Expression and purification of uniformly (15)N-labeled amyloid beta peptide 1-40 in Escherichia coli.

For biophysical studies using heteronuclear NMR analysis of amyloid beta peptide, construction of an efficient and high yield expression system of uniformly isotopic labeled amyloid beta peptide is desirable. Here we succeeded in obtaining (15)N-labeled amyloid beta 1-40 expressed by attachment to hen egg white lysozyme as a fusion protein.

Combination of 22-oxa-1,25-dihydroxyvitamin D(3), a vitamin D(3) derivative, with vitamin K(2) (VK2) synergistically enhances cell differentiation but suppresses VK2-inducing apoptosis in HL-60 cells.

We originally reported that vitamin K(2) (VK2) effectively induces apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. In addition, VK2 was shown to induce differentiation of leukemia cells when the cells were resistant against VK2-inducing apoptosis. A novel synthetic vitamin D(3)derivative, 22-oxa-1,25-dihydroxyvitamin D(3) (OCT: oxacarcitriol) shows a more potent differentiation-inducing ability among myeloid leukemia cells in vitro with much lesser extent of the induction of hypercalcemia in vivo as compared to the effects of 1alpha,25(OH)(2)D(3). In the present study, we focused on the effects of a combination of OCT plus VK2 on leukemia cells. Treatment of HL-60 cells with OCT for 72 h induces monocytic differentiation. A combination of OCT plus VK2 dramatically enhances monocytic differentiation as assessed by morphologic features, positivity for non-specific esterase staining, and cell surface antigen expressions. This combined effect far exceeds the maximum differentiation induction ability at the optimal concentrations of either OCT or VK2 alone. In addition, pronounced accumulation of the cells in the G0/G1 phase is observed by combined treatment with OCT plus VK2 as compared with each vitamin alone. In contrast to cell differentiation, caspase-3 activation and apoptosis induction in response to VK2 are significantly suppressed in the presence of OCT in HL-60 cells. These data suggest that monocytic differentiation and apoptosis induction of HL-60 cells are inversely regulated. Furthermore, pronounced induction of differentiation by combined treatment with VK2 plus OCT was also observed in four out of six cases of primary cultured acute myeloid leukemia cells in vitro, suggesting that VK2 plus OCT might be a potent combination for the differentiation-based therapy for acute myeloid leukemias.

Disparity between the macroglobulin-producing components of 2 cases of follicular lymphoma with Waldenström's macroglobulinemia.

We report 2 patients with follicular lymphoma (FL) which was accompanied by Waldenström's macroglobulinemia (WM). One patient was a 65-year-old woman and the other a 60-year-old man. Both patients showed a high level of circulating macroglobulin (4.6 g/dL and 3.6 g/dL, respectively) and bone marrow involvement of small lymphoid cells. Moreover, in each case, the macroglobulin-producing component and the follicular component were determined to be of the same clone based on their identical light-chain restriction pattern and other factors. However, there was a difference in the histopathological characteristics of the macroglobulin-producing components of the 2 patients, especially the cytoplasmic immunoglobulin (Ig)M+ cell distribution in the biopsied lymph nodes. Test results for the female patient showed intrafollicular proliferation of those cells. The male patient's test results showed that IgM+ cells were located in the narrow extrafollicular areas of the lymph nodes. Our observations suggest that at least 2 different subtypes of FL may also be causative of a WM presentation.

Successful treatment with cyclosporin A for myelodysplastic syndrome with erythroid hypoplasia associated with T-cell receptor gene rearrangements.

Myelodysplastic syndrome (MDS) with erythroid hypoplasia, a rare form of MDS, has not yet been clearly defined. We report four patients with MDS with erythroid hypoplasia who received immunosuppressive therapy. All were elderly, had severe transfusion-dependent anaemia, morphological evidence of myelodysplasia and a low percentage (3.2-13.6%) of erythroid precursors. Administration of cyclosporin A (CsA) improved their anaemia; all transfusion-dependent patients achieved transfusion-independence. An inverted CD4/8 ratio was seen in three patients who also demonstrated T-cell receptor (TCR)-beta and -gamma gene rearrangements by Southern blotting and clonality by polymerase chain reaction. Treatment with CsA can be an attractive alternative treatment for patients with MDS with erythroid hypoplasia, which may be associated with a clonal abnormality in T cells.

