Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation.

To assess the role of cis-acting elements within the smooth muscle alpha-actin gene in smooth muscle cells (SMC), we transfected chicken smooth muscle alpha-actin promoter-chloramphenicol acetyltransferase gene fusion plasmids into SMC derived from rat and chicken aortas. In marked contrast to effects in chicken skeletal myoblasts and fibroblasts, p122CAT (positions -122 to +19), containing two conserved CArG elements, elicited a modest increase in chloramphenicol acetyltransferase reporter activity in chicken SMC. Addition of upstream sequences between -122 and -151 (p151CAT) increased activity in adult chicken SMC.

Public screening for lung disease: experience with the NIH Lung Health Study.

The Lung Health Study is the first major initiative of the National Heart, Lung, and Blood Institute in screening for and intervention in early lung disease. The objective of the study is to identify cigarette smokers who have early disease and determine whether an intervention program of smoking cessation and bronchodilator therapy will alter the course of their disease. After an extensive screening program, 5,887 participants have been randomized to one of three groups: usual care, intervention with smoking cessation and the use of a placebo inhaler, and intervention with smoking cessation and the use of ipratropium bromide.

Schwann cells infected with a recombinant retrovirus expressing myelin-associated glycoprotein antisense RNA do not form myelin.

To elucidate the role of myelin-associated glycoprotein (MAG) in the axon-Schwann cell interaction leading to myelination, neonatal rodent Schwann cells were infected in vitro with a recombinant retrovirus expressing MAG antisense RNA or MAG sense RNA. Stably infected Schwann cells and uninfected cells were then cocultured with purified sensory neurons under conditions permitting extensive myelination in vitro. A proportion of the Schwann cells infected with the MAG antisense virus did not myelinate axons and expressed lower levels of MAG than control myelinating Schwann cells, as measured by immunofluorescence.

Assignment of the human prohibitin gene (PHB) to chromosome 17 and identification of a DNA polymorphism.

Prohibition is a recently identified antiproliferative protein whose exact role in the cell is under investigation. To determine the human chromosomal location of the prohibition gene (PHB) and whether this site corresponds to that of any suspected tumor suppressor gene, we have analyzed DNA from three sources by hybridization analysis: mouse--human hybrid cell lines, hybrid cell lines containing portions of human chromosomes, and human metaphase chromosomes in situ. All three techniques confirm a location in the region 17q21-q22, a region genetically linked to early-onset human breast cancer.

Identification of mRNAs associated with programmed cell death in immature thymocytes.

Programmed cell death is an essential cellular process that occurs in epithelial turnover, neural development, and regulation of cell populations of the immune system. Thymocytes undergo programmed cell death in response to several inductive stimuli, including exposure to glucocorticoids or radiation. This program can be blocked by inhibitors of RNA or protein synthesis; this implies that new proteins are required to execute the death programs.

The effect of obliterative bronchiolitis on breathing pattern during exercise in recipients of heart-lung transplants.

The more rapid and shallow ventilation pattern seen during exercise in patients with obstructive and/or restrictive lung disease has been attributed by some investigators to the effects of vagal afferents from intrapulmonary receptors. Recipients of heart-lung transplants (HLTR) offer a unique opportunity to test this hypothesis since they have denervated lungs and may develop obliterative bronchiolitis after organ rejection. We thus compared the ventilation responses to incremental bicycle ergometry of five HLTR with relatively normal pulmonary function (HLTR-N) and four with bronchiolitis obliterans (HLTR-O).

Expressing antisense P0 RNA in Schwann cells perturbs myelination.

Primary Schwann cells were infected in vitro with a recombinant retrovirus expressing a dominant selectable marker, neomycin phosphotransferase (conferring resistance to the drug G418), and antisense P0 RNA under the control of the human beta-actin promoter. A proportion of the G418-resistant cells failed to form myelin when cocultured with dorsal root ganglion neurons under conditions that promote Schwann cell differentiation. These cells expressed high levels of P0 antisense RNA.

Mechanism of enlargement of major cerebral collateral arteries in rabbits.

Major cerebral collateral arteries enlarge following bilateral ligation of the common and internal carotid arteries. The purpose of this investigation was to determine the relative contribution of cellular hypertrophy versus cellular hyperplasia to this vessel change in a morphometric analysis as well as the functional properties of remodeled vessels in an in vitro study. We assessed cell number and vessel dimensions by morphometric analysis of 16 perfusion-fixed rabbit basilar arteries.

