Nf1 regulates hematopoietic progenitor cell growth and ras signaling in response to multiple cytokines

Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21(ras) (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1-/- hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1-/- progenitors. Nf1-/- fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1-/- progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.     

Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway

The expression of tissue plasminogen activator (tPA) is increased during activity-dependent forms of synaptic plasticity. We have found that inhibitors of tPA inhibit the late phase of long-term potentiation (L-LTP) induced by either forskolin or tetanic stimulation in the hippocampal mossy fiber and Schaffer collateral pathways. Moreover, application of tPA enhances L-LTP induced by a single tetanus. Exposure of granule cells in culture to forskolin results in secretion of tPA, elongation of mossy fiber axons, and formation of new, active presynaptic varicosities contiguous to dendritic clusters of the glutamate receptor R1. These structural changes are blocked by tPA inhibitors and induced by application of tPA. Thus, tPA may be critically involved in the production of L-LTP and specifically in synaptic growth.     

Long-term therapy with the guanine nucleoside analog penciclovir controls chronic duck hepatitis B virus infection in vivo

Ducks congenitally infected with duck hepatitis B virus (DHBV) were treated with the antiviral guanine nucleoside analog penciclovir for 12 or 24 weeks at a dosage of 10 mg/kg of body weight per day. By the completion of both 12 and 24 weeks of therapy, molecular hybridization studies of the liver tissue revealed that the viral DNA, RNA, and protein levels were significantly reduced compared to those in the placebo-treated controls. Penciclovir treatment for 12 or 24 weeks was not associated with any toxicity, establishing the efficacy and safety of long-term penciclovir therapy in chronic DHBV infection.     

Diffusion-weighted imaging in hyperacute stroke--gold standard?

Diffusion-weighted imaging (DWI) is more sensitive than CT and conventional T2-weighted MR imaging in the detection of early cerebral ischemia and infarction. In this case report, we present a case of a 79-year-old woman with symptoms of acute stroke, clinically and radiologically documented progression to complete stroke with an initial negative diffusion-weighted scan.     

Early intrinsic deflection--a marker for successful radiofrequency ablation of overt accessory pathways

Striking morphological similarities exist between T-cell-rich B-cell lymphoma and lymphocyte-predominant Hodgkin's disease (Hodgkin's paragranuloma), making the distinction between them extremely difficult. Immunohistochemistry provides a means of overcoming this difficulty. Immunostaining with UCHL1, L26, MB1, and 4KB5 was performed on five T-cell-rich B-cell lymphomas and 11 Hodgkin's paragranulomas (7/11 nodular, 4/11 diffuse). L26 stained the tumour cells not only of T-cell-rich B-cell lymphomas, but also of L+H Hodgkin's disease. In contrast, MB1 as well as 4KB5 identified all of the neoplastic cells in 3/5 T-cell-rich B-cell lymphomas, but did not react with the L+H cells in 8/11 Hodgkin's paragranulomas. Some overlap of staining patterns became apparent in the remaining cases, with 2/5 T-cell-rich B-cell lymphomas showing the MB1+/4KB5+ phenotype in a tumor cell subset only. Similarly, in 3/11 Hodgkin's paragranulomas, some MB1/4KB5-positive L+H cells occurred in addition to MB1/4KB5-negative L+H cells. These cases, nevertheless, could be distinguished from one another by the numbers of MB1/4KB5-positive background lymphocytes, which were scanty or absent in T-cell-rich B-cell lymphomas and more numerous in Hodgkin's paragranulomas.     

Prevalence and genotypes of alpha- and beta-thalassemia carriers in Hong Kong -- implications for population screening

