TLS-ERG leukemia fusion protein inhibits RNA splicing mediated by serine-arginine proteins.

The translocation liposarcoma (TLS) gene is fused to the ETS-related gene (ERG) in human myeloid leukemia, resulting in the generation of a TLS-ERG protein. We demonstrate that both TLS and the TLS-ERG leukemia fusion protein bind to RNA polymerase II through the TLS N-terminal domain, which is retained in the fusion protein; however, TLS recruits members of the serine-arginine (SR) family of splicing factors through its C-terminal domain, whereas the TLS-ERG fusion protein lacks the ability to recruit SR proteins due to replacement of the C-terminal domain by the fusion partner ERG. In transient-transfection assays, the TLS-ERG fusion protein inhibits E1A pre-mRNA splicing mediated by these TLS-associated SR proteins (TASR), and stable expression of the TLS-ERG fusion protein in K562 cells alters the splicing profile of CD44 mRNA. These results suggest that TLS fusion proteins may lead to cellular abnormalities by interfering with the splicing of important cellular regulators.

The effect of hypoxia on monoamine levels in discrete regions of aged rat brain.

Aged rats were exposed to 10% oxygen for 1, 13, and 36 hr. Norepinephrine levels in cerebral cortex, hypothalamus, hippocampus, midbrain, cerebellum, pons-medulla and dopamine levels in striatum were determined after each exposure. While there was no significant change in monoamine levels in brain regions after 2 hr, norepinephrine concentration in hypothalamus and midbrain decreased significantly after 13 hr of hypoxia. After 36 hr in a hypoxic environment, levels of the monoamines in brain regions were similar to the controls. This would suggest NE metabolism is most vulnerable to hypoxia in two regions of the aged brain. The precise mechanism of these changes is unknown, but they suggest both a vulnerability and an adaptive recovery of central adrenergic metabolism by the aged brain under hypoxia.

Volume infusion produces abdominal distension, lung compression, and chest wall stiffening in pigs.

The effect of severe generalized edema on respiratory system mechanics is not well described. We measured airway pressure, gastric pressure, and four vertical pleural pressures in 13 anesthetized paralyzed pigs ventilated in the upright position. Pressure-volume relationships of the respiratory system, chest wall, and lung were measured on deflation from total lung capacity to residual volume and during tidal breathing both before (control) and 50 min after one of two interventions. In one series of experiments, a volume equal to 15-20% of the pig's body weight was infused intravenously. In a second series, a balloon was placed in the peritoneal space to distend the abdomen to the same gastric pressures as achieved in the first series. Measurements were compared before and after either abdominal balloon inflation or volume infusion. Volume infusion increased the pleural pressure in dependent lung regions, decreased both total lung capacity (34%) and functional residual capacity (62%) (both P less than 0.05), and markedly shifted the respiratory system and chest wall pressure-volume curves to the right, but it only moderately affected the lung deflation curve. Tidal compliances of the respiratory system, chest wall, and lung decreased 36, 31, and 49%, respectively (all P less than 0.05). The effect of abdominal balloon inflation on respiratory system mechanics was similar to that of volume infusion. We conclude that infusing large volumes of fluid markedly alters chest wall mechanics, mainly by causing abdominal distension that prohibits descent of the diaphragm.

Abdominal distension alters regional pleural pressures and chest wall mechanics in pigs in vivo.

Abdominal distension (AD) occurs in pregnancy and is also commonly seen in patients with ascites from various causes. Because the abdomen forms part of the "chest wall," the purpose of this study was to clarify the effects of AD on ventilatory mechanics. Airway pressure, four (vertical) regional pleural pressures, and abdominal pressure were measured in five anesthetized, paralyzed, and ventilated upright pigs. The effects of AD on the lung and chest wall were studied by inflating a liquid-filled balloon placed in the abdominal cavity. Respiratory system, chest wall, and lung pressure-volume (PV) relationships were measured on deflation from total lung capacity to residual volume, as well as in the tidal breathing range, before and 15 min after abdominal pressure was raised. Increasing abdominal pressure from 3 to 15 cmH2O decreased total lung capacity and functional residual capacity by approximately 40% and shifted the respiratory system and chest wall PV curves downward and to the right. Much smaller downward shifts in lung deflation curves were seen, with no change in the transdiaphragmatic PV relationship. All regional pleural pressures increased (became less negative) and, in the dependent region, approached 0 cmH2O at functional residual capacity. Tidal compliances of the respiratory system, chest wall, and lung were decreased 43, 42, and 48%, respectively. AD markedly alters respiratory system mechanics primarily by "stiffening" the diaphragm/abdomen part of the chest wall and secondarily by restricting lung expansion, thus shifting the lung PV curve as seen after chest strapping. The less negative pleural pressures in the dependent lung regions suggest that nonuniformities of ventilation could also be accentuated and gas exchange impaired by AD.

