Expression of different members of the Ly-49 gene family defines distinct natural killer cell subsets and cell adhesion properties.

The murine Ly-49 antigen belongs to a family of type II transmembrane molecules containing lectin-like domains. The original member of this family, Ly-49A, has been demonstrated to be expressed by a subpopulation of natural killer (NK) cells, bind certain class I major histocompatibility complexes (MHC), and act as a negative regulator of lytic activity. The expression patterns and functional activities of the other Ly-49s, however, is unknown. We extended the study of this family by isolating cDNAs encoding two new Ly-49 molecules. The reactivity of these and previously identified Ly-49 molecules with NK antibodies was tested in a COS cell expression system. YE1/32 and YE1/48 bound Ly-49A specifically, and 5E6 reacted only with Ly-49C. Three-color flow cytometric analysis demonstrated Ly-49A and Ly-49C expression defines complex, but distinct subsets within NK1.1+ cells. Some NK1.1-CD3+ as well as NK1.1-CD3- cells expressing Ly-49A or C were also detected. Analysis of MHC congenic strains of mice demonstrated that YE1/32+ and YE1/48+ NK cells are not deleted, as has been shown with the Ly-49A mAb A1. Furthermore, COS cells transfected with Ly-49A bound H-2d and H-2k cell lines, whereas Ly-49C transfectants bound H-2d, H-2k, H-2b, and H-2s. The antibodies 5E6 and 34-1-2S (anti-class I MHC) inhibited the binding of Ly-49C to an H-2s cell line. These results imply that the NK cell antigens Ly-49A and C bind to different repertoires of class I MHC molecules.

Defective development of thymocytes overexpressing the costimulatory molecule, heat-stable antigen.

Heat-stable antigen (HSA) is a small, glycosyl phosphatidylinositol-anchored protein that can act as a costimulatory molecule for antigen-dependent activation of helper T cells. In addition to being expressed on antigen-presenting B cells, HSA is also expressed during the initial stages of T cell development in the thymus. HSA levels are very high on immature CD4-, CD8- double negative thymocytes, but are reduced on CD4+, CD8+ double positive cells undergoing selection in the thymus, and are entirely eliminated when these cells differentiate into immunologically competent CD4+ or CD8+ single positive T cells. To examine the potential roles of this molecule in T cell development and selection, we generated transgenic mice in which HSA was highly expressed on all classes of thymocytes. The consequence of deregulated HSA expression was a pronounced reduction in the numbers of double positive and single positive thymocytes, whereas the numbers of their double negative precursors were largely unaffected. These results demonstrate that downregulation of HSA expression at the double positive stage is a critical event in thymocyte development. The depletion of thymocytes resulting from HSA overexpression begins at the same time as the onset of negative selection, suggesting that HSA may provide signals that contribute to determining the efficiency of this process.

Cross-linking the murine heat-stable antigen induces apoptosis in B cell precursors and suppresses the anti-CD40-induced proliferation of mature resting B lymphocytes.

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes, the co-stimulation of CD4+ T cells, and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells, but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together, these data implicate HSA as a potent negative regulator of B cell development and activation.

Heterogeneity among Ly-49C natural killer (NK) cells: characterization of highly related receptors with differing functions and expression patterns.