BACKGROUND: The thalassemias are common in southern China. We determined the prevalence of heterozygous carriers of these genetic disorders in Hong Kong and assessed the feasibility of a community-based screening program. METHODS: An educational and screening program for the thalassemias was carried out in three high schools with a total of 2420 students. Seventy-five percent of the students agreed to undergo screening, which consisted of blood counts, hemoglobin electrophoresis, serum ferritin measurements, and DNA analyses. RESULTS: Of the 1800 blood samples tested, 150 (8.3 percent) had microcytosis (mean corpuscular volume, <80 microm3). Ninety students (5.0 percent) were carriers of alpha-thalassemia, of whom 81 (4.5 percent) were carriers of the Southeast Asian type of deletion, in which both alpha-globin genes on the same chromosome 16 are deleted. Sixty-one students (3.4 percent) were carriers of either beta-thalassemia or the mutation coding for hemoglobin E. Six students were carriers of both alpha- and beta-thalassemias. On the basis of these figures, the estimated numbers of pregnancies in Hong Kong in which the fetus is at risk for homozygous alpha-thalassemia and beta-thalassemia major or intermedia are 145 and 80 per year, respectively. In Hong Kong the actual numbers of women referred for prenatal diagnoses of these disorders are approximately 95 and 40 per year, respectively. CONCLUSIONS: Despite the availability of hospital-based screening and prenatal diagnosis for many years in Hong Kong, many women carrying fetuses at risk for thalassemia are not referred for genetic counseling. A community-based program of education, screening, and counseling is needed in Hong Kong and southern China.     

Comparison of the Ca2+ movement by activation of alpha1-adrenoceptor subtypes in HEK-293 cells

We studied the Ca2+ movement induced by activation of alpha1A-, alpha1B- and alpha1D-adrenoceptor subtypes in transfected HEK-293 cells with the fura-2 probe. All these alpha1-AR subtypes induced both Ca2+ release and Ca2+ entry. The effect on Ca2+ release in alpha1b transfected HEK-293 cells was bigger than that in alpha1a and alpha1d transfected HEK-293 cells, and the effects on Ca2+ entry were the same in alpha1a, alpha1b and alpha1d transfected HEK-293 cells. The Ca2+ entry was inhibited by 1 mM NiSO4, but not by nifedipine. Cyclopiazonic acid (CPA) produced a biphasic Ca2+ signal response in Ca2+ medium, and only induced a transient response in Ca2+-free medium. After depletion of CPA-sensitive Ca2+ pool by 10 microM CPA in Ca2+-free medium, 10 microM adrenaline (Adr) still transiently increased [Ca2+]i in three different alpha1-adrenoceptor subtype transfected HEK-293 cells. However, after depletion of adrenaline-sensitive Ca2+ pool by 10 microM Adr, CPA transiently elevated [Ca2+]i only in alpha1a and alpha1d transfected HEK-293 cells, not in alpha1b transfected HEK-293 cells. U73122, a phospholipase C (PLC) inhibitor, inhibited both Ca2+ release and Ca2+ entry induced by activation of alpha1A alpha1B and alpha1D subtypes in transfected HEK-293 cells. These results suggest that HEK-293 cell line contains two functionally separate intracellular Ca2+ pools, CPA-sensitive and Adr-sensitive pools. Activation of alpha1B-AR stimulates Ca2+ release from both CPA-sensitive and Adr-sensitive Ca2+ pools. Alpha1A and alpha1D subtypes induce Ca2+ release only from Adr-sensitive Ca2+ pool.     

Random versus selective membrane phospholipid oxidation in apoptosis: role of phosphatidylserine

The formation of reactive oxygen species has been associated with apoptosis. To assess the role of lipid peroxidation in apoptosis, we used 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN) to generate peroxyl radicals within cellular membranes of HL-60 cells. cis-Parinaric acid (cis-PnA) metabolically integrated into phospholipids of HL-60 cells was used as a probe to assess the extent of lipid peroxidation within specific phospholipid classes. Within 2 h, AMVN (500 microM) randomly oxidized more than 85% of cis-PnA contained in all major classes of phospholipids. AMVN-induced lipid peroxidation was followed by apoptosis as determined by nuclear condensation, DNA fragmentation, and annexin V binding to externalized phosphatidylserine (PS). Fluorescamine derivatization of external aminophospholipids revealed that PS, but not phosphatidylethanolamine, was externalized. The vitamin E analogue, 6-hydroxy-2,2,5,7,8-pentamethylchromane (PMC), inhibited overall oxidation of cis-PnA in phospholipids by more than 85%. Not all phospholipids, however, were equally protected. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin were nearly completely protected by PMC, while oxidation of PS was unaffected in whole living cells. The insensitivity of PS to PMC was not an intrinsic property because PMC protected all lipids equally during AMVN oxidation of liposomes prepared from cis-PnA-labeled cells. The potential role for PS oxidation in apoptosis was further suggested by the faithful execution of apoptosis following coexposure of cells to AMVN and PMC.