Apoptosis/differentiation-inducing effects of vitamin K2 on HL-60 cells: dichotomous nature of vitamin K2 in leukemia cells.

We originally reported that vitamin K2 (VK2) analogs, including menaquinone 4 (MK4) but not vitamin K1, effectively induce apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. It has also been reported by others that VK2 showed the differentiation-inducing activity in leukemia cell lines. To investigate the discrepancy between apoptosis- and differentiation-inductions of leukemia cells by VK2 treatment, we used bcl-2 gene transfected HL-60 cells (HL-60-bcl-2) which resulted in five-fold over-expression of BCL-2 protein, and then compared the effects of MK4 to the control HL-60-neo cells. Seventy-two hours of exposure to various concentrations of MK4 resulted in growth inhibition of these cells in a dose-dependent manner (0.1-50 microM), however, HL-60-bcl-2 was less sensitive against MK4. MK4 potently induced apoptosis of HL-60-neo cells along with the depolarization of mitochondrial membrane potential and caspase-3 activation. Notably, HL-60-bcl-2 was almost completely resistant to apoptosis induction in response to MK4, although cell growth inhibition was still observed. In spite of the abrogation of apoptosis induction, about 90% of HL-60-bcl-2 cells were arrested in the G0/G1 phase within 48 h of exposure to 10 microM of MK4 accompanied by up-modulation of p27KIP1 expression. Concomitantly, HL-60-bcl-2 cells underwent monocytic differentiation. These data suggest that VK2 also shows the differentiation inducing effects on leukemia cells which are resistant against VK2-inducing apoptosis. The dichotomous nature of VK2 against leukemia cells appears to have clinical benefits for the treatment of patients with leukemias and myelodysplastic syndromes.

Effects of chronic alcohol consumption on hepatic poly-ADP-ribosylation in the rat.

BACKGROUND: Poly-adenosine diphosphate (ADP)-ribosylation is involved in a variety of biological processes, which include DNA repair, malignant transformation, and apoptosis. It is of interest how this reaction is altered after long-term alcohol intake. Therefore, we determined long-term alcohol effects on hepatic poly-ADP-ribosylation in the rat. METHODS: Male Sprague Dawley(R) rats (four pairs) were pair-fed a nutritionally adequate liquid diet that contained ethanol as 36% of total energy and an isocaloric control diets for 4 weeks. Liver tissue homogenates and nuclear fractions were subjected to ADP-ribosylation with [32P]nicotinamide adenine dinucleotide. The ADP-ribosylated proteins were separated by SDS-PAGE, followed by autoradiography. Expression of poly-ADP-ribose polymerase (PARP) also was evaluated by Western blotting. RESULTS: Incubation of rat liver homogenates in ADP-ribosylation reaction mixture resulted in a radiolabeling of a 116 kDa protein, most likely auto-ribosylation of PARP. This poly-ADP-ribosylation was increased significantly (p < 0.025) after long-term alcohol intake. This alcohol effect was reproducible in nuclear fractions as well. Expression levels of PARP, however, were comparable between alcohol-fed rats and their pair-fed controls. CONCLUSION: Poly-ADP-ribosylation, an important posttranslational modification of nuclear proteins, was increased significantly after chronic alcohol consumption in the rat.

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry.

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and capillary liquid chromatography-electrospray mass spectrometry (cLC-ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC-ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man8GlcNAc2) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC-PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.

Presynaptic inhibition of cerebellar GABAergic transmission by glutamate decarboxylase autoantibodies in progressive cerebellar ataxia.

Autoantibodies against glutamic acid decarboxylase (GAD) have been found in stiff-man syndrome, insulin dependent diabetes mellitus, and progressive cerebellar ataxia. A patient with progressive cerebellar ataxia is described who was positive for GAD autoantibodies, and had Sjögren's syndrome. Immunohistochemical studies using CSF and serum samples from the patient showed immunoreactivities in axon terminals of cerebellar GABAergic neurons. A whole cell patch clamp technique recording from rat cerebellar slices showed that the CSF, presumably through GAD autoantibodies, presynaptically inhibited GABAergic transmission. Intravenous administration of immunoglobulin failed to improve clinical symptoms and immunoreactivities examined after therapy. The findings suggest that GAD autoantibodies play a pathogenic part in reducing GABA release in in vitro slices.

