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.

N-Terminal homology in three cysteinyl proteases from Papaya latex.

Sequences to residue 17 have been determined for the three Papaya cysteinyl proteases, chymopapain and papaya peptidase A and B. Extensive homologies were found for these three enzymes and with papain and bromelain. These results suggest that the five sulphydryl enzymes discussed derive from a common ancestral gene.

Homologies of the N-terminal sequences of asclepains and papain.

Sequences to residue 21 have been determined for the two asclepains, cysteinyl proteases isolated from milkweed (Asclepias syriaca L.). These were compared with the sequence for papain, and extensive homology was found.

Sequence homologies in the N-terminal region of the ribosomal 'A' proteins from Methanobacterium Thermoautotrophicum and Halobacterium cutirubrum.

The ribosomal 'A' protein from the methanogen, Methanobacterium thermoautotrophicum, has been isolated and purified. The amino acid composition and mobility on two-dimensional gels indicates that this protein shows characteristics similar to the equivalent protein from the extreme halophile, Halobacterium cutirubrum, and is significantly different from the equivalent protein from other bacteria. The first 48 residues of the methanogenic 'A' protein were sequenced and showed a large amount of sequence homology to the H. cutirubrum 'A' protein. When the sequences of these two proteins were compared to the 'A' protein from other procaryotes (eubacteria), much less homology was evident. These results support the hypothesis (Woese, C.R. and Fox, G.F., (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5088-5090) of two procaryotic lines of evolutionary descent, the eubacteria and the archaebacteria, the latter including the methanogens and the extreme halophiles. The sequence data from the 'A' proteins also indicate that, phylogenetically, the archaebacteria are much closer to the cytoplasmic components of eucaryotes than they are to the eubacteria (or 'true bacteria').

The N-terminal sequence of the ribosomal 'A' protein from two moderate halophiles, Vibrio costicola and an unidentified moderate (NRCC 11227).

The 'A' protein, equivalent to ribosomal protein EL7/L12 from Escherichia coli, has been isolated and purified from two moderate halophiles Vibrio costicola and NRCC 11227. The 'A' protein from V. costicola contained an N-terminal serine and separated into two forms on DEAE-cellulose and two-dimensional electrophoresis while the equivalent protein in NRCC 11227 contained an N-terminal alanine residue and was present in only one form. The amino acid composition and mobility on two-dimensional gels indicated these proteins were very similar to EL7/L12. The first 38 residues of the 'A' proteins were sequenced and compared to the equivalent protein from E. coli and the extreme halophile Halobacterium cutirubrum. The N-terminal region of the 'A' protein from both moderate halophiles showed substantial homology to EL 12 (75--80%) but no evidence of any homology to the equivalent protein from the extreme halophile. The ribosomal proteins equivalent to ES1A in E. coli were also isolated and their amino acid compositions determined.

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.

Vitamin K2 and its derivatives induce apoptosis in leukemia cells and enhance the effect of all-trans retinoic acid.

Geranylgeraniol, a polyprenylalcohol composing the side chain of vitamin K2 (VK2), was previously reported to be a potent inducer of apoptosis in tumor cell lines (Ohzumi H et al, J Biochem 1995; 117: 11-13). We examined the apoptosis-inducing ability of VK2 (menaquinone 3 (MK3), MK4 and MK5) and its derivatives such as phytonadione (VK1), as well as polyprenylalcohols with side chains of various lengths including farnesol (C15-OH; FO), geranylgeraniol (C20-OH; GGO), and geranylfarnesol (C25-OH; GFO) toward leukemia cells in vitro. MK3, MK4, MK5 and GFO (at 10 microM) showed a potent apoptosis-inducing activity for all freshly isolated leukemia cells tested and for leukemia cell lines such as NB4, an acute promyelocytic leukemia (APL)-derived cell line and MDS92, a cell line derived from a patient with myelodysplastic syndrome, although there were some differences depending on the cells tested. In contrast, VK1 showed no effect on any of the leukemia cells. The combination of MK5 plus all-trans retinoic acid (ATRA) resulted in enhanced induction of apoptosis in both freshly isolated APL cells and NB4 cells as compared to each reagent alone. These data suggest the possibility of using VK2 and its derivatives for the treatment of myelogenous leukemias, including APL.

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.

Interleukin-7 enhances colony growth and induces CD20 antigen of a Ph+ acute lymphoblastic leukemia cell line, OM9;22.

A newly established human leukemia cell line, OM9;22, is reported, with B-precursor immunophenotype (CD10+ CD19+ CD22+ HLA- DR+ C mu-) and CD13 antigen, originated from a 19-year-old female patient with Philadelphia (Ph) chromosome-positive acute lymphoblastic leukemia (ALL). The OM9;22 cells carry a Philadelphia (Ph) translocation and hybrid message detected by a minor-breakpoint cluster region (BCR) exon 1/ABL exon 2 junctional probe using reverse transcriptase polymerase chain reaction. The genetic alterations are consistent with those observed in the donor's leukemia cells, allowing us to conclude that this cell line is a minor-BCR rearranged Ph-positive ALL (Ph+ ALL). Colony formation of the OM9;22 cells in methylcellulose culture is enhanced by the addition of human interleukin 7 (IL-7). In liquid culture, more than 80% of IL-7-treated OM9;22 cells express CD20 antigen but fail to express surface immunoglobulins or cytoplasmic mu-chain, indicating that the cells have a potential of limited maturation by IL-7. By contrast, IL-4 suppresses the colony formation of the OM9;22 cells. These findings suggest that this cell line might be a model of B-precursor human leukemia with proliferative capability by IL-7.

