ALBERT

Description: The CDKN2 gene located on chromosome 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16. This gene is a putative tumor-suppressor gene because of its frequent alterations in many kinds of tumor cell lines. We analyzed the CDKN2 gene to evaluate its alterations in 52 primary specimens of non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) of B-cell origin by Southern blot analysis, polymerase chain reaction-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing. By Southern blot analysis, we showed homozygous deletion of the CDKN2 gene in 3 of 42 patients with B-NHL (7.1%). After screening by PCR-SSCP analysis, direct sequencing identified one missense mutation at codon 72 (nucleotide 233) and two frameshifts due to a 35-bp deletion arising at codon 49 (nucleotides 163 to 175) in patients with B-NHL (3 of 42, 7.1%). In the patient carrying the missense mutation, hemizygous deletion of the CDKN2 gene was also suspected. In this study, we detected alterations in CDKN2 in 6 of 42 patients (14.3%) with B-NHL and in none of 10 patients with B-CLL. Our results suggest that the CDKN2 alterations contribute in tumorigenesis in some patients with B-NHL.

Description: The bacterial superantigens (SAg), toxic shock syndrome toxin-1 (TSST- 1) and staphylococcal enterotoxin B (SEB), are powerful T-cell stimulators, triggering systemic release of lymphokines causing lethal shock in D-galactosamine (D-Gal)-sensitized mice. We show that pretreatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) protects mice against T-cell-mediated SAg-shock. In mice challenged with D-Gal/TSST-1, lethal shock was caused within 30 hours. In contrast, animals pretreated with two consecutive subcutaneous injections of 2 micrograms rhG-CSF with a 12-hour time interval showed only marginal signs of illness and no lethality after challenge with D-Gal/TSST-1. Mice treated with 5 micrograms rhG-CSF either 12 or 6 hours in advance also survived otherwise lethal doses of D-Gal/TSST-1. The protective effects of rhG-CSF pretreatment was also evident against lethal doses of D-Gal/SEB challenge and this protection was accompanied by suppression of systemic interleukin-2. However, rhG- CSF affected neither the proliferative responses of SAg-reactive T cells in vivo or in vitro nor their interleukin-2 production in vitro, implying that rhG-CSF may indirectly interfere with cytokine synthesis in T cells but not with T-cell-SAg binding itself. These results represent another beneficial effect of rhG-CSF as an anti-inflammatory agent against T-cell-mediated toxicity triggered by SAg.

Description: The molecular basis for a hereditary type I protein S (PS) deficiency was investigated. DNA sequence analysis in the proband showed a novel missense mutation substituting Cys (TGT) for Arg474 (CGT) that is a highly conserved amino acid residue among the related proteins. This missense mutation cosegregated with the type I PS deficiency in this family. Transient expression studies showed that the secretion of the recombinant Cys-mutant PS was markedly decreased compared with that of the recombinant wild-type PS, reproducing the observed phenotype of type I deficiency. Stable expression and pulse-chase experiments demonstrated an intracellular degradation and an impaired secretion of the recombinant Cys-mutant PS. Furthermore, the substitution of Arg474 by Ala or Glu, but not by Lys, markedly reduced the secretion of the recombinant PS mutants in transient expression studies, suggesting that a positively charged basic amino acid might be needed at residue 474 and might play a key role in the protein structure and conformation of the sex hormone binding globulin-homology domain of the PS molecule. We postulate that the loss of the highly conserved Arg474 might be responsible for the type I PS deficiency inherited in this family.

Description: Protein C Nagoya, an elongated variant of the human protein C, is retained and degraded within the cells in which it is produced (Yamamoto et al, J Clin Invest 90:2439, 1992). To determine the subcellular localization of the protein C Nagoya, the recombinant protein C bearing this mutation was expressed in Chinese hamster ovary (CHO) cells. The mutant protein C was not secreted from the cells and remained susceptible to endo-beta-N-acetylglucosaminidase H (endo H). Immunoelectron microscopy indicated that protein C Nagoya was retained in the endoplasmic reticulum (ER), whereas wild-type protein C was observed in both the ER and the Golgi apparatus. Metabolic radiolabeling with [35S] methionine in combination with chemical cross- linking showed that the protein C Nagoya existed in the ER as a complex with 78-kD glucose-regulated protein (GRP78) and 94-kD glucose- regulated protein (GRP94). Because both GRP78 and GRP94 associate to a far lesser degree with wild-type protein C than with protein C Nagoya, our data suggest that both stress proteins function as molecular chaperones and work in concert with the folding and assembly of protein C. These findings extend our understanding the molecular pathogenesis of protein C deficiency.