Combination of granulocyte colony-stimulating factor and low-dose cytosine arabinoside further enhances myeloid differentiation in leukemia cells in vitro.

We examined the differentiation-inducing effect on freshly isolated myeloid leukemia cells in liquid suspension culture by combined treatment with granulocyte colony-stimulating factor (G-CSF) plus low-dose cytosine arabinoside (Ara-C; 5-10 ng/ml) in 25 patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in leukemic transformation. Culture with G-CSF alone showed leukemic cell growth stimulation in 15 out of the 25 cases (60%) and induction of cell differentiation in 19 out of the 25 cases (76%), respectively. In 23 cases (92%), either growth stimulation and/or differentiation induction of leukemia cells was observed in response to G-CSF. This suggests that most myeloid leukemia cells are able to respond to G-CSF stimulation. In addition, treatment of cells with low-dose Ara-C alone resulted in the enhancement of myeloid specific antigens expression in 16 cases (64%). Treatment of leukemia cells with higher concentrations of Ara-C (over 50 ng/ml) alone resulted in cytocidal effects but not in the induction of differentiation. Furthermore, 15 cases (60%) showed pronounced myeloid differentiation of leukemia cells after combined exposure to G-CSF plus low-dose Ara-C as compared with cells treated with either G-CSF or Ara-C alone. The enhanced effect of differentiation induction by combining G-CSF plus low-dose Ara-C was also observed in a murine myeloid leukemia cell line WEHI-3B in vitro. These data suggest that treatment with G-CSF plus low-dose Ara-C is capable of inducing differentiation of leukemic cells in vitro, and also appears to be useful for the differentiation-based therapy of patients with AML and MDS.

Enhancement of poly-adenosine diphosphate-ribosylation in human hepatocellular carcinoma.

BACKGROUND: Poly-adenosine diphosphate (ADP)-ribosylation, catalysed by poly(ADP-ribose) polymerase (PARP), is a post-translational modification of nuclear proteins and is involved in a wide range of biological processes including DNA repair, cell proliferation and malignant transformation. Alteration of this reaction in human hepatocellular carcinoma (HCC) is of interest, but has not yet been explored. The aim of this study was to evaluate poly-ADP-ribosylation and to compare the expression of PARP in HCC and adjacent non-tumour tissues. METHODS: Tumorous and adjacent non-tumorous tissues were obtained from five consecutive patients with HCC during surgery for tumour resection. Tissue homogenates were subjected to ADP-ribosylation with [32P]-nicotinamide adenine dinucleotide. The ADP-ribosylated proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by autoradiography. Expression of PARP was also evaluated by western blotting. RESULTS: Several proteins were ADP-ribosylated in human HCC tissues. Notably, the radiolabelling of a 116-kDa protein was remarkably greater than that in adjacent non-tumorous tissues (86.5 +/- 35.2 arbitrary units by densitometry vs 12.2 +/- 9.9, mean +/- SD, n = 5, P < 0.02). The radiolabelling of the 116-kDa protein was decreased in the presence of PARP inhibitors in a concentration-dependent manner. Immunoblot analyses revealed that the radiolabelled protein was PARP and that its expression was significantly greater in HCC than in adjacent non-tumorous tissues (333 +/- 204% of non-tumorous tissue, P < 0.05). CONCLUSIONS: We found that poly-ADP-ribosylation and PARP expression were significantly increased in human HCC compared with those in adjacent non-tumorous tissues in surgically obtained specimens.

Refractory anemia with ringed sideroblasts with a low IPSS score progressed rapidly with de novo appearance of multiple karyotypic abnormalities and into acute erythroleukemia (AML-M6A).

We report here a case of refractory anemia with ringed sideroblasts (RARS) with a low risk group by the International Prognostic Scoring System (IPSS) at the time of diagnosis but had a rapid disease progression. Although the patient showed a normal male karyotype at the time of RARS diagnosis, his marrow cells had del(5)(q14) and add(17)(p12) abnormalities 2 months after the diagnosis, and later the marrow cells had multiple abnormalities and the patient expired 6 months after the initial diagnosis of RARS. The patient was diagnosed as having RARS with a low risk group by the IPSS classification, however, one should keep in mind that some patients with myelodysplastic syndromes with low risks by either the French-American-British (FAB) classification or the IPSS classification may have progressive disease and subsequential cytogenetic analysis could predict the disease progression.

