Restriction analysis of the plasmid and chromosomal DNA of the strain Streptomyces fradiae--a producer of neomycin

Plasmid DNA with a molecular weight of 53-55 Md was isolated from Streptomyces fradiae strain 676 producing neomycin on centrifugation of the DNA preparation at the density gradient of cesium chloride-ethidium bromide. The extrachromosomal DNA had 6 recognition sites for Bgl II, more than 13 recognition sites for Bam HI, more than 14 recognition sites for Kpn I, more than 18 recognition sites for Pst I, more than 20 recognition sites for Sac I, more than 21 recognition sites for Pvu II and no recognition sites for Eco RI, Eco RV, Hind III and Sla I.     

Structure/function studies of the common acute lymphoblastic leukemia antigen (CALLA/CD10) expressed on human neutrophils

The common acute lymphoblastic leukemia antigen (CALLA/CD10) is a nonintegral membrane glycoprotein expressed on normal and neoplastic cells of hematopoietic and nonhematopoietic origin. We have undertaken a series of experiments to examine 1) the structural homology between leukemia cell and neutrophil CALLA/CD10 and 2) the putative function CALLA/CD10 subserves to human neutrophils. Biosynthetic labeling, peptide mapping, and two-dimensional gel electrophoresis indicate that neutrophils synthesize and express a CALLA/CD10 molecule that is similar, but not identical, to leukemic cell CALLA/CD10. The level of CALLA/CD10 expression is similar on the two cell populations, and neutrophil CALLA/CD10 (like its leukemic cell counterpart) undergoes antigenic modulation. Finally, we report that neutrophil cell surface-bound anti-CALLA/CD10 monoclonal antibodies inhibit the chemotactic response to both N-Formyl-methionyl-leucyl-phenylalanine (F-mlp) and zymosan-activated sera (ZAS), but had no inhibitory effect on random migration, degranulation, or aggregation. The anti-class I monoclonal antibody W6/32 exerted a similar effect on chemotaxis. We conclude that CALLA/CD10 has no clearly defined role in neutrophil function but may play a role in some distal event in chemotaxis.     

Distribution and modulation of a human leukemia-associated antigen (CALLA)

CALLA is a 100,000-dalton surface glycoprotein expressed by malignant cells of patients with clinically important subtypes of acute leukemia. Incubation of human leukemic cells expressing CALLA with specific monoclonal antibody (J5) at 37 degrees C causes rapid and selective internalization and degradation of this antigen (antigenic modulation). In these studies we show that CALLA-specific monoclonal antibodies also identify a cell surface glycoprotein having a m. w. of approximately 100,000 on 2 to 6% of nonmyeloid nucleated cells of normal adult bone marrow, on normal fibroblasts in tissue culture, and on cells of several nonhematopoietic human tumor cell lines. J5 antibody similarly modulates the surface expression of CALLA on nonleukemic cell populations, although the extent of modulation at a given concentration of antibody varied considerably. Modulation was almost complete for CALLA on cells of normal bone marrow, but was highly variable for cells of nonhematopoietic cell lines, possibly reflecting variability in antibody access to surface antigen. Using fluoresceinated or iodinated J5 antibody to modulate expression of CALLA on cells of leukemic cell lines, we show that antibody-antigen complexes undergo a temperature-dependent redistribution on the cell surface during modulation to form microaggregates. Antibody as well as antigen is then internalized. Studies of [35S]methionine-labeled cells indicate that synthesis of CALLA continues despite modulation of its surface expression by specific antibody, implying that the presence of CALLA on the cell surface reflects a dynamic equilibrium between the processes of surface expression of newly synthesized glycoprotein and its spontaneous and antibody-mediated clearance. The implications of these observations for immunotherapy are discussed.     

Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells

In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.     

Expression of metastasis associated proteins,CD44v6 and NM23-H1, in pediatric acute lymphoblastic leukemia

Two metastasis associated proteins, CD44v6 and NM23-H1, are expressed by normal lymphoid cells, the former serving as activation marker and the later as a constitutive protein. CD44v6 is considered as a marker of poor prognosis of various hematological cancers but its expression was not demonstrated in childhood acute lymphoblastic leukemia (ALL). On the other hand, NM23-H1 is considered as a differentiation inhibitory factor in various hematological cancers and as a marker of poor prognosis. Therefore we have analyzed the expression of CD44v6 and NM23-H1 in bone marrow of sixteen pediatric ALL patients using immunocytochemistry. For the first time, we have demonstrated the expression of CD44v6 protein epitopes on leukemic cells in a proportion of ALL cases (6/16), primarily in the medium/high risk group (except one case), suggesting a possible association to an unfavorable outcome. On the other hand, NM23-H1 protein expression was maintained in leukemic cells in 50% of both low and medium/high risk ALL cases. The majority of the pediatric ALL cases expressed only one of the metastasis associated proteins (10/16). This feature is highly similar to the observations made in several adult solid cancers. The potential of CD44v6 expression in leukemic cells as prognosticator in pediatric ALL has to be evaluated in a larger clinical trial.     

