Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphoryl

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (h beta c). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of h beta c. We report here a comprehensive screen of the entire h beta c molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of h beta c that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most h beta c mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together; these results highlight key regions involved in h beta c activation, dissociate h beta c tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by h beta c. (C) 1998 by The American Society of Hematology.

Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice

Previously we described activating mutations of h beta(c), the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, h beta(c)FI Delta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the h beta(c)FI Delta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in h beta(c) may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic h beta(c) activation has the potential to contribute to pathological events in the central nervous system.

Suicide and recency of health care contacts - A systematic review

Background Many countries have set targets for suicide reduction, and suggested that mental health care providers and general practitioners have a key role to play. Method Asystematic review of the literature. Results Among those in the general population who commit suicide, up to 41% may have contact with psychiatric inpatient care in the year prior to death and up-to 9% may commit suicide within one day of discharge. The corresponding figures are I I and 4% for community-based psychiatric care and 83 and 20% for general practitioners. Conclusions Among those who die by suicide. contact with health services is common before death. This is a necessary but not sufficient condition for clinicians to intervene. More work is needed to determine whether these people show characteristic patterns of care and/or particular risk factors which would enable a targeted approach to be developed to assist clinicians in detecting and managing high-risk patients.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Urban and rural suicide differentials in migrants and the Australian-born, New South Wales, Australia 1985-1994

We estimated risk of suicide in adults in New South Wales (NSW) by sex, country of birth and rural/urban residence, after adjusting for age; we also examined youth suicide (age 15-24 years). The study population was the entire population of NSW, Australia, aged greater than or equal to 15 years during the period 1985-1994. Poisson regression was used to examine the relationship between predictor variables and the risk of suicide, with the focus on migrant status and area of residence. A significantly higher risk of suicide was found in male migrants from Northern Europe and Eastern Europe/former USSR, compared to Australian-born males; a significantly lower suicide risk occurred in males from Southern Europe, the Middle East and Asia. In female migrants, those from UK/Eire, Northern Europe, Eastern Europe/former USSR and New Zealand exhibited a significantly higher risk of suicide compared to Australian-born females. A significantly lower risk of suicide occurred in females from the Middle East. Male migrants overall were at significantly lower risk of suicide than the Australian-born, while female migrants overall had a significantly higher risk of suicide than Australian-born females. Among migrant males overall, the rural-urban suicide risk differential was significantly higher for those living in non-metropolitan areas (RR = 1.9; 95% CI: 1.7-2.1). Suicide risk was significantly higher in non-metropolitan male immigrants from the UK/Eire (RR = 1.4; 95% CI: 1.1-1.7), Southern Europe (RR = 1.7; 95% CI: 1.2-2.4), Northern/Western Europe (1.5; 95% CI: 1.2-1.9), the Middle East (RR = 3.8; 95% CI: 1.9-7.8), New :Zealand (RR = 1.4; 95% CI: 1.0-1.8) and 'other' (RR = 2.6; 95% CI: 1.9-3.5), when compared to their urban counterparts. There was no statistically significant difference in suicide risk between rural and urban Australian-born males. For female suicide, significantly lower risk was found in female immigrants living in non-metropolitan areas who were from Northern/Western Europe (RR = 0.7; 95% CI: 0.4-0.96), as well as the Australian-born (RR = 0.7; 95% CI: 0.6-0.8), when compared to their urban counterparts. The non-metropolitan/metropolitan relative risk for suicide in female migrants overall was not significantly different from one. Among male youth there was a significantly higher suicide risk in non-metropolitan areas, with a relative risk estimate of 1.4 for Australian-born youth (95% CI: 1.2-1.5) and 1.7 for migrant youth (95% CI: 1.2-2.4), when compared with metropolitan counterparts. We conclude that suicide among migrant males living in non-metropolitan areas accounts for most of the excess of male suicide in rural NSW, and the significantly lower risk of suicide for non-metropolitan Australian-born women does not apply to migrant women. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.