[A case of brain stem infarction with bilateral hearing loss].

The study case was a 66-year-old man who had bilateral neurosensory hearing impairment due to brain stem infarctions. He noticed mild hearing loss, frequent vertigo and tinnitus. About one month later, his hearing took a sudden turn for the worse, and he suffered from dysarthria, dysphagea and abasia. Neurological examination revealed pseudobulber palsy, left hemiparesis, cerebeller ataxia, disturbance of pain and temperature sensation on the right face and left side of the body. Brain stem auditory evoked potentials (BAEPs) showed a delayed small wave V with the abscence of previous waves on the right side and no significant waves on the left side. Brain magnetic resonance images (MRI) revealed infarctions in the bilateral middle cerebellar peduncles, including in the right lateral portions of pons, and the right lower pontine base. We believe that not only peripheral, but also central auditory pathways adjacent to infarctions were damaged. Magnetic resonance angiography (MRA) showed severe stenosis or occlusion of left vertebral artery and basilar artery. We concluded that hypoperfusion of the vertebrobasilar artery territories caused ischemia of the cochlear nerve and the auditory tracts in the brain stem, which resulted in bilateral hearing loss.

IgA-lambda/IgG-kappa biclonal myeloma in which two clones proliferated in individual sites.

A 72-year-old man was admitted to our hospital because of lumbago and numbness of legs. Tumor invasion at the fourth lumbar vertebra was revealed. Immunohistochemistry using specific antibodies against each heavy and light chain of immunoglobulin revealed that the myeloma cells in bone marrow were all IgA-lambda type whereas they were all positive for IgG-kappa type in a tumor of the fourth lumbar vertebra. These data indicate that the patient had IgG-kappa/IgA-lambda biclonal myeloma. Different phenotypes of M-proteins and distinct proliferating sites for two clones suggest that they may have resulted from two independent transforming events.

Vitamin K2 induces apoptosis of a novel cell line established from a patient with myelodysplastic syndrome in blastic transformation.

We have previously reported that vitamin K2 (VK2) has a potent apoptosis inducing activity toward various types of primary cultured leukemia cells including acute myelogenous leukemia arising from myelodysplastic syndromes (MDS). We established a novel cell line, designated MDS-KZ, from a patient with MDS in blastic transformation, and further investigated the effects of VK2 using this novel cell line. MDS-KZ shows complex chromosomal anomaly including -4, 5q-, -7, 13q+, 20q-, consistent with that seen in the original patient. Culture of MDS-KZ cells in RPMI1640 medium containing 10% FBS lead to steady but very slow proliferation with a doubling time of 14 days. However, the cellular growth rate was significantly accelerated in the presence of various growth factors such as granulocyte colony-stimulating factor, stem cell factor, granulocyte-macrophage colony-stimulating factor, interleukin-3, and thrombopoietin. Most of the cultured cells show the morphological features of myeloblasts. They are positive for CD7, CD33, CD34, CD45, CD117, and HLA-DR. However, about 10% of the cells are more mature metamyelocytes and neutrophils with various dysplastic characteristics such as pseudo-Pelger nuclear anomaly and hypersegmentation, suggesting a potential for differentiation in this cell line. As previously reported for cultured primary leukemia cells, exposure to VK2, but not to VK1, resulted in induction of apoptosis of MDS-KZ cells in a dose-dependent manner (IC50: 5 microM). In addition, VK2 treatment induced down-regulation of BCL-2 and up-regulation of BAX protein expression with concomitant activation of caspase-3 (CPP32). A tetrapeptide functioning as antagonist of caspase-3, Ac-DEVD-H, suppressed the VK2-induced inhibition of cell growth, suggesting that caspase-3 is, at least in part, involved in VK2-induced apoptosis. These observations suggest that the MDS-KZ cell line can serve as a model for the study of the molecular mechanisms of VK2-induced apoptosis.

[Clinical effects of combination therapy with cefozopran and tobramycin for severe infections in patients with hematologic diseases].

