A simple and highly repeatable colorimetric toxicity assay method using 2,6-dichlorophenolindophenol as the redox color indicator and whole eukaryote cells.

A simple and highly reproducible toxicity assay method was studied by employing 2,6-dichlorophenolindophenol (DCIP) as a redox color indicator, baker's yeast Saccharomyces cerevisiae, and a thermostable three-consecutive-stir unit. The absorbance of DCIP was decreased by increasing the metabolism activity of S. cerevisiae to intake glucose as an organic substance. By optimizing the measurement conditions, we obtained highly sensitive responses to glucose between 0.75 and 30 mg/L (eight points, n = 3) with an incubation time of the reaction mixture of 10 min at 30 degrees C. An excellent value of 1.15% was obtained as the average of the repeatability from eight points. Next, for the characterization of this method, we investigated the influence on the colorimetric response of dissolved substances, such as inorganic ions and surfactants, in natural water. Furthermore, the colorimetric responses to several toxicants were examined using Cu2+, Mn2+, Zn2+, Cr3+, and Fe3+ as heavy-metal ions and simazine as an agricultural chemical. As a result, notable colorimetric responses were obtained for Cu2+ and Mn2+ at several concentrations, and the results were compared with those obtained using river water as a real sample. In the stability test, responses to 30 mg/L glucose were obtained for 28 days when the yeast cell suspension was stored at 4 degrees C (response reduction, 43.9%; average of the relative standard deviation for nine testing days, 22.7%; average of repeatability, 1.01%).

Peripheral blood stem cell mobilization following CHOP plus rituximab therapy combined with G-CSF in patients with B-cell non-Hodgkin's lymphoma.

We mobilized peripheral blood stem cells (PBSC) following CHOP plus rituximab (CHOP-R) therapy, and compared with the findings following CHOP therapy without rituximab. All patients were given G-CSF starting from day 11 after CHOP therapy. Patients in the CHOP-R group (n=8) were given rituximab on day 12. Target CD34(+) cells number was collected in a single leukapheresis on day 14, from all the eight patients in the CHOP-R group. PBSC mobilization kinetics, CD34(+) cells yield and colony-forming ability in the graft collection, toxicity during mobilization, and engraftment after transplantation of CHOP-R group were not significantly different from those in the CHOP group (n=8). In all patients given CHOP-R therapy, CD20(+) cells and immunoglobulin heavy chain (IgH) rearrangement in the graft collection were undetectable by flow-cytometric analysis and Southern blot analysis, respectively, but with PCR analysis two of eight grafts were positive for IgH rearrangement. While further studies are needed to evaluate the efficacy of purging and the outcome of patients undergoing autologous transplantation, CHOP-R therapy can be safely and effectively used in the mobilization phase of PBSC collection, without excess clinical toxicity or deleterious effect on PBSC mobilization kinetics or engraftment time.

Comparison of sensitivity to ultraviolet B irradiation between human lymphocytes and hematopoietic stem cells.

To investigate the clinical applicability of prophylaxis of post-transplant graft-versus-host disease by UV-B irradiation of stem cell preparations, the UV-B sensitivities of human lymphocytes and primitive hematopoietic progenitors were compared. The mononuclear cell fractions (MNC) derived from human cord blood and granulocyte-colony-stimulating factor-mobilized peripheral blood were used. After UV-B irradiation, lymphocyte proliferation ability, hematopoietic colony-forming cells, and apoptotic cells were analyzed. At a dose of 33 J/m(2), significant differences were observed in the residual percentages of hematopoietic progenitors and lymphocyte functions [ANOVA, F (5,46) = 19.4; P <.0001], and the difference between CFU-C (85.2% + 24.0%; n = 8) and MLR (12.7% + 12. 6%; n = 10) was significant (P <.0001). There were no significant differences in the residual percentages of CFU-C, HPP-CFC, and LTC-IC. Percentages of annexin V-positive cells in the total MNC and the CD34(+) cell population in MNC after UV-B irradiation were 69.8% and 18.7%, respectively. In conclusion, there was a range of UV-B doses over which T lymphocytes were inactivated but hematopoietic progenitors, including HPP-CFC and LTC-IC, were preserved.

A case of diffuse large B-cell lymphoma transformed from immunoglobulin A-producing marginal zone B-cell lymphoma.

