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J Sklar

Publications and source records attributed to J Sklar.

At least 19 recordsLinked to original sources
PubMed2000

Intracellular forms of human NOTCH1 interact at distinctly different levels with RBP-jkappa in human B and T cells.

The cellular transcriptional repressor RBP-Jkappa associates with the Epstein-Barr virus nuclear antigens (EBNAs) determined to be essential for transformation of human primary B lymphocytes. It was demonstrated through genetic analysis that interaction between the viral transactivator EBNA2 and RBP-Jkappa is essential for EBV immortalization of primary B lymphocytes. We have shown that the association of RBP-Jkappa with intracellular NOTCH1 differs significantly in B and T cells. Immunoprecipitation analyses with antibodies to both the intracellular forms of NOTCH1 and to RBP-Jkappa demonstrated that little or no RBP-Jkappa is associated with NOTCH1 in B cell lines compared to the RBP-Jkappa associated with NOTCH1 in T cell lines and was further demonstrated in human primary lymphocytes. Additionally, EBNA2 can compete with intracellular NOTCH1 for binding to GST-RBP-Jkappa in vitro. Northern blot for the cellular gene hairy enhancer of split (HES1) demonstrated that HES1 is upregulated in the EBV transformed lymphoblastoid cells expressing high levels of EBNA2 and in a T cell line SupT1 overexpressing intracellular activated NOTCH1. Hence, EBNA2 may be able to compete for the available pool of RBP-Jkappa more effectively in human B cells than in T cells and provides a possible explanation for the ability of EBV to potently and efficiently infect and immortalize human B cells. Leukemia (2000) 14, 84-92.

B-Lymphocytes↗
PubMed2000

Postoperative analgesia for outpatient arthroscopic knee surgery with intraarticular clonidine and/or morphine.

Both clonidine, an alpha(2) agonist, and morphine, an opioid agonist, provide enhanced patient analgesia after arthroscopic knee surgery when administered via the intraarticular (IA) route. Clonidine potentiates morphine analgesia in the animal model. We designed this study to determine whether clonidine or morphine results in better analgesia and whether their combination would provide superior analgesia to either drug alone. We evaluated 60 patients undergoing arthroscopic knee meniscus repair under local anesthesia with sedation. After surgery, patients were randomized into four IA groups: Group B received 30 mL 0.25% bupivacaine; Group BC received 30 mL 0.25% bupivacaine and clonidine 1 microg/kg; Group BM received 30 mL 0.25% bupivacaine and morphine 3 mg; and Group BCM received 30 mL 0.25% bupivacaine, clonidine 1 microg/kg, and morphine 3 mg. This study revealed a significant benefit from the individual IA administration of both clonidine and morphine. The combination of these drugs resulted in decreased postoperative pain and analgesic use, as well as an increased analgesic duration compared with either drug alone. We conclude that IA clonidine and morphine improved comfort compared with either drug alone in patients undergoing knee arthroscopy.

Adult↗
PubMed2000

Calcium depletion dissociates and activates heterodimeric notch receptors.

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.

3T3 Cells↗
PubMed1999

Breakpoints of the t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma lie within or near the previously undescribed gene MALT1 in chromosome 18.

Lymphomas arising in mucosa-associated lymphoid tissue (MALT) are indolent B-cell tumors that have a predilection for epithelial sites and often develop in a setting of chronic inflammation or autoimmunity. As many as 50% of low-grade MALT lymphomas contain an (11;18)(q21; q21) chromosomal translocation. Using fluorescence in situ hybridization, we have analyzed the position of recombination within chromosome 18 DNA in three examples of MALT lymphoma bearing this translocation. In all three cases, the breakpoint maps to DNA in BAC b357H2, covering about 150 kb of sequence. A previously undescribed, ubiquitously expressed gene, which we refer to as MALT1, was identified within this sequence and was found to be broken in one case for which we have definitively located the position of recombination between chromosomes 18 and 11. The sequence of this gene indicates the presence of two immunoglobulin-like C2 domains and a region of partial homology to caspases, suggesting a possible role for MALT1 in the regulation of apoptosis.

Amino Acid Sequence↗
PubMed1999

Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics.