The ability of bacterial DNA methyltransferases to use methylcobalamine as a cofactor in DNA methylation reactions

The ability of bacterial DNA methyltransferases Alu I, Cfr I, Cfr 6, Cfr 10, Eco RI, Eco RII, Msp I, Mva I, Pvu I, Pvu II, and Sau 3A to use methylcobalamine and methylmethionine as cofactors of DNA methylation in vitro has been investigated. These bacterial DNA methyltransferases used methylcobalamine, but not methylmethionine for DNA methylation.     

Down-regulation and inactivation of neutral endopeptidase 24.11 (enkephalinase) in human neutrophils

Neutral endopeptidase (EC 3.4.24.11, NEP) is an integral membrane protein of human neutrophils. NEP is identical with the common acute lymphoblastic leukemia antigen (CALLA) of leukemic cells. The expression of NEP on the surface of neutrophils is down-regulated by endocytosis which can be induced by phorbol 12-myristate 13-acetate (PMA) at 37 degrees C. The activity of the enzyme on the surface of intact cells decreases by 76% within 5 min. The activity can be recovered, however, if the cells are lysed within 5 min of the endocytosis. After 30 min, only 32% of the NEP activity is present in the neutrophil lysates. The loss of activity is presumably due to proteolytic inactivation. Diacylglycerol and monoclonal antibody to CALLA/NEP also induce internalization of NEP. PMA induces endocytosis even at 4 degrees C, but NEP is not inactivated at that temperature. The disappearance of NEP activity after adding PMA was inhibited by various agents. Among the most active were the phospholipase inhibitor 4-bromophenacyl bromide and a combination of the serine protease and cathepsin inhibitors, diisopropylfluorophosphate and N-ethylmaleimide. The employment of fluorescent monoclonal antibody confirmed the down-regulation and internalization of NEP antigen on the neutrophils. Since NEP inactivates chemotactic peptides and thereby affects chemotaxis of neutrophils (Painter, R. G., Dukes, R., Sullivan, J., Carter, R., Erd?s, E. G., and Johnson, A. R. (1988) J. Biol. Chem. 263, 9456-9461), the down-regulation of NEP activity on the cell membrane may modulate the function of these cells in inflammation.     

Neutral endopeptidase 24.11 and angiotensin converting enzyme like activity in CALLA positive and CALLA negative lymphoid cells

Taking advantage of the recently demonstrated identity of common acute lymphoblastic leukemia antigen (CALLA) and neutral endopeptidase EC.24.11 (NEP) the presence of this ectoenzyme on lymphoid cells has been reassessed using highly sensitive assays (cleavage of [3H]-D-Ala2-leucine-enkephalin and binding of the inhibitor [3H]HACBO-Gly. NEP activity was found not only on already classified CALLA + ve cells but also on numerous cells (including mature B and polyclonal T cells) previously considered as CALLA-ve. This suggests that CALLA/NEP is expressed all along the differentiation pathway in B and T cell lineage. Moreover substantial ACE-like activity was also detected in three tested cells, all of the pre-B phenotype. The availability of specific inhibitors for these enzymes should help clarify their role in cell-differentiation.     

Stromal endothelial cells establish a bidirectional crosstalk with chronic lymphocytic leukemia cells through the TNF-related factors BAFF, APRIL, and CD40L

Chronic lymphocytic leukemia (CLL) is a clonal B cell disorder of unknown origin. Accessory signals from the microenvironment are critical for the survival, expansion, and progression of malignant B cells. We found that the CLL stroma included microvascular endothelial cells (MVECs) expressing BAFF and APRIL, two TNF family members related to the T cell-associated B cell-stimulating molecule CD40L. Constitutive release of soluble BAFF and APRIL increased upon engagement of CD40 on MVECs by CD40L aberrantly expressed on CLL cells. In addition to enhancing MVEC expression of CD40, leukemic CD40L induced cleavases that elicited intracellular processing of pro-BAFF and pro-APRIL proteins in MVECs. The resulting soluble BAFF and APRIL proteins delivered survival, activation, Ig gene remodeling, and differentiation signals by stimulating CLL cells through TACI, BAFF-R, and BCMA receptors. BAFF and APRIL further amplified CLL cell survival by upregulating the expression of leukemic CD40L. Inhibition of TACI, BCMA, and BAFF-R expression on CLL cells; abrogation of CD40 expression in MVECs; or suppression of BAFF and APRIL cleavases in MVECs reduced the survival and diversification of malignant B cells. These data indicate that BAFF, APRIL, and CD40L form a CLL-enhancing bidirectional signaling network linking neoplastic B cells with the microvascular stroma.     