Myeloproliferative disorder and leukaemia in mice induced by different classes of constitutive mutants of the human IL-3/IL-5/GM-CSF receptor common beta subunit

Several constitutively active mutant forms of the common β subunit of the human IL-3, IL-5 and GM-CSF receptors (hβc), which enable it to signal in the absence of ligand, have recently been described. Two of these, V449E and I374N, are amino acid substitutions in the transmembrane and extracellular regions of hβc, respectively. A third, FIΔ, contains a 37 amino acid duplication in the extracellular domain. We have shown previously that when expressed in primary murine haemopoietic cells, the extracellular mutants confer factor-independence on cells of the neutrophil and monocyte lineages only, whereas V449E does so on all cell types of the myeloid and erythroid compartments. To study the in vivo effects and leukaemic potential of these mutants, we have expressed all three in mice by bone marrow reconstitution using retrovirally infected donor cells. Expression of the extracellular mutants leads to an early onset, chronic myeloproliferative disorder marked by elevations in the neutrophil, monocyte, erythrocyte and platelet lineages. In contrast, expression of V449E leads to an acute leukaemia-like syndrome of anaemia, thrombocytopaenia and blast cell expansion. These data support the possibility that activating mutations in hβc are involved in haemopoietic disorders in man.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.

Tension regulation during lengthening and shortening actions of the human soleus muscle

In the present study we investigated tension regulation in the human soleus (SOL) muscle during controlled lengthening and shortening actions. Eleven subjects performed plantar flexor efforts on an ankle torque motor through 30 degrees of ankle displacement (75 degrees-105 degrees internal ankle angle) at lengthening and shortening velocities of 5, 15 and 30 degrees s(-1). To isolate the SOL from the remainder of the triceps surae, the subject's knee was flexed to 60 degrees during all trials. Voluntary plantar flexor efforts were performed under two test conditions: (1) maximal voluntary activation (MVA) of the SOL, and (2) constant submaximal voluntary activation (SVA) of the SOL. SVA trials were performed with direct visual feedback of the SOL electromyogram (EMG) at a level resulting in a torque output of 30% of isometric maximum. Angle-specific (90 degrees ankle angle) torque and EMG of the SOL, medial gastrocnemius (MG) and tibialis anterior (TA) were recorded. In seven subjects from the initial group, the test protocol was repeated under submaximal percutaneous electrical activation (SEA) of SOL (to 30% isometric maximal effort). Lengthening torques were significantly greater than shortening torques in all test conditions. Lengthening torques in MVA and SVA were independent of velocity and remained at the isometric level, whereas SEA torques were greater than isometric torques and increased at higher lengthening velocities. Shortening torques were lower than the isometric level for all conditions. However, whereas SVA and SEA torques decreased at higher velocities of shortening, MVA torques were independent of velocity. These results indicate velocity- and activation-type-specific tension regulation in the human SOL muscle.

Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

We have generated transgenic mice that harbor a 140 kb genomic fragment of the human BRCA1 locus (TgN.BRCA1(GEN)). We find that the transgene directs appropriate expression of human BRCA1 transcripts in multiple mouse tissues, and that human BRCA1 protein is expressed and stabilized following exposure to DIVA damage, Such mice are completely normal, with no overt signs of BRCA1 toxicity commonly observed when BRCA1 is expressed from heterologous promoters. Most importantly, however, the transgene rescues the otherwise lethal phenotype associated with the targeted hypomorphic allele (Brca1(Delta exIISA)). Brca1(-/-); TgN.BRCA1(GEN) bigenic animals develop normally and can be maintained as a distinct line. These results show that a 140 kb fragment of chromosome 17 contains all elements necessary for the correct expression, localization, and function of the BRCA1 protein, Further, the model provides evidence that function and regulation of the human BRCA1 gene can be studied and manipulated in a genetically tractable mammalian system.