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells.

L-3,4-dihydroxyphenylalanine synthesis by genetically modified Schwann cells.

We have investigated whether Schwann cells can be modified by gene transfer to synthesize L-3,4-dihydroxyphenylalanine (L-DOPA), the immediate precursor in the formation of dopamine. By using a retrovirus containing a rat tyrosine hydroxylase (TH) cDNA, we established an immortalized rodent Schwann cell line that stably expressed high levels of TH and secreted L-DOPA in vitro when supplied with tyrosine and the essential cofactor biopterin. We also infected primary Schwann cells and demonstrated that cells expressing TH secreted L-DOPA while maintaining their capacity to myelinate neurons in vitro.

Mechanisms of angiotensin II- and arginine vasopressin-induced increases in protein synthesis and content in cultured rat aortic smooth muscle cells. Evidence for selective increases in smooth muscle isoactin expression.

Previous studies from this laboratory have demonstrated that angiotensin II (Ang II) and arginine vasopressin (AVP) are potent hypertrophic agents in cultured rat aortic smooth muscle cells. The present study identified major proteins that accumulate in Ang II-induced and AVP-induced hypertrophic cells and initiated studies of the mechanisms that contribute to their accumulation. Smooth muscle cell hypertrophy induced by Ang II and/or AVP (1 microM each) was associated with widespread increases in the content of many cellular proteins that were resolved by one- and two-dimensional gel electrophoresis.

Role of contractile agonists in growth regulation of vascular smooth muscle cells.

There is now clear evidence demonstrating that contractile agonists such as angiotensin II and arginine vasopressin are potent hypertrophic agents for cultured vascular smooth muscle cells. Furthermore, there is circumstantial evidence supporting a role for these factors in mediation of smooth muscle cell hypertrophy in hypertensive animal models as well as in maintenance of contractile mass in normotensive animals. At least part of the hypertrophic effect of angiotensin II and arginine vasopressin appears to involve a generalized increase in protein synthesis since the synthesis of most if not all proteins is increased to some extent.

Platelet-derived growth factor-induced destabilization of smooth muscle alpha-actin mRNA.

We have previously shown that treatment of postconfluent, quiescent rat vascular smooth muscle cells (SMC) with platelet-derived growth factor (PDGF) dramatically reduced smooth muscle (SM) alpha-actin synthesis and SM alpha-actin mRNA abundance, suggesting a role for this mitogen in the control of SMC differentiation. In the present studies, we explored the molecular mechanisms whereby PDGF decreases SM alpha-actin mRNA levels. Treatment of postconfluent SMC with both platelet PDGF and recombinant PDGF-BB resulted in a dramatic and concentration-dependent decrease in SM alpha-actin mRNA levels.

The myelin-associated glycoproteins: membrane disposition, evidence of a novel disulfide linkage between immunoglobulin-like domains, and posttranslational palmitylation.

The myelin-associated glycoproteins (MAG) are members of the immunoglobulin gene superfamily that function in the cell interactions of myelinating glial cells with axons. In this paper, we have characterized the structural features of these proteins. The disposition of MAG in the bilayer as a type 1 integral membrane protein (with an extracellularly disposed amino terminus, single transmembrane segment, and cytoplasmic carboxy terminus) was demonstrated in protease protection studies of MAG cotranslationally inserted into microsomes in vitro and in immunofluorescent studies with site specific antibodies.

Expression of recombinant myelin-associated glycoprotein in primary Schwann cells promotes the initial investment of axons by myelinating Schwann cells.

Myelin-associated glycoprotein (MAG) is an integral membrane protein expressed by myelinating glial cells that occurs in two developmentally regulated forms with different carboxyterminal cytoplasmic domains (L-MAG and S-MAG). To investigate the role of MAG in myelination a recombinant retrovirus was used to introduce a MAG cDNA (L-MAG form) into primary Schwann cells in vitro. Stably infected populations of cells were obtained that constitutively expressed MAG at the cell surface without the normal requirement for neuronal contact to induce expression.

The short-term effects of smoke exposure on the pulmonary function of firefighters.

The short-term effects of smoke inhalation have been little studied. This study evaluated whether firefighters experience a significant change in spirometric values following exposure to smoke from a fire. Sixty firefighters from the city of Pittsburgh completed a questionnaire (Medical Research Council) and underwent spirometric testing following exposure to house fires.