Hematopoiesis is a balance between proliferation and differentiation that may be modulated by environmental signals. Notch receptors and their ligands are highly conserved during evolution and have been shown to regulate cell fate decisions in multiple developmental systems. To assess whether Notch1 signaling may regulate human hematopoiesis to maintain cells in an immature state, we transduced a vesicular stomatitis virus G-protein (VSV-G) pseudo-typed bicistronic murine stem cell virus (MSCV)-based retroviral vector expressing a constitutively active form of Notch1 (ICN) and green fluorescence protein into the differentiation competent HL-60 cell line and primary cord blood-derived CD34(+) cells. In addition, we observed endogenous Notch1 expression on the surface of both HL-60 cells and primary CD34(+) cells, and therefore exposed cells to Notch ligand Jagged2, expressed on NIH3T3 cells. Both ligand-independent and ligand-dependent activation of Notch resulted in delayed acquisition of differentiation markers by HL-60 cells and cord blood CD34(+) cells. In addition, primary CD34(+) cells retained their ability to form immature colonies, colony-forming unit-mix (CFU-mix), whereas control cells lost this capacity. Activation of Notch1 correlated with a decrease in the fraction of HL-60 cells that were in G0/G1 phase before acquisition of a mature cell phenotype. This enhanced progression through G1 was noted despite preservation of the proliferative rate of the cells and the overall length of the cell cycle. These findings show that Notch1 activation delays human hematopoietic differentiation and suggest a link of Notch differentiation effects with altered cell cycle kinetics.

3T3 Cells↗
PubMed1998

Safety and efficacy of closed-loop arbutamine stress echocardiography for detection of coronary artery disease. International Arbutamine Study Group.

Closed-loop arbutamine stress echocardiography has been shown to be safe and effective for detecting coronary artery disease (CAD) using a standardized infusion protocol and centralized core laboratory analyses. However, the accuracy of arbutamine stress echocardiography using local test interpretation has not been previously tested in a large population. The present study reports the safety, sensitivity, and specificity of arbutamine stress echocardiography in a multicenter trial allowing user-defined, nonstandardized protocols and local test interpretation. In all, 1,070 patients underwent arbutamine stress testing at 81 sites. Heart rate increased from 73 +/- 13 to 124 +/- 15 beats/min, systolic blood pressure from 144 +/- 24 to 174 +/- 25 mm Hg, and pressure rate product x 10(3) from 10.5 +/- 2.8 to 19.6 +/- 3.9. Among 1,070 patients, there were only 2 (0.2%) significant adverse events related to arbutamine, both of which resolved completely with appropriate therapy. There were no incidents of ventricular fibrillation, sustained ventricular tachycardia, or death related to testing. Among 242 patients who underwent arbutamine stress echocardiography and diagnostic coronary angiography within 12 weeks, sensitivity and specificity for detection of CAD were 71% (95% confidence interval 64% to 77%) and 67% (95% confidence interval 52% to 80%), respectively. Closed-loop arbutamine stress echocardiography is a safe and effective method for evaluating CAD in clinical practice.

Adult↗
PubMed1998

Psychosis.

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Adult↗
PubMed1997

Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

Amino Acid Sequence↗
PubMed1997

Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription.

Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (DeltaE) or consisting only of the intracellular domain (ICN) were found to activate transcription in cultured cells, presumably through RBP-Jkappa response elements within DNA. Both truncated forms also bound to the transcription factor RBP-Jkappa in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation required the presence of a weak RBP-Jkappa-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jkappa-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jkappa, was not needed for oncogenesis or for transcriptional activation. While ICN appeared primarily in the nucleus, DeltaE localized to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreased nuclear localization and increased transcriptional activation by membrane-bound DeltaE. Transcriptional activation by this mutant form of membrane-bound DeltaE was approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jkappa at promoter sites.

Amino Acid Sequence↗
PubMed1997

A simplified polymerase chain reaction assay for detection of chromosomal translocations in hematologic malignancies.

The polymerase chain reaction (PCR) is a rapid and highly sensitive method for detection of a variety of chromosomal translocations in malignant tissues. Detection of each different type of translocation, or even DNA rearrangements at different breakpoint cluster regions within the same type of translocation, usually requires separate thermocycling parameters and/or buffer conditions. In this report, we describe a single set of reaction conditions, making use of progressively decreasing annealing temperatures and a standardized reaction buffer, that permits the detection of several different translocations simultaneously. Specificity equal to or better than current procedures and sensitivity equivalent to one malignant cell in 1 x 10(5) normal cells was achieved for translocations t(14;18)(q32;q21), t(9;22)(q34;q11), and t(4;11)(q21;q23). For PCRs formerly requiring different, fixed annealing temperatures, the new technology allows batching or multiplexing of PCR samples. Thus, shorter turnaround time, decreased cost per sample, and simplified mechanization of PCR may be attainable using this assay.

Hematologic Neoplasms↗
PubMed1997

Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor.

