Patients with intestinal histologic type (10/12) and good performance status ([PS], 13/18) showed good response rate (83.3%, and 72.2%, respectively) compared to patients with diffuse histologic type (4/18) and poor PS [(1/12) 22.2%, and 8. 3%, respectively]. The response rate of VEGF-positive cases and VEGF-negative cases was 75% (12/16), and 16.7% (2/14), respectively. Multivarite analysis revealed that VEGF-positive and good PS had a significant impact on chemotherapy response in this treatment. The most common garde 3 or higher toxicities were myelosuppression (30%) and diarrhea (13.3%). Continuous infusion of 5-FU and low dose cisplatin infusion is an effective treatment for patients with unresectable gastric carcinoma, and VEGF expression may be a useful predictor of chemotherapy response in this regimen.

Cholesterol sulfate, an activator of protein kinase C mediating squamous cell differentiation: a review.

Activity of protein kinase C (PKC) depends on the interaction with polar head-groups of two membrane lipids, i.e., phosphatidylserine and diacylglycerol. We demonstrated that cholesterol metabolism is directly involved in activation of the eta isoform of protein kinase C (PKCeta), which is predominantly expressed in epithelial tissues in close association with epithelial differentiation. We found that PKCeta was activated by cholesterol sulfate (CS), a metabolite of cholesterol formed during squamous cell differentiation. In the presence of CS, phorbol ester only weakly enhanced the activity of PKCeta. CS also activated PKCeta, PKCdelta and PKCepsilon in a dose-dependent manner, when assayed using purified recombinant materials. However, when partially purified materials were used from overexpressing normal human keratinocytes, only PKCeta was activated by CS among the isoforms examined. All the existing lines of evidence, mainly supplied from our laboratory, suggest that CS is involved in a signal transduction of squamous cell differentiation and thereby modifying squamous cell carcinogenesis.

Regioselective acylation of flavonoid glucoside with aromatic acid by an enzymatic reaction system from cultured cells of Ipomoea batatas.

The regioselective acylation of naturally occurring plant pigments with aromatic acids such as caffeic acid and p-coumaric acid was examined by an enzymatic reaction system from cultured cells of Ipomoea batatas. Isoquercitrin, a flavonoid glucoside, was converted to isoquercitrin 6''-O-caffeate by the enzymatic system. Chrysanthemin, an anthocyanin, was also acylated to the corresponding caffeate ester. This enzymatic method should prove useful for the synthesis of acylated flavonoid glucosides such as isoquercitrin caffeate, whose productivity is low using lipase-catalyzed transesterification.

Simultaneous determination of the urinary metabolites of toluene, xylene and styrene using high-performance capillary electrophoresis. Comparison with high-performance liquid chromatography.

A simple and rapid method using high-performance capillary electrophoresis (HPCE) for the simultaneous determination of the urinary metabolites of toluene, xylene and styrene, plus creatinine and uric acid in human urine specimens and standard solutions is described. The compounds were well separated from each other on a fused-silica capillary utilizing a 20 mM sodium tetraborate buffer (pH 9.65) with 15 mM beta-cyclodextrin and UV detection at 200 and 225 nm. The total analysis time was less than 6 min per sample. The capillary zone electrophoresis (CZE) method shows a good correlation with the high-performance liquid chromatography (HPLC) method with respect to urinary hippuric acid concentrations in the urine specimens of subjects exposed to the vapors of a solvent mixture of toluene and xylene. In comparing these two techniques, HPCE was found to be superior to HPLC because the analysis time is shorter, and the separation of m-MHA and p-MHA takes a long time with HPLC.

Low E-cadherin and beta-catenin expression correlates with increased spontaneous and artificial lung metastases of murine carcinomas.