We present a rare case of diffuse large B-cell lymphoma transformed from immunoglobulin (Ig) A-secreting marginal zone B-cell lymphoma. A 62-year-old woman was admitted to our hospital for examination of a disseminated pulmonary shadow. Gradual swelling of bilateral axilla and right inguinal lymph nodes were noted after admission. Histological examination of the lymph node biopsy specimen revealed the appearance of marginal zone B-cell lymphoma. The surface Ig of lymphoma cells was IgA-kappa, which coincided with the class of monoclonal Ig found in the patient's serum. The lymph node swelling and pulmonary shadow subsided, and the serum IgA level was normalized by 3 courses of systemic chemotherapy. However, after 4 courses of treatment, new tumor lesions at the right chest wall and left arm progressively became apparent. The biopsy specimen of the tumor showed a feature of diffuse large B-cell lymphoma. Despite intensive chemotherapy, the patient died of spreading tumor burden into the central nervous system.

Suppressive effect of ethanol on the expression of hepatic asialoglycoprotein receptors augmented by interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha.

Blood levels of inflammatory-related cytokines, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, are elevated in patients with alcoholic liver diseases. We investigated the effects of these cytokines and ethanol on the expression of hepatic asialoglycoprotein receptors (AGPRs) in a human hepatoblastoma cell line, HepG2. An [125I]-asialo-orosomucoid binding assay showed significant increases in surface AGPR numbers in HepG2 cells by treatment with IL-1beta, IL-6, and TNF-alpha, to levels which were approximately 130% of the values in untreated control cells. However, the enhanced AGPR numbers induced by treatment with these cytokines were markedly suppressed, to 70%-80% of the number in the untreated cells, by treatment with ethanol. Immunological detection of AGPR with a specific antibody demonstrated that the modulation of surface AGPR numbers was correlated with the cellular expression levels of AGPR. These results suggest that, although IL-1beta, IL-6, and TNF-alpha stimulate the synthesis of hepatic AGPR, ethanol suppresses the expression of AGPR augmented by these cytokines. This leads to an increase in serum asialo-orosomucoid levels caused by the disordered catabolism mediated by AGPR in patients with alcoholic liver disease.

Different adhesive characteristics and VLA-4 expression of CD34(+) progenitors in G0/G1 versus S+G2/M phases of the cell cycle.

We identified the cell cycle status of CD34(+) cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34(+) progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.

Retroperitoneal extrarenal angiomyolipoma with early gastric carcinoma.

We present a very rare case of a retroperitoneal extrarenal angiomyolipoma accompanied by early gastric cancer. A 41-year-old Japanese man, who had undergone surgery for a type IIc early gastric cancer 2 years earlier, was admitted to hospital presenting with back pain and abdominal fullness. Computed tomographic scanning and magnetic resonance imaging of the abdomen disclosed a massive fatty tumor extending from the hepatic hilus to the retroperitoneum. A large retroperitoneal tumor mass with no sign of involvement in the kidney was totally resected by radical surgery. Histologically, the tumor was classified as an angiomyolipoma.

Adenocarcinoma mimicking primary gastric lymphoma demonstrated by I-123 IMP scintigraphy.

A poorly differentiated adenocarcinoma of the stomach showed an unusual type of diffuse wall thickening of the entire stomach, similar to that seen in malignant lymphoma. The pathologic specimen revealed a "lymphoepithelioma"-like pattern of undifferentiated carcinoma with intense lymphocyte and plasma cell infiltration. I-123 IMP scintigraphy incidentally demonstrated increased uptake in the entire stomach on early imaging and moderate washout on delayed studies. SPECT images demonstrated diffusely increased accumulation in the entire gastric wall.

Long-term survival and differentiation of human peripheral blood CD34+ cells in SCID mice.

Human peripheral blood progenitor cells (PBPC) are currently used as a source of hematopoietic reconstitution by autologous transplantation after myeloabrative chemotherapy for malignancies. PBPC would also be useful for allogeneic transplantation since the collection of PBPC is much safer than that of bone marrow stem cells (BM). For allogeneic transplantation, it is imperative to confirm that PBPC contains self-renewable stem cells that can sustain a long lasting hematopoiesis. In the present study, we examined the reconstitution of human hematopoiesis in severe combined immunodeficiency (SCID) mice by transplanting peripheral blood CD34+ cells in which the neo gene was transduced as a marker. In 2 of 4 mice receiving PB-CD34+ cell transplantation, the neo gene appeared as early as 4 weeks and lasted as long as 24 weeks in all DNA preparations of bone marrow, peripheral blood and spleen cells from the SCID mice, while in 2 of 4 mice receiving BM-CD34+ cell transplantation, although the neo gene also lasted as late as 24 weeks, it did not appear as early as in the mice receiving PB-CD34+ cell transplantation. A similar observation was noted in clinical trials, i.e. the white blood cell and platelet recovered earlier by transplantation of PBPC than of BM. In mice who had the neo gene, we were also able to demonstrate by FACS the presence of human lineage specific antigen in the cells as late as 24 weeks after transplantation with PB-CD34+ cells, and the presence of human IgG in the sera 10 weeks after transplantation. These findings indicate that PB-CD34+ cells contain long-term repopulating stem cells which undergo differentiation in SCID mice.