Signaling through Notch receptors has been implicated in the control of cellular differentiation in animals ranging from nematodes to humans. Starting from a human expressed sequence tag-containing sequence resembling that of Serrate, the gene for a ligand of Drosophila melanogaster Notch, we assembled a full-length cDNA, now called human Jagged2, from overlapping cDNA clones. The full-length cDNA encodes a polypeptide having extensive sequence homology to Serrate (40.6% identity and 58.7% similarity) and even greater homology to several putative mammalian Notch ligands that have subsequently been described. When in situ hybridization was performed, expression of the murine Jagged2 homolog was found to be highest in fetal thymus, epidermis, foregut, dorsal root ganglia, and inner ear. In Northern blot analysis of RNA from tissues of 2-week-old mice, the 5.0-kb Jagged2 transcript was most abundant in heart, lung, thymus, skeletal muscle, brain, and testis. Immunohistochemistry revealed coexpression of Jagged2 and Notch1 within thymus and other fetal murine tissues, consistent with interaction of the two proteins in vivo. Coculture of fibroblasts expressing human Jagged2 with murine C2C12 myoblasts inhibited myogenic differentiation, accompanied by increased Notch1 and the appearance of a novel 115-kDa Notch1 fragment. Exposure of C2C12 cells to Jagged2 led to increased amounts of Notch mRNA as well as mRNAs for a second Notch receptor, Notch3, and a second Notch ligand, Jagged1. Constitutively active forms of Notchl in C2C12 cells also induced increased levels of the same set of mRNAs, suggesting positive feedback control of these genes initiated by binding of Jagged2 to Notch1. This feedback control may function in vivo to coordinate differentiation across certain groups of progenitor cells adopting identical cell fates.

Amino Acid Sequence↗
PubMed1997

Detection of circulating tumor cells in patients with Ewing's sarcoma and peripheral primitive neuroectodermal tumor.

PURPOSE: To determine the feasibility of detecting Ewing's sarcoma (ES) or peripheral primitive neuroectodermal tumor (PNET) through a reverse-transcriptase polymerase chain reaction (RT-PCR) of the t(11;22)(q24;q12) fusion transcript in blood and bone marrow samples from patients with these neoplasms. PATIENTS AND METHODS: Peripheral-blood (PB) and/or bone marrow aspirate (BM) samples were obtained from 28 patients with ES or PNET at initial presentation or at relapse. Patients were divided into two groups: newly diagnosed patients with nonmetastatic disease and those with metastatic/relapsed disease. RNA was extracted from fractionated BM and PB samples, and RT-PCR was performed for the EWS/HumFLI1 fusion mRNA was transcribed across the t(11;22) breakpoint. RESULTS: Among the 16 patients with nonmetastatic disease, three of 16 were RT-PCR positive for EWS/HumFLI1 RNA in BM and three of 10 were positive in PB. The total number of nonmetastatic patients who were positive in either PB or BM was four of 16 (25%). Among patients with metastatic/relapsed disease, two of six were positive in BM and five of 10 were positive in PB. The total fraction of patients with metastatic/relapsed disease that was positive in either BM or PB was six of 12 (50%). CONCLUSION: In this study, we show that it is possible to amplify the EWS/HumFLI1 RNA by RT-PCR from the BM and PB of a subset of patients with both nonmetastatic and metastatic ES or PNET, which implies that occult tumor cells are present at these sites. The true biologic and clinical meaning of this information is unknown. However, it does suggest a possible application of RT-PCR for the monitoring of residual disease in patients who are undergoing therapy for ES or PNET. This approach may permit early identification of patients who may benefit from alternative therapy or who may be spared possible overtreatment.

Chromosomes, Human, Pair 11↗
PubMed1996

Modulated expression of notch1 during thymocyte development.

The Notch gene family encodes transmembrane proteins that have been implicated in control of diverse cellular differentiation events in the fly, frog, and mouse. Mammalian Notch1 is expressed at high levels in thymus and is mutated in a subset of human T-cell acute lymphoblastic neoplasms, suggesting a role in T-cell differentiation. To investigate the patterns of expression of NOTCH1 protein in thymocytes of the developing and mature thymus, antibodies raised against NOTCH1 were used to perform immunohistochemical and flow cytometric analyses. Strong staining for NOTCH1 within the fetal murine thymus was observed as early as 13.5 days postcoitum. By 17.5 days postcoitum, preferential staining of superficial cortical thymocytes was observed, with weak staining of developing medulla. Flow cytometric analysis and immunohistochemical staining of flow-sorted cells confirmed that the highest levels of NOTCH1 expression in adult murine thymus were present in immature cortical thymocytes (CD24high, CD4-CD8-). In contrast, NOTCH1 expression was low or absent in more mature cortical thymocytes (CD24low, CD4+CD8+), whereas intermediate levels of expression were observed in CD4+CD8- and CD4-CD8+ cells. These data indicate a dynamic pattern of NOTCH1 expression during T-cell differentiation and suggest that downregulation of NOTCH1 may be required for maturation of cortical thymocytes.