This study examined the relationship between the expression of E-cadherin or beta-catenin in murine adenocarcinomas and their hematogenous metastatic propensity, assessed by both spontaneous and artificial lung metastasis. Seven different carcinomas, syngeneic to C3Hf/Kam mice were used: 4 mammary carcinomas (MCa-4, MCa-29, MCa-35, and MCa-K), ovarian carcinoma OCa-I, hepatocarcinoma HCa-I, and adenosquamous carcinoma ACa-SG. These tumors vary widely in their ability to spontaneously metastasize to the lung (from 0 to 100% metastatic incidence), and their cells greatly differ in their ability to form artificial lung nodules when injected i.v. Primary tumors in the leg were assessed for E-cadherin and beta-catenin expression by western blotting. The expression of both proteins showed wide variation among the tumors; however, the expression of E-cadherin correlated well with that of beta-catenin. There was significant inverse correlation between the expression of E-cadherin, as well as beta-catenin, and the incidence of both spontaneous and artificial lung metastases from these tumors. Spontaneous metastases of highly metastatic HCa-I and moderately metastatic MCa-35 were significantly lower in E-cadherin and beta-catenin expression than their corresponding primary tumors were. Thus, the propensity of murine carcinomas for hematogenous spread is highly related to E-cadherin and beta-catenin levels in primary tumors. The inverse correlation between the expression of these molecules and spontaneous and artificial metastases implies that tumor cells with low E-cadherin and beta-catenin content have increased ability to enter the vascular circulation at the primary tumor site and to colonize distant tissues.

Cholesterol sulfate activates transcription of transglutaminase 1 gene in normal human keratinocytes.

Cholesterol sulfate and transglutaminase 1 are essential for the process of keratinization. Cholesterol sulfate is formed during keratinization and activates the eta isoform of protein kinase C. Transglutaminase 1 is a key enzyme for formation of the cornified envelope in terminally differentiated keratinocytes. In this study, we demonstrated that cholesterol sulfate acts as a transcriptional activator of the transglutaminase 1 gene in normal human keratinocytes. Growth of normal human keratinocytes was inhibited by cholesterol sulfate, but not by its parental cholesterol. Treatment of normal human keratinocytes with cholesterol sulfate induced activity of transglutaminase 1 in a dose- and time-dependent manner. Activation of transcription of transglutaminase 1 by cholesterol sulfate was demonstrated by northern blotting analysis, whereas that by cholesterol was not. In order to identify a cholesterol sulfate responsive region in the transglutaminase 1 gene, plasmids were constructed containing a luciferase reporter gene ligated to deletion fragments of the 5' upstream region of the tranglutaminase 1 gene and were transfected into normal human keratinocytes. Transfected cells were treated with cholesterol sulfate, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and a high concentration of Ca2+. Our results indicate that the responsive element(s) for cholesterol sulfate and phorbol ester is located upstream of the human transglutaminase 1 gene at a position(s) between -819 and -549, whereas the responsive element for Ca2+ is located at a position between -79 and -49.

Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C.

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha, delta, eta, and zeta isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha, delta, and eta isoforms. Overexpression of the eta isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha and zeta isoforms did not. These findings indicate that the eta and delta isoforms of PKC are involved crucially in squamous cell differentiation.

Phorbol ester-induced G1 arrest in BALB/MK-2 mouse keratinocytes is mediated by delta and eta isoforms of protein kinase C.

We investigated the possible negative regulation of the cell cycle by protein kinase C (PKC) isoforms in synchronously grown BALB/MK-2 mouse keratinocytes, in which PKC isoforms were overexpressed by using the adenovirus vector Ax. Cells at the G1/S boundary of the cell cycle were the most sensitive to the inhibitory effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC agonist, resulting in G1 arrest. TPA-induced inhibition of DNA synthesis was augmented by overexpression of the eta and delta isoforms, but rescued by the dominant-negative and antisense eta isoforms. In contrast, the alpha and zeta isoforms showed no effect on DNA synthesis with or without TPA treatment. Immunoblotting indicated cell cycle-dependent expression of the eta isoform, being highest in cells at the G1/S boundary. The present study provides evidence that the eta and delta isoforms of PKC are involved in negative regulation of cell cycle at the G1/S boundary in mouse keratinocytes.