Ribosomal DNA restriction analysis and synthetic oligonucleotide probing in the identification of genera of lower trypanosomatids.

Fifty-four species or isolates of insect trypanosomatids were examined for the presence of selected restriction enzyme sites in the small (SSU) and large (LSU) rRNA coding units of ribosomal genes. In the SSU, sites for Eco RI, Bgl II, Pst I, and Hind III were found to occur at the same location for all species examined, thus displaying a universal distribution among trypanosomatids. In the LSU, a site for Bgl II in the 24S-alpha sequence and sites for Hind III and Pst I in the 24S-beta sequence were found in all species examined. In contrast, a site for Pvu II in the SSU exhibited a genus-related distribution, being present in Crithidia and Herpetomonas but absent in Phytomonas. A site for Hind III in the 24S-alpha sequence of the LSU also exhibited genus-restricted distribution. The site was present in Crithidia but absent in Phytomonas and Herpetomonas. These findings were confirmed by dot hybridization with a synthetic oligonucleotide complementary to the 18S rRNA sequence containing the Pvu II site. Results point to the usefulness of restriction markers as diagnostic tools for distinguishing the lower trypanosomatid genera Crithidia, Herpetomonas, and Phytomonas at the same time revealing a marked complexity within the genus Leptomonas.     

Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor.

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.     

Two types of triplicated alpha-globin loci in humans.

DNA from healthy Malaysian newborns was studied on gene maps after digestion with different restriction endonucleases. Of 65 newborns, two were found to be carriers of two different variants of triplicated alpha-globin loci. In variant no. 1, found in an Malay, the three alpha-globin genes are in an elongated DNA fragment on digestion with Eco RI and Bam HI. The third alpha-globin gene was found in a additional 3.7-kb fragment on digestion with Hpa I, Bgl II and Hind III. In variant no. 2, a new type of triplicated alpha-globin loci, found in a Chinese, the three alpha-globin genes reside in an elongated DNA fragment longer than that of variant no. 1 on digestion with Eco RI and Bam HI. The third alpha-globin gene was found in an additional 4.2-kb fragment on digestion with Hpa I and Hind III. Digestion of this variant DNA with Bg1 II produced an abnormal 16.7-kb fragment in addition to the normal 7.0-kb Bgl-II fragment. The locations of the restriction sites in the two types of triplicated alpha-globin loci are compatible with a mechanism of unequal crossing over following two different modes of misalignment.     

CD200: a putative therapeutic target in cancer

CD200 was recently described as a new prognosis factor in multiple myeloma and acute myeloid leukemia. CD200 is a membrane glycoprotein that imparts an immunoregulatory signal through CD200R, leading to the suppression of T-cell-mediated immune responses. We investigated the expression of CD200 in cancer using publicly available gene expression data. CD200 gene expression in normal or malignant human tissues or cell lines was obtained from the Oncomine Cancer Microarray database, Amazonia database and the ITTACA database. We found significant overexpression of CD200 in renal carcinoma, head and neck carcinoma, testicular cancer, malignant mesothelioma, colon carcinoma, MGUS/smoldering myeloma, and in chronic lymphocytic leukemia compared to their normal cells or their tissue counterparts. Moreover, we show that CD200 expression is associated with tumor progression in various cancers. Taken together, these data suggest that CD200 is a potential therapeutic target and prognostic factor for a large array of malignancies.     

Molecular and biological diversity of HIV-1 in Brazil.

To determine the genomic polymorphism and biological properties present in HIV-1 Brazilian isolates, we analyzed five viral isolates obtained from patients residing in Rio de Janeiro (P1 and P5), S?o Paulo (P3) and Bahia (P2 and P4) states. For each viral isolate in vitro characteristics such as replication rate, syncytium-inducing capacity and cell death were observed in lymphoblastoid (H9, CEM and peripheral blood mononuclear cells) as well as monocytoid (U937) cells. In addition, the evaluation of the restriction fragment length polymorphism of these isolates was also performed using a panel of endonucleases such as Hind III, Bgl II, Sac I, Pst I, Kpn I and Eco RI. One of the isolates (P1), showed the highest phenotypic and genotypic divergence, when compared to others. The results found suggest a HIV heterogeneity in Brazil similar to that already described in other regions of the world.     