[Hemolytic anemia and thrombocytopenia induced by cimetidine: recurrence with ranitidine administration].

A 69-year-old woman was admitted with apoplexy after operation of mitral valve stenosis and gastrectomy due to a gastric ulcer. In June 1994, her condition gradually worsened after acute pneumoniae in her right lung. Intravenous hyperalimentation with cimetidine administration was started to improve her undernourishment, because she had a history of gastric ulcer. However, after 10 days from the start of cimetidine therapy, anemia progressed rapidly. Biochemical examinations revealed that the serum indirect bilirubin and LDH levels were elevated and no serum haptoglobin was detected. These results indicated the development of hemolytic anemia, but at that time we could not clarify the reason. In October 1994, thrombocytopenia gradually progressed, and we halted the administration of cimetidine to ranitidine. Both hemolytic anemia and thrombocytopenia was dramatically improved after cessation of cimetidine administration. We then changed the drug from cimetidine, however the same phenomena have appeared again. The patient was in stable condition, after cessation of H2-blockers administration. The complication of hemolytic anemia and thrombocytopenia associated with H2-blocker administration in Japan.

[Functions of the transforming growth factor-beta superfamily in eyes].

One human body is composed of 6 x 10(13) cells, and eyes are also composed of many cells of different functions. The cellular functions and intercellular interaction are regulated by many regulators including cytokines and growth factors to maintain the homeostasis. The transforming growth factor-beta (TGF-beta) superfamily, a large family of multifunctional factors, regulates various cellular functions, including cellular proliferation, migration, differentiation, apoptosis and extracellular matrix production. The TGF-beta superfamily contains about 30 multifunctional factors, and is divided into several families according to the sequence homology. The TGF-beta family, the activin family, and bone morphogenic proteins belong to the TGF-beta superfamily. TGF-beta superfamily members transduce signals through type I and type II serine/threonine type transmembrane receptors. The signals are transduced from receptors through nuclei by Smad family members, which are phosphorylated by the activated type I receptors and translocate from cytoplasm into nuclei. TGF-beta family members and the TGF-beta superfamily receptor family are expressed in ocular tissues including the cornea, ciliary epithelium, lens epithelium, retina, and blood vessels. This observation suggests the importance of the TGF-beta superfamily in eyes. Smad family members (Smad 1, Smad 2, Smad 3 and Smad 4) are expressed in the cultured retinal pigmant epithelial cell line (D407), in which TGF-beta and activin A stimulate the translocation of Smad 2, but not Smad 1 into nuclei, whereas bone morphogenetic protein (BMP) stimulates that of Smad 1, but not Smad 2. TGF-beta superfamily members play important roles in the pathogenesis of retinal neovascularization and in the wound healing process of corneal tissue. TGF-beta inhibits the endothelial functions, but, stimulates angiogenesis in vivo. TGF-beta is involved in the formation of abnormal connective tissue in corneal wound healing. In these processes, many cytokines and growth factors are involved, interacting with each other and forming networks. It is mandatory to clarify the networks to investigate molecular pathogenesis and new therapeutic agents.

[Thrombocytopenia associated with sodium polystyrene sulfonate].

A 84-year-old man was admitted with diabetes mellitus, hypertension and chronic renal failure in September 1994. In October 1995, his renal function gradually worsened with hyperkalemia. Sodium polystyrene sulfonate (Kayexalate) was administered orally for the treatment of hyperkalemia. However, after 7 days from the start of Kayexalate therapy, thrombocytopenia progressed gradually, and 12 days later the initial platelet count of 20.7 x 10(4)/microliter decreased to 8.6 x 10(4)/microliter. This thrombocytopenia rapidly improved after cessation of Kayexalate administration. In December 1995, readministration of Kayexalate for the treatment of hyperkalemia induced thrombocytopenia again. Bone marrow aspiration biopsy revealed normal counts of nucleated cells and megakaryocytes with no increase in blasts. No other disorders which cause thrombocytopenia were seen in this patient. The complication of thrombocytopenia associated with Kayexalate has not been reported. This is the first reported case of thrombocytopenia caused by Kayexalate administration.

Crosslinking of a 28-residue N-terminal peptide of actin to myosin subfragment 1.