Ly-49C is a member of the polymorphic family of murine NK cell inhibitory receptors. The 5E6 antibody that defines a subset of NK cells responsible for the rejection of parental H-2d bone marrow by F1 mice has been shown previously to react with Ly-49C. Here, the 5E6 antibody was found to detect two Ly-49C-related molecules in B6 mice. Two cDNA clones were isolated from B6 NK cells, one identical to previously reported Ly-49CB6 and the other a novel cDNA. The deduced amino acid sequence of the latter differs from that of Ly-49CBALB at only 4 residues, whereas the previously reported Ly-49CB6 differs at 22 residues. Flow cytometric analyses of COS cells transfected with the two cDNAs showed that the 5E6 antibody binds to both Ly-49 molecules, while another anti-Ly-49C antibody, 4LO3311, binds to the newly described Ly-49C but not the previously reported Ly-49CB6. Two-color flow cytometric analysis detected 5E6+4LO3311- as well as 5E6+4LO3311+ subsets of NK cells from B6, but not BALB/c, mice. The level of Ly-49C expression on B6 NK cells detected by the 4LO3311 antibody was substantially lower than that on BALB/c NK cells. Binding specificity of the novel Ly-49CB6 was indistinguishable from that of Ly-49CBALB, whereas no binding was detectable with previously reported Ly-49CB6. These results demonstrate that the newly described Ly-49CB6, not the previously reported Ly-49CB6, is the probable B6 allelic form of Ly-49C. The previously reported Ly-49CB6 must be encoded by a separate gene and should be renamed Ly-49I. The implication of these results with respect to the role of Ly-49C in hybrid resistance is discussed.

Recognition of class I major histocompatibility complex molecules by Ly-49: specificities and domain interactions.

Ly-49 is a family type II transmembrane proteins encoded by a gene cluster on murine chromosome 6. One member of this family, Ly-49A, is expressed by a natural killer (NK) cell subset, binds to class I major histocompatibility complex (MHC) molecules, and blocks the killing of target cells bearing the appropriate H-2 antigens. Here we show that another member of this family which is expressed by an NK cell subset, Ly-49C, recognizes H-2b and H-2d structures which are distinct from and overlapping with those recognized by Ly-49A. Interactions between Ly-49A and C and their class I ligands are entirely blocked by the antibodies 5E6, YE1/48, YE1/32, and A1, all of which were found to recognize epitopes contained within the carbohydrate recognition domain (CRD). However, cell-cell binding assays revealed that class I binding specificity is conferred by a combination of sequences within both the CRD and a 19-amino acid adjacent region. We also investigated the question of whether Ly-49A and C form dimers on cells which express both receptors. When coexpressed on COS cells, sequential immunoprecipitation demonstrated that these receptors pair exclusively as homodimers, with no evidence for heterodimeric structures. These observations provide insight into both the biochemical nature of the Ly-49 family as well as the receptor functions of Ly-49C on NK cells.

Further evidence for importance of the subunit combination of silk fibroin in its efficient secretion from the posterior silk gland cells.

A locus responsible for the Nd-s mutation of the silkworm, Bombyx mori, has been mapped very close to or within the fibroin light (L) chain gene on the 14th chromosome (Takei, F., K. Kimura, S. Mizuno, T. Yamamoto, and K. Shimura, 1984, Jpn. J. Genet., 59:307-313). A strain of B. mori carrying the homozygous Nd-sD mutation (Nd-sD/Nd-sD; Nd-sD is allelic to Nd-s) secretes less than 0.3% of fibroin into the lumen of the posterior silk gland compared with a strain carrying the homozygous wild-type alleles (+/+). The small amount of fibroin that is secreted in the Nd-sD/Nd-sD strain consists of the heavy (H) chain only and lacks the L chain, although the L chain mRNA and the proteins that are cross-reactable with the anti-L chain serum are present in the posterior silk gland cells. In the hybrid silkworm, Nd-sD/+, the H chain derived from either the Nd-sD or + allele forms disulfide linkage with the L chain derived from the + allele and these fibroins are secreted into the lumen with an equal efficiency, but the L chain derived from the Nd-sD allele remains in the cell unbound to the H chain. Some evidence suggesting structural abnormality of the L chain derived from the Nd-sD allele is presented. These results, together with the previous results on the effect of the H chain gene-linked Nd(2) mutation (Takei, F., F. Oyama, K. Kimura, A. Hyodo, S. Mizuno, and K. Shimura, 1984, J. Cell Biol., 99:2005-2010), strongly suggest that the H-L subunit combination of silk fibroin is important for its efficient secretion.

Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd(2).

Fibroin is normally composed of one H chain (350 kd) and one L chain (25 kd) which are connected by disulfide bond(s). However, the small amount of fibroin secreted into the lumen of the posterior silk gland of the Nd(2) (naked pupa) mutant does not contain L chain, although L chain mRNA is present and L chain is synthesized in the posterior silk gland cells of the mutant. In a hybrid silkworm, Nd(2)/Tamanashikasuri, where Tamanashikasuri is a normal producer of fibroin, L chain from the two alleles are distinguishable electrophoretically. It is demonstrated using this system that the L chain from the Nd(2) allele can combine normally with the H chain from Tamanashikasuri and the H-L complex is secreted normally. In another hybrid system, Nd(2)/J-131, where J-131 is a normal producer of fibroin, fibroin derived from the two alleles are distinguishable due to the different electrophoretic mobility of H chain. The fibroin derived from the J-131 allele is composed of H chain and L chain, while the fibroin derived from the Nd(2) allele is devoid of L chain, and its secretion is greatly reduced. We present evidence suggesting that the H chain derived from the Nd(2) allele is structurally abnormal and discuss how the H-L subunit structure is advantageous in the secretion of fibroin.

Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice.

This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM-1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/1 (75%). No inhibition was observed in the ICAM-1 mutant compared to wild-type mice. These data show that antisense oligonucleotides targeted to ICAM-1 inhibit the endotoxin-induced upregulation of ICAM-1 in the lung and are as effective as anti-ICAM-1 antibodies in preventing neutrophil emigration. The incomplete inhibition by either antisense oligonucleotides or antibodies suggests that alternative adhesion pathways that do not require ICAM-1 are important in neutrophil emigration in the lungs. The disparity in the role of ICAM-1 when evaluated using antisense or antibodies compared to mutant mice suggests that either these inhibitors are exerting additional effects on endothelial cells other than blockade of ICAM-1 or mutant mice have upregulated the ICAM-1-independent pathways to compensate for the long-term loss of ICAM-1.

Fluorescence turn-on hairpin-probe PCR.

A new fluorescence turn-on type of PCR monitoring system (Hpro-PCR) using a hairpin probe and a primer having a tag sequence at the 5' end with the fluorescent molecule 2,7-diamino-1,8-naphthyridine derivative (DANP) has been developed. The Hpro-PCR exploited the modulation of the equilibrium states between the DANP-bound hairpin structure and probe-tag duplex, and the PCR progress alternated the equilibrium state, resulting in the change of fluorescent intensity.

Two monoclonal antibodies defining unique antigenic determinants on human B-lymphoma cells.

The non-Hodgkin's lymphomas are a clinically, morphologically, and immunologically heterogeneous group of diseases. Why lymphoma cells are unresponsive to normal regulatory growth controls and how they differ from normal lymphocytes are not well understood. In order to address these questions we have raised monoclonal antibodies to neoplastic B-cells. Two of these, LM-26 and LM-155, show a high degree of specificity for B-cell lymphomas. When tested by fluorescence activated cell sorter analysis, LM-26 reacted with 80% (18 of 23) of B-cell lymphomas freshly explanted from patients and LM-155 reacted with 20% (5 of 23). The antigenic determinant detected by LM-26 was also found to be present on four of seven neoplastic large cell B-lymphoma lines. LM-155 detected a determinant present on all seven of these lines. For neither monoclonal antibody was there any association between antibody reactivity and the morphological subtype of lymphoma examined or the type of cell surface immunoglobulin expressed. LM-155 reacted with one case of B-cell acute lymphoblastic leukemia. Neither antibody reacted with normal B-cell blasts, normal peripheral blood mononuclear or marrow cells, T-cell leukemias or lymphomas, or chronic lymphocytic leukemia cells. Lymphocytes from reactive lymphoid hyperplasias involving lymph nodes, spleen, peripheral blood, and lung were also negative for LM-26 and LM-155 binding or showed only a small percentage of cells positive (4-8%). Both monoclonals were unreactive with non-B-lymphoid neoplastic cell lines, nine of ten Epstein-Barr virus transformed B-cell lines, and cells freshly explanted from patients with cancers of diverse cellular origins. Fluorescence activated cell sorter analysis of the expression of the antigens defined by LM-26 and LM-155 on lymphoma cells and normal B-cell blasts suggests that they are not normal differentiation antigens associated with lymphocyte activation or proliferation. The highly restricted expression of detectable levels of antigens reactive with monoclonal antibodies LM-26 and LM-155 on non-Hodgkin's lymphoma cells suggests a possible relation to their neoplastic properties. From a practical viewpoint these monoclonals may prove useful in the diagnosis, classification, detection of residual disease, and treatment of the non-Hodgkin's lymphomas.