Changes in intra-abdominal pressure during postural and respiratory activation of the human diaphragm

In humans, when the stability of the trunk is challenged in a controlled manner by repetitive movement of a limb, activity of the diaphragm becomes tonic but is also modulated at the frequency of limb movement. In addition, the tonic activity is modulated by respiration. This study investigated the mechanical output of these components of diaphragm activity. Recordings were made of costal diaphragm, abdominal, and erector spinae muscle electromyographic activity; intra-abdominal, intrathoracic, and transdiaphragmatic pressures; and motion of the rib cage, abdomen, and arm. During limb movement the diaphragm and transversus abdominis were tonically active with added phasic modulation at the frequencies of both respiration and limb movement. Activity of the other trunk muscles was not modulated by respiration. Intra-abdominal pressure was increased during the period of limb movement in proportion to the reactive forces from the movement. These results show that coactivation of the diaphragm and abdominal muscles causes a sustained increase in intra-abdominal pressure, whereas inspiration and expiration are controlled by opposing activity of the diaphragm and abdominal muscles to vary the shape of the pressurized abdominal cavity.

The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-alpha

It is now well recognized that cervical cancer is caused by infection with certain human papillomavirus (HPV) subtypes and while interferon-alpha (IFN-alpha) is used to treat HPV-infected lesions, HPV appears to have developed a means to avoid the effects of IFN-alpha. Clinically, resistance appears to be associated with the expression of the E7 oncoprotein. Here we investigated the effects of expression in cells of the E7 protein from high- and low-risk papillomavirus subtypes on a range of responses to IFN-alpha. 2fTGH, a cell line dependent on IFN-alpha for growth in selection medium, grew significantly less well in the presence of E7, and the antiproliferative effects of IFN-alpha upon epithelial cells was lost upon E7 expression. The antiviral effects of IFN-alpha were abrogated in E7-expressing cells. Loss of response to IFN-alpha was found to occur in both high- and low-risk papillomaviruses. Finally, deletion of amino acids 21-24 of HPV type 16 E7 protein partially reversed repression. We conclude that E7 inhibits the functional effects of IFN-alpha and that this property is shared by all HPV subtypes tested. (C) 2000 Academic Press.

H-reflex modulation during passive lengthening and shortening of the human triceps surae

1. The present study investigated the effects of lengthening and shortening actions on IT-reflex amplitude. H-reflexes were evoked in the soleus (SOL) and medial gastroenemius (MG) of human subject, during passive isometric, lengthening and shortening actions performed at angular velocities of 0, +/-2, +/-5 and +/- 15 deg s(-1). 2. H-reflex amplitude, in froth SOL and MG were significantly depressed during passive lengthening actions and facilitated during passive shortening actions, when compared with the isometric R-reflex amplitude. 3. Four experiments were performed in which the latencies front the onset of movement to delivery of the stimulus were altered. Passive H-reflex modulation during lengthening actions was found tee begin at latencies of less than 60 ms suggesting that this inhibition was due to peripheral and/or spinal mechanisms. 4. It is postulated that, the H-reflex modulation seen in the present study is related to the tunic discharge of muscle spindle afferents and the consequent effects of transmission within the la pathway. Inhibition of the H-reflex at less than 60 ms after the onset of muscle lengthening may he attributed to several mechanisms, which cannot be distinguished using the current protocol. These may include the inability to evoke volleys in la fibres that are refractory following muscle spindle discharge during; rapid muscle lengthening, a reduced probability of transmitter release front the presynaptic terminal (homosynaptic post.-activation depression) and presynaptic inhibition of la afferents from plantar flexor agonists. Short latency facilitation of the H-reflex may be attributed to temporal summation of excitatory postsynaptic potentials arising from muscle spindle afferents during rapid muscle lengthening. At longer latencies, presynaptic inhibition of Ia afferents cannot be excluded as a potential inhibitory mechanism.