[A case of recurrent advanced colon cancer treated with CPT-11 for second-line chemotherapy].

A 59-year old male patient had been operated on for sigmoid colon cancer in July, 1990. Operative findings were, P0, H0, S1, N (-), Stage I, and histological findings were ss, ly 2, v0, n (-). In July 1994, the CEA level elevated, and be was diagnoted as having para-aortic LN swelling and stenosis of anastomosis of colon. He was admitted for treatment of recurrent colon cancer. Initially, he was treated with continuous injection of 5-FU, low-dose CDDP and Leucovorin. His CEA level decreased and para-aortic LN diminished in size. But, in December 1995, the CEA level and para-aortic LN relapsed. 5-FU, CDDP and Leucovorin were administered, but the CEA level became more and more elevated. This regimen was not considered responsible for drug resistance. CPT-11 was administered at 60 mg/week 6 times, and 80 mg/week 3 times. The side effects disappeared, LN sightly diminished in size, and the CEA level decreased. Judging by the anticancer effect without severe side effect, we found CPT-11 a useful drug for second-line chemotherapy.

Biotransformation of 4-androstene-3,17-dione by green cell suspension of Marchantia polymorpha: stereoselective reduction at carbon 17.

The biotransformation of 4-androstene-3,17-dione by a green cell suspension of Marchantia polymorpha was studied. It was found that the cultured cells stereoselectively reduce the carbonyl group of 4-androstene-3,17-dione from the re-face at C-17 to form testosterone as the primary metabolite.

[The effects of r-HuEPO on platelet function and coagulation factors in hemodialysis patients].

To evaluate the effects of correction of anemia with recombinant human erythropoietin (r-HuEPO) on the hemostatic defects in uremia, hemostatic parameters were examined in 18 patients with renal anemia receiving hemodialysis (HD). During the study, hematocrit (Ht) increased from 22.9 +/- 3.1% (mean +/- SD) at pre-treatment (stage-I) to 31.0 +/- 3.0% 12 weeks after 3000 IU intravenous r-HuEPO administration at the end of every HD (stage-II), and decreased to 26.2 +/- 4.2% 6 weeks after r-HuEPO discontinuation (stage III). Platelet count did not change among these three stages, however, mean platelet volume significantly increased at stage II compared to stage I. Ivy bleeding time (Ivy-BT) significantly shortened at stage II (I; 14.3 +/- 6.0, II; 10.1 +/- 6.5 min, p less than 0.01), and prolonged again at stage III (p less than 0.05 vs stage II). Among the patients, 6 out of 18 patients did not show any reduction in Ivy-BT (unchanged group). Though there were no significant changes in platelet aggregation rates, plasma TxB2, 6-keto-PGF1 alpha, F. VIII: C, and F. VIII: Ag levels throughout the study, platelet adhesion rate was significantly improved at stage II (I; 11.8 +/- 6.8, II; 19.6 +/- 12.8%, p less than 0.05), and similar augmentation in vWf: Ag was observed. Improvement in these two parameters were more remarkable in shortened Ivy-BT group (n = 12) than in unchanged group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of force output and preparatory set on premotor time of simultaneous bilateral responses.

Premotor times in simultaneous bilateral elbow-flexion movements were measured for 20 subjects when loads (light or heavy) were applied to both elbow joints. Premotor times for the heavy load were longer than those for the light load on both sides. The difference in premotor time between the two loads was larger for bilateral response than for unilateral response, suggesting that exertion of strength combines with response modality to increase the difference between the two loads. Preparatory set did not affect premotor time under the heavy load but affected premotor time under the light load. Possible mechanisms subserving these findings are discussed.

The reductive transformation of decanones with immobilized cells of Nicotiana tabacum.

The biotransformation of decanones with immobilized cells of Nicotiana tabacum led to the formation of their corresponding alcohols of high optical purity.