Induction of sensitivity to NK-mediated cytotoxicity by TNF-alpha treatment: possible role of ICAM-3 and CD44.

SR-91 is a natural killer (NK)-resistant leukemic cell line expressing a low level of ICAM-1. Pre-treatment of SR-91 cells with TNF-alpha or IFN-gamma, increased both ICAM-1 (CD54) expression on SR-91 cells and binding to the human NK cell line NK-92. However, only TNF-alpha-treated SR-91 cells became sensitive to killing by NK-92 cells. The increased binding induced by both cytokines and the TNF-alpha-induced sensitivity of SR-91 cells to NK-92 cell killing were abrogated by anti-LFA-1 mAb as well as by a combination of antibodies against the three ligands of LFA-1 (CD11a/CD18), ICAM-1 (CD54), ICAM-2 (CD102) and ICAM-3 (CD50). This indicated that LFA-1 interaction with the three ICAMs on SR-91 cells is essential for effector-target cell binding (which is a prerequisite for subsequent target cell lysis), but is insufficient to render the SR-91 cells sensitive to killing by NK-92 cells. TNF-alpha, but not IFN-gamma also induced the activation of LFA-1, CD44 and beta1 integrins on SR-91 cells. Based on these observations we propose that the differential effect of TNF-alpha and IFN-gamma could be related to the activation of certain adhesion molecules on the surface of SR-91 cells by TNF-alpha that, upon interaction with their counter-receptors on NK-92 cells, lead to the activation of the NK-92 cells.

Monoclonal antibodies to HLA-DP, DQ, and DR determinants: functional effects on the activation and proliferation of normal and EBV-transformed B cells.

To investigate the function of HLA class II molecules in B-cell activation, we generated three new anti-HLA class II monoclonal antibodies with differing specificities for DP, DQ, and DR determinants. These were tested for their ability to inhibit various B- and T-lymphocyte responses. Each of these antibodies (NB-29, DH-84, and DH-224) immunoprecipitates a heterodimer of approximately 35,000 and 28,000 mol wt from 125I-surface-labeled B-lymphoma cells, as shown by SDS-PAGE. NB-29 (IgG1) detects a polymorphic DQ determinant, while DH-224 (IgG1) is reactive with monomorphic DR determinants, and DH-84 (IgG2a) has specificity for DP, DQ, and DR. Both DH-224 and DH-84, but not NB-29, were found to inhibit significantly the stimulation of peripheral blood mononuclear cells (PBMC) by anti-mu (70%-90% inhibition) and by lipopolysaccharide (80%-90% inhibition), as measured by incorporation of tritiated thymidine. When added to highly purified populations of peripheral blood B cells, none of these anti-class II monoclonal antibodies inhibited anti-mu-induced stimulation. This suggests that the inhibitory effect that DH-224 and DH-84 have on the stimulation of unfractionated PBMC may be due to their ability to interfere with the action of accessory cells. Epstein-Barr-virus (EBV)-transformed B-cell lines, in contrast, showed substantial inhibition of growth when cultured in the presence of any of the three antibodies. With respect to T cells, DH-84 and DH-224 strongly inhibited the mixed lymphocyte response; NB-29 did not. None of these antibodies inhibited stimulation of PBMC by phytohemagglutinin (PHA). These findings suggest that DQ and DR HLA class II molecules have differing roles in B-cell activation and document a direct antiproliferative effect of anti-HLA class II monoclonal antibodies on the growth of EBV-transformed cell lines.