Animals↗
PubMed1996

Lymphocytic progenitor cell origin and clonal evolution of human B-lineage acute lymphoblastic leukemia.

At presentation, bone marrow specimens from over 25% of B-lineage acute lymphoblastic leukemias (ALL) display more than two clonal rearrangements of immunoglobulin heavy chain (IgH) genes in Southern blot analyses. Nucleotide sequence analysis has shown predominantly different V(H)DJ(H) junctions among these genes, leading to the frequent description of such cases as oligoclonal leukemias. In the present study, we have analyzed the lgH genes from four patients whose leukemic cells contained different patterns of lgH gene rearrangements between presentation and relapse. Nucleotide sequence analysis of the lgH genes showed that three mechanisms could account for these differences: de novo V(H)DJ(H) rearrangement, V(H) to DJ(H) recombination, and V(H) replacement. In all cases, more than two totally different V(H)DJ(H) rearrangements appeared during evolution of the disease, formally consistent with the conclusion that these tumors were composed of apparently unrelated clones. However, the retention of some of the antigen receptor gene rearrangements, as well as the persistence of a chromosomal marker in two cases, indicated that these leukemias had a monoclonal origin. These findings support the hypothesis that some ALLs arise from a lymphoid progenitor cell at a stage of lymphocyte development before the onset of IgH gene rearrangement. These leukemic lymphocyte progenitors generate malignant daughter cells capable of an in vivo maturation that involves the completion of multiple different lgH rearrangements as well as the modification of preexisting rearrangements by V(H) to DJ(H) recombination or by a V(H) replacement.

Base Sequence↗
PubMed1996

Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles.

Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

Animals↗
PubMed1996

Loss of heterozygosity at chromosome regions 22q11-12 and 11p15.5 in renal rhabdoid tumors.

Rhabdoid tumors of the kidney are highly malignant neoplasms that occur primarily within the first 3 years of life. Although they are regarded as distinct from Wilms' tumors, their pathogenesis remains unclear. Whereas most cytogenetic studies of these tumors have revealed normal karyotypes, a few reports have indicated abnormalities at chromosome regions 22q and 11p15.5. We analyzed 30 primary renal rhabdoid tumors for loss of heterozygosity (LOH) at both regions and found that 24 of 30 tumors (80%) had LOH at chromosome arm 22q and that 5 of 30 (17%) had LOH at chromosome band 11p15.5. All of the five tumors with LOH at chromosome arm 11p also had LOH at chromosome arm 22q. The data suggest that there is a gene in chromosome 22, probably a tumor suppressor, inactivation of which may be involved in the genesis of renal rhabdoid tumors. A second gene in chromosome segment 11p15.5, in the region of the putative WT2 gene, may also be involved, in at least a subset of rhabdoid tumors. In addition, five tumors were characterized by microsatellite instability at three or more of 21 loci examined, suggesting a possible role for a replicative error in tumorigenesis or progression in some cases of renal rhabdoid tumors. Genes Chromosom Cancer 15:10-17 (1996).

Chromosome Deletion↗
PubMed1996

Hemophagocytosis as a para-neoplastic syndrome in NK cell leukemia.

Hemophagocytic syndrome is a proliferative disorder of an activated monocyte-macrophage system and is characterized by fever, hepato-splenomegaly and pancytopenia. The serum level of interferon-gamma in the syndrome is increased but its origin is unknown. Here we describe a case of NK cell leukemia with hemophagocytic syndrome with elevated serum level of interferon-gamma. The levels of various cytokines were monitored during the course and statistic analysis was performed. To identify the clonal component, the NK cell fraction was sorted from the mononuclear layer and was subjected to Southern blot hybridization with a probe for EB virus tandem repeats. The fraction was also stimulated with interleukin-2 and the level of interferon-gamma in the conditioned medium was measured. Levels of M-CSF and interferon-gamma were significantly correlated with the degree of clinical manifestations and laboratory data. Southern blot hybridization revealed monoclonality of an NK cell fraction. The fraction also released interferon-gamma. Since macrophage can be activated through cytokines, the hemophagocytosis might have been triggered by factor(s) released from the abnormal NK cell clone at least in this case.

Adolescent↗