Raf-1 independent stimulation of mitogen-activated protein kinase by leukemia inhibitory factor in 3T3-L1 cells

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulated Raf-1 activity in a time- and dose-dependent manner. Although phorbol ester failed to activate Raf-1 directly, a protein kinase C-stimulated signal was found to be necessary, but not sufficient, for LIF-mediated activation of Raf-1. Elevation of intracellular cAMP levels completely blocked Raf-1 activation by LIF, but was without effect on the magnitude of mitogen-activated protein kinase (MAPK) stimulation by the cytokine, suggesting the presence of a Raf-1-independent, cAMP-insensitive MAPK kinase kinase (MAPKKK) pathway in 3T3-L1 cells. Mono Q-fractionation of LIF-stimulated 3T3-L1 extracts identified a single peak of MAPKKK activity that was largely insensitive to elevated intracellular levels of cAMP, and that failed to correlate with stimulation of either Raf-1 or MEKK1 protein kinases. Our results demonstrate that LIF-mediated activation of the MAP kinase cascade in 3T3-L1 cells proceeds through both Raf-1-dependent and -independent pathways which differ in their sensitivity to inhibition by intracellular cAMP.     

The frequency of illegitimate V(D)J recombinase-mediated mutations in children treated with etoposide-containing antileukemic therapy

Etoposide is among the most widely used anti-cancer drugs. Its use, however, has been associated with increased risk of secondary acute myeloid leukemia (AML) which is characterized by chromosomal translocations suggesting involvement of recombination-associated motifs at the breakpoints. A PCR-based assay was developed to quantitate the frequency of two illegitimate V(D)J recombinase-mediated genomic rearrangements-a 20-kb deletion in the hprt gene and the bcl2/IgH translocation (t(14;18)) found in non-Hodgkin's lymphoma. We examined both lymphocyte and non-lymphocyte blood cell DNA of children with acute lymphoblastic leukemia (ALL) for changes in the frequencies of these biomarkers during etoposide therapy to determine the level of illegitimate V(D)J recombination changes during therapy. A low level of t(14;18) was found in the lymphocytes before etoposide treatment, which was significantly reduced during etoposide therapy. In before-etoposide samples, no t(14;18) were found among 7.72x107 non-lymphocytes; during treatment none were found among 1.87x108 non-lymphocytes. Deletions were not found before etoposide treatment in either the lymphocytes (6.67x107) or non-lymphocytes (5.43x107) and were non-significantly elevated during etoposide therapy (1 in 1.4x108 lymphocytes and 1 in 1.39x108 non-lymphocytes). It is interesting to note the one patient with an hprt deletion mutation in non-lymphocytes; V(D)J recombination is not normally found in this cell type, but is the cell type from which AML derives. Several patients had clones of t(14;18)-bearing cells as determined by DNA sequence analysis. These results suggest that this etoposide-based chemotherapy was ineffective in producing genomic rearrangements mediated by illegitimate V(D)J recombination in these patients.     

Vascular hyporesponsiveness in aortic rings from cirrhotic rats: role of nitric oxide and endothelium

1. The role of nitric oxide as mediator of the vascular alterations present in different models of experimental liver cirrhosis is controversial. In the present study, we evaluated the role of nitric oxide and that of the endothelium in the response to phenylephrine and acetylcholine of isolated aortic rings from chronic bile duct-ligated (29 days) rats and their corresponding controls. Experiments were performed in rings with or without endothelium, in rings pretreated with N-omega-nitro-L-arginine methyl ester (10(-4) mol/l) to inhibit nitric oxide synthesis and in rings pretreated with aminoguanidine (10(-4) mol/l) to inhibit inducible nitric oxide synthesis. 2. Under basal conditions, the maximum absolute tension developed in response to cumulative addition of phenylephrine was significantly decreased in rings from bile duct-ligated animals (1.62 +/- 0.06 g) compared with the control rings (2.15 +/- 0.099). This hyporesponsiveness to phenylephrine of rings from bile duct-ligated animals was corrected after treatment with N-omega-nitro-L-arginine methyl ester and reduced, but not completely eliminated, in rings without endothelium. In contrast, aminoguanidine did not modify the lower response to phenylephrine rings from bile duct-ligated animals. ED50 values were not different between groups under any experimental conditions. 3. The endothelium-dependent vasodilatation to acetylcholine in phenylephrine-constricted rings was similar in both groups of animals, control and bile duct ligated, under all experimental conditions. N-omega-nitro-L-arginine methyl ester pretreatment and removal of the endothelium completely abolished the response to acetylcholine in cirrhotic and control rings. 4. These results demonstrate that in aortic rings from cirrhotic, bile duct-ligated rats, increased production of nitric oxide, mainly of endothelial origin, is responsible for the lower contractile response to phenylephrine. Our data, however, do not support the involvement of the inducible nitric oxide synthase isoform in this alteration. In contrast, endothelial vasodilatory response to acetylcholine is not altered in this model of cirrhosis, which indicates that not all mechanisms of nitric oxide release are abnormal.     

Cerebral haemodynamics and embolization during carotid angioplasty in high-risk patients

The genomes of 10 equine herpesvirus 1 (EHV-1) strains isolated in Argentina from 1979 to 1991, and a Japanese HH1 reference strain were compared by restriction endonuclease analysis. Two restriction enzymes, BamHI and BglII, were used and analysis of the electropherotypes did not show significant differences among isolates obtained from horses with different clinical signs. This suggests that the EHV-1 isolates studied, which circulated in Argentina for more than 10 years, belong to a single genotype.     

Remote fetal medicine consultation using stored ultrasound video images

Fifty-four (54) unrelated patients with Mediterranean Kaposi's sarcoma (MKS) and 8 patients members of 4 unrelated families with familial MKS were serotyped for HLA-A,B and DR antigens. The diagnosis was histologically confirmed and all patients were negative for anti-HIV antibodies. An increased frequency of HLA-B18 (44.4% vs 14.2% in the controls, p < 0.001, RR = 4.8) and HLA-DR5 (57.6% vs 37.2% in the controls, p < 0.025, RR = 2.29) was observed in the group of patients with MKS. Seven (7) of the 8 family members with FMKS possessed HLA-DR5, and the affected members in the 3 families shared a common haplotype which included HLA-DR5. These findings support the hypothesis that genetic factors linked to HLA-DR5 antigen may contribute to the pathogenesis of MKS.     

Activation and cell cycle antigens in CD4+ and CD8+ T cells correlate with plasma human immunodeficiency virus (HIV-1) RNA level in HIV-1 infection

gamma-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine gamma-sarcoglycan gene was disrupted using homologous recombination. Mice lacking gamma-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of gamma-sarcoglycan produced secondary reduction of beta- and delta-sarcoglycan with partial retention of alpha- and epsilon-sarcoglycan, suggesting that beta-, gamma-, and delta-sarcoglycan function as a unit. Importantly, mice lacking gamma-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, beta-dystroglycan and laminin were left intact, implying that the dystrophin-dystroglycan-laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking gamma-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking gamma-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.     

Molecular mechanisms for the regulation of the expression and function of muscarinic acetylcholine receptors

The regulation of muscarinic acetylcholine receptor expression and function was investigated in cultured cells and in knockout mice. Muscarinic agonist exposure causes m2 receptor desensitization and sequestration and decreases the expression of cardiac potassium channels. The expression of m2 receptors in chick retina is regulated by a developmentally regulated secreted factor. Mice lacking the m1 receptor exhibit a loss of muscarinic regulation of M-current potassium channel activity and pilocarpine-induced seizures.