Dual role of IL-7 in the growth and differentiation of immature thymocytes.

The effects of interleukin 7 (IL-7) on subpopulations of CD4-CD8- thymocytes from young adult mice were tested in vitro. When highly purified CD3-CD4-CD8-thymocytes were cultured in the presence of recombinant IL-7, significant proportions of them became CD4+ and/or CD8+ within a day. CD3+ cells were also detected after 2 days. CD3-CD4-CD8- thymocytes were further subdivided into interleukin 2 receptor (IL-2R)- and IL-2R+ populations. The majority of the IL-2R- cells became CD4+ and/or CD8+ in 1 day in the presence of IL-7, and a substantial proportion of them also became CD3+ in 2-3 days. No significant number of CD4+ or CD8+ cells were generated from the IL-2R+ population under the same conditions. However, a small but significant proportion of them became CD3+ in 3-day cultures with IL-7. Although CD4+/CD8+ cells were also generated from the IL-2R- population in 1-day cultures in the absence of IL-7, the viability of the cells declined rapidly, and no significant numbers of CD3+ cells were generated. In proliferation assays, IL-7 alone vigorously stimulated relatively minor subpopulations of CD4-CD8- thymocytes. The IL-7-responsive cells were CD3+, did not express the IL-2R or the heat-stable antigen M1/69, and included both T-cell receptor (TCR)alpha beta + and TCR alpha beta- populations, the latter most likely TCR gamma delta +. The CD3+CD4-CD8- thymocytes, stimulated with IL-7 for 3 days, remained CD4-CD8-. These results demonstrate important roles of IL-7 in the growth and differentiation of CD4-CD8- thymocytes in vitro. It functions as a survival factor and allows CD3-CD4-CD8- cells to undergo their precommitted differentiation without inducing their proliferation, and it also stimulates CD3+CD4-CD8-thymocytes to proliferate without inducing their differentiation.

Sequence polymorphisms in the 5'-upstream region of the fibroin H-chain gene in the silkworm, Bombyx mori.

DNA fragments containing the fibroin H-chain gene from two different strains of Bombyx mori, J-139 and Nd (2) were cloned into phage lambda Charon 4A. Comparison of the restriction sites in these cloned DNAs revealed that in addition to the known polymorphism in the region coding for the repetitive amino acid sequence of the fibroin H-chain [Manning and Gage, J. Biol. Chem. 255 (1980) 9451-9457], at least two other types of polymorphism were present, one around the 5' end of the structural gene, and the other in the far upstream region of the gene. Restriction sites around the 5' end of the gene were well conserved between these strains, but some heterogeneity, suggesting the presence of small insertions, deletions or base changes, was noted. In contrast, DNA sequences of the region 2-4 kb upstream from the 5' end of the gene were markedly different between these two strains, indicating that either a deletion or an insertion of a DNA sequence longer than 2 kb had occurred in this region. Comparison with several other strains suggested that the observed changes in the far-upstream region were unique to the Nd(2) strain.

Novel technique for isolation of human lung mast cells.