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.

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.

Hematopoietic supportive function of human bone marrow stromal cell lines established by a recombinant SV40-adenovirus vector.

We have previously reported the establishment of a variety of human bone marrow stromal cell lines using a recombinant SV40-adenovirus vector. Using this vector, we obtained more clonal stromal cells. Here, we have characterized these cells and analyzed their capacity to support the proliferation and differentiation of hematopoietic cells. The stromal cells were cocultured with nonadherent human bone marrow cells used as hematopoietic cells. The total numbers of hematopoietic cells and CFU-GM in culture were counted every week. Two of the six stromal cell lines, AA101 and HAS303, supported the proliferation and differentiation of hematopoietic cells and CFU-GM for more than 9 weeks. Further, granulocytes, macrophages, and megakaryocytes were detected when cocultured with these cells. When hematopoietic cells were cocultured but separated from the two stromal cell lines by a 0.45-microns millipore membrane to prevent their attachment, almost all CFU-GM disappeared within 7 weeks. The supportive stromal cells produced GM-CSF and IL-6. However, other cell lines producing these humoral factors did not support hematopoietic cell proliferation for such a long time. These findings suggest that these established human bone marrow stromal cell lines will be useful, in that analysis of their supportive function in hematopoietic cell proliferation and differentiation through cell-to-cell interaction will shed some light on this area.

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.

Sequences of three genes specifying xylanases in Streptomyces lividans.

The entire nucleotide (nt) sequences of three genes (xlnA, xlnB and xlnC) of Streptomyces lividans encoding three distinct xylanases (Xln) have been determined. The nt sequences were confirmed by comparing the deduced amino acid (aa) sequences with the ones derived from the N-terminal aa sequences of the mature purified proteins. The N-terminus of the XlnA showed some homology with either the N-termini or the C-termini of eight other Xln and of two exo-glucanases. The N-terminus of XlnB is homologous to that of XlnC and to Xln of seven other microorganisms.

Identification of the catalytic nucleophile in the cellulase from Schizophyllum commune and assignment of the enzyme to Family 5, subtype 5 of the glycosidases.

Differential chemical modification of the cellulase from Schizophyllum commune with [N-methyl-3H]1-ethyl-3(4-azonia-4,4-dimethylpentyl)-carbodiimide in the presence and absence of substrate identified an active site glutamate residue within the peptide: Leu-Gln-Ala-Ala-Thr-Glu-Trp-Leu-(Lys). This Glu residue is proposed to participate in binding of substrate as amino acid sequence homology studies combined with mechanism-based inhibition of the cellulase with 4',5'-epoxypentyl-beta-D-cellobioside identified a neighboring Glu residue, which conforms to the Glu-X-Gly motif of Family 5 glycosidases, as the catalytic nucleophile. These data allow the assignment of the S. commune cellulase to Family 5, subtype 5 of the glycosidases.

Amino acid sequence and thermostability of xylanase A from Schizophyllum commune.

The amino acid sequence (197 residues) of xylanase A from the fungus, Schizophyllum commune, was determined by automated analysis of peptides from proteolytic and acid cleavage. The sequence is similar to two Trichoderma xylanases (approximately 56% identical amino acids), but also shows at least 40% identities with xylanases from Bacillus subtilis, B. pumilus and B. circulans. The conserved regions of the enzyme contain only two glutamic acid residues which implicates their possible involvement in catalysis. The disulfide bond in xylanase A is not conserved in this family. In spite of this, the B. subtilis xylanase was found to be more thermostable than xylanase A.

Applications of recombinant DNA technology to the pulp and paper industry.

New gene selection techniques (Recombinant DNA) are currently available to exploit useful properties of various biological systems hitherto regarded as interesting but of little or no immediate commercial value. The application of genetic engineering techniques to problems in the Pulp and Paper Industry are many. As a first step these techniques are being used to provide much needed fundamental information on the cellular and molecular mechanisms involved in the expression of extra-cellular enzymes that degrade lignocellulosic pulping wastes. The information gleaned from the studies on cellulolytic fungi and bacteria can be used to genetically engineer a yeast or bacterium capable of converting pulping wastes into ethanol and other useful by-products.

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.

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.

Establishment of a novel B-lymphoma cell line, CTB-1, with strong Pas antigen expression having chromosomal translocation (14;22).

We established a new lymphoma cell line, designated CTB-1, from pericardial effusion of a patient with diffuse large B-cell lymphoma. This cell line showing vigorous growth ability has undergone 260 passages over a period of 34 months in suspension culture, and is heterotransplantable to nude mice. The cultured cells were positive for CD10, CD19, CD20, CD21, HLA-DR, and surface IgG kappa, and negative for T cell antigens. Chromosomal analysis revealed a t(14;22)(q32;q11) that is consistent with original lymphoma cells. CTB-1 cells show the high cell surface expression level of Fas antigen/APO-1. However, ligation of Fas antigen with anti-Fas monoclonal antibody (clone CH-11) did not induce apoptosis of CTB-1 cells. This suggests that Fas itself or the downstream signaling pathways of Fas may be impaired in this cell line. This new cell line may provide a useful in vitro system to study the biology and pathogenesis of B-cell lymphoma which is independent of Fas-mediated apoptosis.