Herpes zoster myelitis occurring during treatment for systemic lupus erythematosus.

A 15-year-old girl with systemic lupus erythematosus suddenly developed fever, meningismus, and herpes zoster. Within 48 hours, transverse myelitis developed at the level of the nerve root involvement of the herpes zoster. Since both systemic lupus erythematosus and varicella-zoster have been reported to cause myelitis, therapy was initiated for both. The rapid and simultaneous resolution of both the herpes zoster and the neurologic deficits strongly supports the causal association of both with varicella-zoster. This is the second reported case of herpes zoster-associated transverse myelitis in a patient with systemic lupus erythematosus.

Post mortem stability of catecholamines in discrete regions of rat brain.

Male Sprague-Dawley rats (200-300 g) were sacrificed by cervical dislocation and left either at room temperature (21-23 degrees C) for periods of 1, 2, and 4 hours or in the refrigerator (4-5 degrees C) for periods of 1, 2, 4, 8, 16, and 24 hours. The brains were left in the intact animals in order to stimulate human autopsy materials. At the end of each period, the rat was decapitated and the brain removed and immediately dissected into the following regions: cerebral cortex, striatum, midbrain, pons and medulla, and cerebellum. The levels of norepinephrine (NE) and dopamine (DA) in brain regions were determined fluorometrically. In animals kept at room temperature the level of NE decreased gradually with time reaching 61%, 76%, 68%, and 68% of control in the cerebral cortex, midbrain, pons and medulla, and cerebellum at 4 hours post mortem respectively. Striatal DA reached 61% of control at 2 hours and remained the same at 4 hours post mortem. The NE level declined at a slower rate in brain regions obtained from refrigerated animals, reaching 65%, 81%, 77%, and 77% of control in cerebral cortex, midbrain, pons and medulla, and cerebellum at 24 hours post mortem. Striatal DA reached 54% of control at 16 hours and showed no further decline at 54 hours post mortem. There was a significant post mortem decrease in NE and DA levels in all brain regions examined, however, the rate of this decline varied from region to region. The tissues obtained from refrigerated animals showed statistically less of a decline in amine levels.

Changes in catecholamine levels in discrete regions of rat brain during aging.

The effect of aging on the levels of dopamine (DA) in striatum and noradrenaline (NA) in cerebral cortex, cerebellum, hypothalamus, midbrain and pons-medulla from young adult and five groups of aging rats was evaluated. Determinations of the levels of these amines were conducted. Age-related changes which commenced at 12 months were observed in specific brain regions. The endogenous content of NA decreased significantly in hypothalamus, midbrain, and pons-medulla when compared with the same regions from young adult animals. These results show that central noradrenergic system is markedly vulnerable to the aging process in rats.

Subacute brain-stem encephalitis.

A 65 year old man developed progressive signs of pontine and medullary dysfunction with striking bilateral paralysis of lateral gaze, dysarthria, dysphagia, and ataxia. A respiratory death occurred seven months from the onset. Pathological examination revealed focal brain-stem changes of perivascular lymphocytic cuffing, microglial infiltration, glial nodules, and neuronophagia. No underlying malignancy or general disease impairing immunity mechanisms was discovered.

Expression and function of beta 2 integrins on alveolar macrophages from human and nonhuman primates.