Quantification of the leukocyte common antigen (CD45) in mature B-cell malignancies.

CD45 is a glycoprotein expressed on all lymphohematopoietic cells. Its expression increases during normal B-cell differentiation and remains stable on mature cells. Although it is widely known that CD45 antigen expression is decreased in B-acute lymphocytic leukemia (ALL), only scarce and contradictory information is available on CD45 expression on mature B-cell malignancies. In healthy adults (n = 15), CD45 expression on B lymphocytes was lower than that on T cells. In patients with chronic lymphocytic leukemia (CLL; n = 22), CD45 expression on malignant cells was lower than that on the whole lymphocyte population of healthy adults (n = 28) and on normal B lymphocytes (n = 15). In 6 of the 22 CLL patients, the malignant cell population could be separated from the normal lymphocyte population on the CD45-side scatter (SSC) plot. In 16 CLL patients, there was some degree of overlap between the malignant and normal cells with respect to CD45 expression. For these patients, there was an inverse correlation between CD45 expression on the whole lymphocyte population and the percentage of malignant cells in this population. In two patients with mantle cell lymphoma (MCL), CD45 expression on the malignant cells appeared lower than that on normal B cells and on the whole lymphocyte population. In six patients with hairy cell leukemia (HCL), CD45 expression on hairy cells was comparable to that on the whole lymphocyte population of healthy adults, but slightly higher than that of normal B cells. Evaluation of CD45 expression may help to characterize mature B-cell malignancies.     

Primitive AML progenitors from most CD34(+) patients lack CD33 expression but progenitors from many CD34(-) AML patients express CD33

BACKGROUND: AML blast populations are heterogeneous in their phenotype and functional properties, and contain a small subset of cells that regenerate leukemia in immunocompromised mice or produce clonogenic progeny in long-term cultures. This suggests the existence of a hierarchy of AML progenitor cells. CD33 is a myeloid marker absent on normal hematopoietic stem cells but expressed in about 75% of AML patients, and has been used for BM purging strategies and Ab-targeted therapies. These CD33 Ab therapies benefit only a minority of AML patients, suggesting that AML stem cells are heterogeneous in their CD33 expression. METHODS: In order to evaluate this question, we determined expression levels of CD34 and CD33 on AML progenitors with long-term in vitro proliferative ability and NOD/SCID engrafting ability. RESULTS: The CD34(+) CD33(-) subfraction contained the majority of progenitors detected in vitro and most often engrafted the mice. This proliferation was leukemic from the CD34(+) AML patients, however from the CD34(-) AML patients only normal progenitors were detected in this fraction in some cases. DISCUSSION: These data suggest that most leukemic progenitors of CD34(+) patients do not express CD33. In contrast, CD34(-) AML primitive leukemic progenitors may be CD33(+). CD34(-) AML patients could potentially benefit most from CD33-targeted therapies or purging.     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

MYCN-related suppression of functional CD44 expression enhances tumorigenic properties of human neuroblastoma cells

Highly malignant neuroblastoma tumors with MYCN amplification have been shown to downregulate the expression of the CD44 adhesion receptor. We have previously shown that MYCN amplified neuroblastoma cell lines either lack CD44 expression or express a nonfunctional, nonhyaluronic acid-binding CD44 receptor. By analysis of cells with manipulated expression of either CD44 or MYCN, we demonstrate that transfection of cells with a CD44 full-length cDNA construct produced a functional receptor in single copy MYCN cells and a nonfunctional CD44 receptor in MYCN amplified cells, similar to the CD44 receptor expressed by cells with enforced MYCN. Analysis of the in vivo growth properties of the transfectants revealed that the restoration of a functional CD44 receptor in nonamplified cells resulted in the suppression of in vivo cell growth, therefore linking the MYCN-related lack of hyaluronic acid-binding function of CD44 to the highly tumorigenic properties of a subset of neuroblastoma cells.     

Restriction endonuclease analysis of human globin genes in cellular DNA

Using samples of human cellular DNA digested with restriction endonucleases Eco RI, Hind III, Hinc II, Bam HI, Alu I, or Hae III, we were able to localize globin gene fragments separated by agarose gel electrophoresis. The fragments were transferred to nitro-cellulose filters and identified by hybridization to [³²P] cDNA for total adult globin mRNA. The α-globin gene fragments were specifically identified by their presence in normal controls and absence in DNA from homozygous α-thalassemia, a genetic disorder due to deletion of α-globin genes. In addition, the patterns with Hind III indicate a 4.1 kb distance between the centers of the normal duplicated α-globin gene loci.