Platelet-derived growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells.

The role of platelet-derived growth factor (PDGF) in the control of smooth muscle cell (SMC) differentiation was explored in vitro by examining its effects on expression of the smooth muscle (SM) specific contractile protein SM alpha actin in cultured rat aortic SMC. Quiescent, postconfluent SMC express maximal levels of alpha actin and responded to human platelet-derived growth factor (partially purified from platelets) by entering the cell cycle and undergoing approximately one synchronous round of DNA synthesis. Concomitantly, these cultures exhibited a marked reduction in alpha actin synthesis.

Studies of the initiation of myelination by Schwann cells.

The rapid morphologic changes in Schwann cells and in their relationships to axons during the transition from the premyelinating to the myelinating state have been known for more than 15 years. The sorting of axons by dividing Schwann cells, the establishment of a 1:1 relationship between a postmitotic Schwann cell, and the onset of myelin sheath formation have all been described in detail. However, the chain of molecular events and mechanisms by which these morphologic changes are regulated has not been elucidated.

Resolution of the pathway taken by implanted Schwann cells to a spinal cord lesion by prior infection with a retrovirus encoding beta-galactosidase.

This series of experiments is designed to follow the fate of implanted Schwann cells by first labeling them with a recombinant retrovirus encoding the bacterial beta-galactosidase gene, then injecting them into the spinal cord after a demyelinating lesion has been produced. The label provides a means of distinguishing the exogenous Schwann cells from endogenous ones and of determining their travel pattern and myelinating or ensheathing behavior in the central nervous system (CNS). Neonatal rat primary Schwann cells were stimulated to divide by administering glial growth factor and forskolin.

Neurons and glia arise from a common progenitor in chicken optic tectum: demonstration with two retroviruses and cell type-specific antibodies.

We used a recombinant retrovirus to study cell lineage in the chicken optic tectum. The virus inserts the Escherichia coli lacZ (beta-galactosidase) gene into the genome of an infected cell; a histochemical stain marks the progeny of infected cells with a blue precipitate. We had previously shown that individual clones frequently contain diverse neuronal types.

Schwann cells depleted of galactocerebroside express myelin-associated glycoprotein and initiate but do not continue the process of myelination.

Two peripheral myelin components, galactocerebroside (GalC) and myelin-associated glycoprotein (MAG), are known to be expressed early in Schwann cell differentiation, prior to the formation of definitive myelin segments containing compacted membrane. To discern the relative roles of these myelin components, cultures of Schwann cells and dorsal root ganglion neurons were treated with antigalactocerebroside mAbs in order to remove GalC from the Schwann cell surface (Ranscht et al., 1987).

Control of hypertrophic versus hyperplastic growth of vascular smooth muscle cells.

A long-term objective of my laboratory has been to understand the mechanisms that regulate both normal and developmental growth of vascular smooth muscle as well as the accelerated growth of smooth muscle that occurs in atherosclerotic lesions or arteries of hypertensive patients and animals. Previous studies in this and other laboratories have demonstrated that smooth muscle cells are capable of two distinct growth responses in vivo, depending on the nature of the growth stimulus. Smooth muscle cell growth in large vessels of chronically hypertensive animals appears to occur primarily by enlargement of preexisting cells (i.

Growth response of tetraploid smooth muscle cells to balloon embolectomy-induced vascular injury in the spontaneously hypertensive rat.

Previous studies in this laboratory have demonstrated that smooth muscle hypertrophy in large arteries of hypertensive animals is accompanied by development of polyploidy in a large fraction of the smooth muscle cell population. The present studies address whether there is an inherent loss in the ability of tetraploid cells to respond to proliferative stimuli by examining whether these cells initiate DNA replication after balloon catheter-induced vascular injury. Left carotid arteries of 5- to 7-month-old spontaneously hypertensive rats were subjected to balloon injury, and the animals were perfused intravenously with [3H]thymidine for 32 h.

Sequence and expression of the murine diazepam binding inhibitor.

Previous studies suggest that a diazepam binding inhibitor (DBI, also referred to as endozepine) present in the brain may function anxiogenically as a modulator of the gamma-aminobutyric acid receptor complex (GABAA). An expression library representing mouse brain mRNA was screened using antisera that recognizes the 11 kDa DBI protein. A cDNA clone was isolated and sequenced.