The alveolar macrophage (AM) participates in diverse, adherence-related activities required for host defense and the inflammatory response. The beta 2 integrins (the CD11/CD18 heterodimer) mediate some of these activities on circulating leukocytes and peritoneal macrophages. We investigated expression of the CD11/CD18 leukocyte integrin subunits on AMs obtained by bronchoalveolar lavage of human and nonhuman primates. We also determined the role of the CD11/CD18 complex in AM chemotaxis and adherence to A549 alveolar epithelial cell monolayers. Immunofluorescence flow cytometry indicated that the CD11a/CD18 complex was expressed in high levels and CD11b/CD18 and CD11c/CD18 in lower levels on the AM surface. Northern blot analysis indicated the presence of CD11a, CD11c, and CD18 mRNA in the AMs. Smaller quantities of CD11b mRNA were also found. AM chemotaxis to zymosan-activated serum was markedly inhibited by a monoclonal antibody to CD18. In addition, adherence of AMs to A549 cells (stimulated by tumor necrosis factor to upregulate intercellular adhesion molecule-1 expression) was decreased from 30.3 +/- 5.0 to 20.8 +/- 2.4% (P less than 0.05) by the same monoclonal antibody. We conclude that: (1) AMs obtained from human and nonhuman primates constitutively express predominantly CD11a/CD18 surface antigen and mRNA, (2) chemotaxis of AMs is CD18 dependent, and (3) adhesion of AMs to an alveolar epithelial cell monolayer is partly but not completely dependent on the beta 2 integrins.

Oncoprotein TLS interacts with serine-arginine proteins involved in RNA splicing.

The gene encoding the human TLS protein, also termed FUS, is located at the site of chromosomal translocations in human leukemias and sarcomas where it forms a chimeric fusion gene with one of several different genes. To identify interacting partners of TLS, we screened a yeast two-hybrid cDNA library constructed from mouse hematopoietic cells using the C-terminal region of TLS in the bait plasmid. Two cDNAs encoding members of the serine-arginine (SR) family of proteins were isolated. The first SR protein is the mouse homolog of human splicing factor SC35, and the second SR member is a novel 183-amino acid protein that we term TASR (TLS-associated serine-arginine protein). cDNA cloning of human TASR indicated that mouse and human TASR have identical amino acid sequences. The interactions between TLS and these two SR proteins were confirmed by co-transfection and immunoprecipitation studies. In vivo splicing assays indicated that SC35 and TASR influence splice site selection of adenovirus E1A pre-mRNA. TLS may recruit SR splicing factors to specific target genes through interaction with its C-terminal region, and chromosomal translocations that truncate the C-terminal region of TLS may prevent this interaction. Thus TLS translocations may alter RNA processing and play a role in malignant transformation.

Human leukocyte integrin CD18 promoter directs low levels of expression of a mutated human CD4 reporter gene in leukocytes of transgenic mice.

The human leukocyte integrin CD18 molecule exists on the leukocyte surface in heterodimeric complexes with individual CD11 subunits, which mediate important leukocyte adhesion reactions. The CD18 subunit is developmentally regulated with the highest levels present on mature leukocytes of all lineages. To identify the regulatory sequences responsible for the tissue- and stage-specific expression of the CD18 subunit, we used 3.5 kb of regulatory sequence upstream from the human CD18 gene transcription start site to drive expression of a modified human CD4 reporter gene in transgenic mice. Despite the inclusion of Sp1 and PU.1 sites in the construct, and the generation of founder lines possessing multiple copies of the transgene, the reporter gene was expressed in low levels in the leukocytes of the transgenic mice. These studies indicate that although PU.1 and Sp1 sites are required for CD18 promoter activity in vitro, additional regulatory regions appear to be required for high levels of copy number dependent expression in vivo.

Aging effect on the noradrenaline content of rat brain microvessels.

Microvessels were isolated by sucrose gradient centrifugation and molecular seiving from forebrains of aged and young adult male Sprague-Dawley rats. Determination of noradrenaline levels in preparations obtained from both groups of animals indicated that the level of NA in cerebral microvessels isolated from aged animals (120 /+- 28 pg NA/mg protein) was significantly reduced when compared with the corresponding value from control (young adult) animals (226 /+- 35 pg NA/mg protein). This decrease in NA may indicate a deficiency in the neural control of the microcirculation and predispose to metabolic and functional disturbances of brain tissue.