The N-terminal 28-residue peptide of actin whose Cys10 was labeled with 5-iodoacetamido-fluorescein (F3K peptide) was isolated from the fluorescently labeled thrombin digest of actin. The effect of myosin subfragment 1 (S1) on the fluorescence of F3K peptide was examined in the absence of ATP. With increasing concentration of S1 added, the fluorescence intensity of F3K peptide increased by maximally 7.3% with an apparent dissociation constant of 5.7 microM, suggesting a role of this peptide region of actin in acto-S1 binding in rigor. F3K peptide was crosslinked with S1 at 10 mM NaCl using a zero-length crosslinker by the method of Grabarek and Gergely [Anal. Biochem. 185, 131-135 (1990)]. The crosslinking was greatly inhibited by the presence of either 0.2 M NaCl or 5 mM MgATP. The analyses of amino acid compositions and sequences of the fluorescent peptides isolated from a lysylendopeptidase digest of the crosslinked S1 indicated that F3K peptide was mainly crosslinked to residues 637-642 of the S1 heavy chain. The crosslinked S1 was isolated by selectively pelleting the uncrosslinked S1 with F-actin. ATPase activity of the isolated crosslinked S1 alone was twice as high as that of control S1. The actin-activated ATPase activity of the crosslinked S1 was much lower than that of uncrosslinked S1. The estimated Vm and Km values were 1.72 s-1 and 125 microM, respectively. The Vm decreased to less than 1/8, while Km increased only twofold. The results suggest that the N-terminal 28-residue segment of actin may be implicated in the rigor binding of actomyosin and in the actin-activation of myosin ATPase, but may not be the main determinant of actomyosin binding in the presence of ATP.

Identification of a transforming growth factor beta-1 activator derived from a human gastric cancer cell line.

It has been shown that some types of tumour cells produce activated transforming growth factor beta-1 (TGF-beta 1). However, the mechanism for the activation of TGF-beta 1 derived from tumour cells has not been fully elucidated. The present study was undertaken to characterise an activator of latent TGF-beta 1 secreted from a human gastric cancer cell line, KATO-III. Western blot analyses using antibodies for TGF-beta 1, latency associated peptide (LAP) and latent TGF-beta 1-binding protein (LTBP) revealed that, in the cell lysate of KATO-III, TGF-beta 1 protein was expressed as a small latent complex of TGF-beta 1 and LAP. This was also confirmed by a gel chromatographic analysis of the cell lysate obtained from KATO-III. A 2.5 kb transcript of TGF-beta 1 mRNA was detected in KATO-III cells by Northern blot analysis. A gel chromatographic analysis of the conditioned medium from KATO-III cells revealed, in addition to the active form of TGF-beta 1, a factor which activated latent TGF-beta 1 from NRK-49F cells at fractions near a molecular size of 65,000. This factor was inactivated by heat (100 degrees C), acidification, trypsin and serine protease inhibitors. TGF-beta 1 activity in KATO-III cell lysate was not detected in the untreated state, but potent TGF-beta 1 activity was detected after acid treatment. These results suggest that KATO-III releases not only a latent TGF-beta 1 complex but also a type of serine protease, different from plasmin, plasminogen activator, cathepsin D, endoglycosidase F or sialidase, which activates the latent TGF-beta 1 complex as effectively as acid treatment.

Incidental finding of herpes zoster infection by Tc-99m HMPAO.

Tc-99m HMPAO brain SPECT study incidentally demonstrated a large area of increased accumulation in the soft tissue of the face and fronto-parietal region of the head, corresponding to the distribution of the ophthalmic and maxillary branches of the trigeminal nerve, which had been infected by herpes zoster.

Ethanol inhibits asialoglycoprotein receptor synthesis but augments its mRNA expression in a human hepatoma cell line.

The effect of ethanol on the expression of asialoglycoprotein receptor protein and its mRNA was studied in a human hepatoma cell line, HepG2. The number of asialoglycoprotein receptors on the cell surface was decreased to 60% of the control level, without a loss in affinity, by incubating the cells with 100 mM ethanol. The decrease in cell surface asialoglycoprotein receptors was paralleled by a decrease in total receptor numbers, including intracellular and surface receptors. The internalization of asialoglycoprotein was also diminished, to 44% of that in control cells. The decreases in cell surface receptors and total receptor numbers were partially restored by 2 mM 4-methylpyrazole, suggesting that ethanol metabolites participated in the diminution of asialoglycoprotein receptor expression. However, the steady-state expression of asialoglycoprotein receptor mRNA was increased in ethanol-treated cells and further augmented by a longer ethanol exposure. These paradoxical results, i.e., the decrease of asialoglycoprotein receptor protein and the increase of its mRNA expression, suggest that the reduction in the asialoglycoprotein receptor protein is a post-transcriptional event and that a possible feedback regulatory mechanism may control asialoglycoprotein receptor gene transcription and/or impair the degradation of its mRNA.