We studied clinical effect of a combination therapy with cefozopran (CZOP) and tobramycin (TOB) for infections in 80 patients with hematologic diseases in 15 institutes. Combined doses with CZOP 2 g and TOB 60-90 mg twice a day had been given intravenously. Of the 80 patients, 61 patients (42 with acute leukemia, 10 with malignant lymphoma, 3 with aplastic anemia, 2 with chronic myeloid leukemia, 2 with multiple myeloma, and 2 with myelodysplastic syndrome) were evaluable. Those consisted of 6 patients with septicemia, 49 with suspected septicemia, 3 with pneumonia, and 3 with other infections. Clinical efficacy by the treatment was excellent in 24, good in 17, fair in 9, and poor in 11 patients, and the overall efficacy rate including excellent and good was 67.2%. Microbiologically, 5 of the 6 patients with septicemia (1 coagulase negative Staphylococcus, 2 S. pneumoniae, 1 S. oralis, and 1 E. coli) were responded. The efficacy rate in patients with severe granulocytopenia showing 100/microliter or lesser neutrophil counts during the drug administration was 57.1% (12/21). Side effects and abnormal changes of clinical laboratory findings were observed in 5 patients, and 16 patients, respectively, but most of them were mild. The findings above suggested that the combination therapy with CZOP and TOB is useful as an empiric therapy for severe infections in patients with hematologic diseases.

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro.

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5-10 days of culture, but declined to low levels at 15-20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34 + cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

Vitamin K2 selectively induces apoptosis of blastic cells in myelodysplastic syndrome: flow cytometric detection of apoptotic cells using APO2.7 monoclonal antibody.

We have previously reported that vitamin K2 (VK2) but not VK1 has a potent apoptosis-inducing effect on freshly isolated leukemia cells from patients with various types of leukemia. By multi-color flow cytometric analysis using monoclonal antibody (mAb), APO2.7, which detects mitochondrial 7A6 antigen specifically expressed by cells undergoing apoptosis, we further investigated the apoptosis-inducing effect of VK2 on minor populations of leukemic blast cells in bone marrow from patients with myelodysplastic syndrome (MDS) and overt myeloid leukemia (post-MDS AML). Limiting dilution of CD95 (anti-Fas) mAb-treated apoptotic Jurkat cells with nonapoptotic CTB-1 cells revealed that APO2.7-positive Jurkat cells were consistently detectable by flow cytometry when present at levels of at least 5% in the CTB-1 suspension. In patient samples the gating area for leukemic clone was determined using cell surface antigen-specific mAbs conjugated with either fluorescein isothionate (FITC) or phycoerythrin (PE) and subsequently the cells stained with phycoerythrin cyanine (PE-Cy5)-conjugated APO2.7 mAb were assessed within the gating area of the leukemic clone for monitoring apoptosis. Treatment of the bone marrow mononuclear cells with 3-10 microM of VK2 (menaquinone-3, -4 and -5) in vitro potently induced apoptosis of the leukemic blast cells as compared with the untreated control cells in all 15 MDS patients tested. This effect was more prominent on blastic cells than that on mature myeloid cells such as CD34-/CD33+ gated cells. In addition, VK2 performed much less effectively on CD3-positive lymphoid cells. In contrast to VK2, VK1 did not show apoptosis-inducing activity. These data suggest that VK2 may be used for treatment of patients with MDS in blastic transformation.

[Induction of Ph-negative normal clone and long-term survival by combined treatment with G-CSF plus middle dose cytosine arabinoside for patients with chronic myeloid leukemia in blastic transformation].

Three patients with chronic myeloid leukemia (CML) in blastic transformation were treated with G-CSF plus middle dose cytosine arabinoside (Ara-C). G-CSF was administered (150 mg, s.c. or 300 mg, d.i.v./day) 24 hr prior to Ara-C (2-3 g/body, 6 hour d.i.v. for 2-5 days) and continued until the peripheral neutrophil count rose above 1,000/microlitre. As a supplement, VP-16 (80 mg/m2, for 2 days) was administered as warranted to control the growth of blastic cells. All 3 patients survived for more than 12 months with a favorable performance status. Normal karyotypes were detected in 2 of the patients after chemotherapy. One of those patients in paticular demonstrated normal bone marrow findings with the almost complete disappearance of the Ph-positive clone. In vitro cultures of peroxidase-negative CML blastic cells revealed that G-CSF stimulated the induction of blastic cells into the cell cycle and that blastic cell apoptosis was more pronounced in cells cultured with G-CSF plus Ara-C than with G-CSF or Ara-C alone. G-CSF plus middle dose Ara-C therapy appears to be a strong candidate for the treatment of CML in blastic transformation with a poor prognosis.