Description: The CDKN2 gene located on chromosome 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16. This gene is a putative tumor-suppressor gene because of its frequent alterations in many kinds of tumor cell lines. We analyzed the CDKN2 gene to evaluate its alterations in 52 primary specimens of non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) of B-cell origin by Southern blot analysis, polymerase chain reaction-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing. By Southern blot analysis, we showed homozygous deletion of the CDKN2 gene in 3 of 42 patients with B-NHL (7.1%). After screening by PCR-SSCP analysis, direct sequencing identified one missense mutation at codon 72 (nucleotide 233) and two frameshifts due to a 35-bp deletion arising at codon 49 (nucleotides 163 to 175) in patients with B-NHL (3 of 42, 7.1%). In the patient carrying the missense mutation, hemizygous deletion of the CDKN2 gene was also suspected. In this study, we detected alterations in CDKN2 in 6 of 42 patients (14.3%) with B-NHL and in none of 10 patients with B-CLL. Our results suggest that the CDKN2 alterations contribute in tumorigenesis in some patients with B-NHL.

Description: The inhibition mechanism of a polysaccharide anticoagulant, depolymerized holothurian glycosaminoglycan (DHG), was examined by analyzing its effects on the clotting time of human plasma depleted of antithrombin III (ATIII), of heparin cofactor II (HCII), or of both heparin cofactors. The effect exerted by this agent on the activation of prothrombin and factor X in purified human components were also examined and all effects were compared with those of other glycosaminoglycans (GAGs). The capacity of DHG to prolong activated partial thromboplastin time was not reduced in ATIII-depleted, HCII- depleted, HCII-depleted, or ATIII- and HCII-depleted plasma, whereas its capacity to prolong prothrombin time and thrombin clotting time was reduced in HCII-depleted plasma. DHG inhibited the amidolytic activity of thrombin in the presence of HCII with a second order rate constant of 1.2 x 10(8) (mol/L)-1 min-1. These results indicated that DHG has two different inhibitory activities, one being an HCII-dependent thrombin inhibition and the other an ATIII- and HCII-independent inhibition of the coagulation cascade. The heparin cofactors- independent inhibitory activity of DHG was investigated in the activation of prothrombin by factor Xa and in the activation of factor X by tissue factor-factor VIIa complex or by factor IXa. DHG significantly inhibited the activation of factor X by factor IXa in the presence of factor VIIIa, but not in the absence of factor VIIIa. The interaction between DHG and factors IXa, VIIIa, and X was investigated with a DHG-cellulofine column, on which DHG had strong affinity for factors IXa and VIIIa. These findings show that the heparin cofactors- independent inhibition exhibited by DHG was caused by inhibition of the interaction of factor X with the intrinsic factor Xase complex, probably by binding to the factor IXa-factor VIIIa complex.

Description: Fas antigen (CD95) can induce apoptosis of cells such as lymphocytes and neutrophils. To determine whether Fas antigen is involved in eosinophil apoptosis, we examined its expression and function on eosinophils in vitro. Purified human eosinophils expressed low but consistently detectable levels of Fas antigen. Culture of eosinophils in up to 10 ng/mL interleukin-5 (IL-5) prolonged eosinophil survival; incorporation of 1 to 1,000 ng/mL Fas antibody led to significant reductions in IL-5-induced eosinophil viability after 48 to 72 hours of culture. Reductions in survival could not be overcome by IL-5 and also occurred in the absence of exogenous IL-5. Preactivation of eosinophils with platelet-activating factor (PAF) significantly reduced eosinophil viability without altering the survival-reducing effects of Fas antibody treatment. In contrast, RANTES did not affect eosinophil viability or Fas antibody-induced reductions in eosinophil survival. After treatment with Fas antibody, electron microscopy of eosinophils and gel electrophoresis of DNA extracted from eosinophils demonstrated changes consistent with apoptosis. These data demonstrate that Fas antigen can modify eosinophil survival by inducing apoptosis through a pathway that is, at least in part, independent of the survival- promoting effects of IL-5.

Description: Although stem cell factor (SCF) has been identified as a critical cytokine for the development of human mast cells from their progenitors, the effects of other cytokines on human mast cells are less well understood. We examined the effects of several cytokines on the survival of human mast cells of 100% purity generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of 100 ng/mL recombinant human (rh) SCF and interleukin-6 (IL-6). Mast cells suspended in conventional serum-containing medium died over a period of 2 to 6 days after the withdrawal of SCF and IL-6. The cells became pyknotic and underwent DNA fragmentation characteristic of apoptosis. The addition of SCF, IL-3, IL-4, IL-5, or IL-6 to the cultures in both serum-containing and serum-free medium prolonged their survival in a dose-dependent manner. Some other cytokines, such as IL- 2, IL-9, IL-10, IL-11, tumor necrosis factor-alpha, transforming growth factor-beta 1, and nerve growth factor, had no survival-promoting effect at 100 ng/mL. Preincubation of mast cells with SCF, IL-4, IL-5, or IL-6 for 24 hours during sensitization with IgE enhanced IgE/anti- IgE antibody-induced histamine release from mast cells, whereas IL-3 showed a negligible effect. Polymerase chain reaction amplification of alpha-chains of IL-3 receptor (R), IL-4 R, IL-5 R, and IL-6 R yielded products of the correct size predicted from the sequence of each receptor. The binding assay using 125I-labeled IL-3 indicated that these mast cells bear receptors for IL-3. These findings suggest that IL-3, Il-4, IL-5, and IL-6, which are mainly produced by T-helper 2 lymphocytes, might regulate the functions of human mast cells in vivo via specific receptors in allergic reactions.