A novel technique for isolation of human lung mast cells is developed. Human lung tissue was enzymatically digested and the cells were partially purified by centrifugation on Percoll density gradient. Cells obtained at the Percoll density of 1.05-1.09 g/ml were then subjected to a cell sorter equipped with a single argon laser beam (FACS 440). Using four criteria as density, granularity, size and autofluorescence, four major cell populations were identified. One of the major populations contained 70-95% mast cells with a mean and SE values of 88 +/- 11% purity, n = 18 as determined by the measurement of total histamine content, light microscopic observation of stained cells with toluidine blue and estrase, and surface-stained IgE fluorescence antibody. Approximately less than 10% mast cells were identified in the three other major cell populations. Mast cells isolated by FACS were found to be intact, viable (approximately equal to 90%) and functionally normal as determined by the release of histamine evoked after stimulation with ionophone A23187, or challenged with anti-human IgE.

Reduction in the severity of graft-versus-host disease and increased survival in allogenic mice by treatment with monoclonal antibodies to cell adhesion antigens LFA-1 alpha and MALA-2.

Bone marrow transplantation is a therapeutic treatment for many life-threatening hematologic disorders, especially leukemia and certain immune deficiency diseases. However, acute graft-versus-host disease is often associated with bone marrow transplantation. In mice, allogeneic GVHD appears to be mediated by both host natural killer cells and donor T cells. In vitro and in vivo experiments demonstrate that treatment with either YN1/1.7 or M17/4.2 mabs is immunomodulatory and inhibits both the mixed lymphocyte reaction and natural killer cell activity. In addition, utilizing an allogeneic model of acute, lethal GVHD with C57B1/6 mice as donors and sublethally irradiated BDF1 mice as recipients, treatment of host mice with anti-LFA-1 alpha (M17/4.2) or anti-MALA-2 (YN1/1.7) mabs at a dose of 10 mg/kg/day for 10 days significantly reduced GVHD and enhanced survival. Mabs to lymphocyte adhesion molecules such as LFA-1 alpha and MALA-2 may provide a useful therapy for the treatment of GVHD.

Adhesion molecules on murine brain microvascular endothelial cells: expression and regulation of ICAM-1 and Lgp 55.

The mechanisms for the initiation of immune reactions in the central nervous system are poorly understood. In this report, we describe the presence of intercellular adhesion molecule-1 (ICAM-1) and Lgp 55 (suggested mouse homologue of human intercellular adhesion molecule-2, ICAM-2) on the surface of brain microvessel endothelium (EN) cells and show in vitro induction of ICAM-1 molecules on EN cells with pro-inflammatory cytokines. ICAM-1 expression was detected using flow cytometry analysis with biotinylated anti-ICAM-1 antibody (YN1/1.7.4). Lgp 55 expression was characterized using PA3 monoclonal antibody. According to our results, 30-40% of the non-activated brain EN cells expressed ICAM-1 and 15-20% expressed Lgp 55 molecules. The ICAM-1 molecule expression was increased after the activation of the cells with recombinant murine gamma interferon (IFN-gamma), tumor necrosis factor (TNF-alpha), and interleukin-1 alpha (IL1-alpha) in a dose-dependent manner. The increased ICAM-1 expression was detected as early as 2 h following the cytokine treatment and reached its maximum after 24 h. Transforming growth factor-beta (TGF-beta) did not influence the expression of ICAM-1 molecule. Lgp 55 molecule does not seem to be regulated by pro-inflammatory cytokines. ICAM-1 and Lgp 55 expression was found to be polarized on the luminal surface of EN by confocal laser microscopy suggesting accessibility for leukocytes. Inducible ICAM-1 expression may play a critical role in formation of inflammatory reactions inside the central nervous system.

NHL-30.5: a monoclonal antibody reactive with an acute myeloid leukemia (AML)-associated antigen.

The reactivity of a murine IgG1 monoclonal antibody, NHL-30.5, that detects a surface antigen expressed on acute myeloid leukemia (AML) cells has been studied. Initially raised against the HL-60 cell line, NHL-30.5 has subsequently reacted with blood and/or bone marrow cells from 15 of 19 AML patients studied at presentation or in relapse, 1 patient with chronic myelomonocytic leukemia (CMML), 1 patient with myelofibrosis (MF) who subsequently developed AML, and 1 of 5 patients with acute lymphoblastic leukemia (ALL). It has shown no detectable binding to cells from AML patients in remission (0/3), patients with chronic myelogenous leukemia in chronic phase (CML) (0/7), normal bone marrow (0/9), normal peripheral blood mononuclear cells, granulocytes, platelets, erythrocytes, monocytes, or splenocytes by radioimmunoassay or fluorescence analysis using flow cytometry. HL-60 cells induced to differentiate following incubation in the presence of dimethylsulfoxide (DMSO) lost their ability to bind NHL-30.5. Immunoprecipitation of iodinated HL-60 cell surface components showed the antigen to have an apparent mol./wt of 180,000 under reducing conditions. These results suggest that the antigen is different from any other myeloid antigens reported to date, and may be useful in further studies of leukemic cell phenotypes.

A pharmacologic study on CO2 responsiveness of intracranial pressure in rats with chronic hypercapnia.

STUDY OBJECTIVES: To investigate intracranial pressure (ICP) changes and their mechanisms in chronic hypercapnia. DESIGN: After 12 male Wistar rats were maintained in CO2-mixed air (mean PaCO2, 71.0 mm Hg) for 21 weeks, their ICP levels were measured during the breathing of 0, 5, 10, 12, and 14% CO2-mixed air before and after the i.v. administration of nitro-L-arginine methyl ester (L-NAME). The ICP responses to i.v. norepinephrine and i.v. adenosine were also tested. Ten rats that were maintained in room air served as the control group. RESULTS: The mean ICP in the study group (5.9 mm Hg) was not significantly different from the mean ICP in the control group. In the study group, the ICP response to changes in PaCO2 was significantly blunted when compared to the response seen in the control group. In both groups, i.v. norepinephrine significantly increased the ICP. In the control group but not in the study group, i.v. L-NAME suppressed the ICP response to changes in PaCO2. In both groups, i.v. adenosine significantly increased the ICP. CONCLUSIONS: The ICP response to PaCO2 was blunted in rats with chronic hypercapnia, and the mechanism of this reduced response may involve nitric oxide.

Screw-sense-selective polymerization of aryl isocyanides initiated by a Pd-Pt mu-ethynediyl dinuclear complex: a novel method for the synthesis of single-handed helical poly(isocyanide)s with the block copolymerization technique

Living polymerization of chiral aryl isocyanides, such as m- and p-menthoxycarbonylphenyl isocyanides 2 and 5, initiated by the Pd-Pt mu-ethynediyl dinuclear complex 1, proceeds with a high screw-sense selectivity to give the poly(isocyanide)s 3 and 6, which exhibit a large specific rotation and an intense CD band at lambda = 364 nm as a consequence of a helical chirality. The molar optical rotation and molar circular dichroism of the resulting polymers 3 and 6 reach a constant value at a degree of polymerization (Pn) of more than 30. Screw-sense-selective polymerization of achiral aryl isocyanides that bear very bulky substituents, such as 3,5-di(propoxycarbonyl)phenyl isocyanide (11), 3,5-di(butoxycarbonyl)phenyl isocyanide (13), and 3,5-di(cyclohexyloxycarbonyl)phenyl isocyanide (15), is achieved by the use of chiral oligomer complexes 3(30) and 6(30), prepared from the reaction of 1 with 30 equivalents of 2 or 5, as an initiator to give predominantly single-handed helical polymers. In contrast, smaller aryl isocyanides are also polymerized by 3(30) and 6(30) with screw-sense selectivity in the initial stage of the reaction, but the single-handed helix is not preserved up to high molecular weight. Kinetic studies of the polymerization of (L)- and (D)-2, or (L)- and (D)-5 with chiral oligomer complexes (L)-3(50) or (L)-6(100) suggests that the screw sense of the polymer backbone is not controlled kinetically, but rather that the thermodynamically stable screw sense is produced.