Adenovirus-mediated expression of interleukin-1 receptor antagonist in swine cells in vitro and in vivo.

Adenovirus-mediated gene delivery of anticytokines is a powerful tool for modulating the cytokine environment under conditions of respiratory disease. In order to determine the feasibility of cytokine modulation in the context of respiratory disease in swine, nonreplicating E1- and E3-deficient adenovirus constructs expressing a model protein, beta-galactosidase, and an anticytokine, the IL-1 receptor antagonist (IL-1Ra), were evaluated for in vitro expression in porcine PK15 cells, and in vivo following endotracheal instillation into the lungs. beta-Galactosidase and IL-1Ra were readily expressed in vitro in swine cells. Endotracheal administration of lacZ-containing adenovirus demonstrated that endothelial and epithelial cells in the alveolar spaces and bronchi of the middle and lower lobes were the principal sites of infection and expression, whereas beta-galactosidase staining was not observed in the upper lobe. Endotracheal administration of IL-1Ra recombinant adenovirus resulted in sustained expression of IL-1Ra into the alveolar spaces, where it was recovered in a concentration of 660 pg/ml in 500 ml of lavage fluid, equivalent to 330 ng IL-1Ra, in the lungs 7 days after treatment. Moreover, in vivo instillation of nonreplicating adenovirus did not induce an inflammatory response in the 1-week time frame of the study period. Lung weight as a percent of body weight, serum zinc, serum amyloid A, leukocyte differentials, neutrophil activity, and TNF levels all were the same between untreated pigs and pigs treated with either recombinant adenovirus. The results indicate that the delivery of IL-1Ra to swine lungs via nonreplicating, recombinant adenovirus may be an effective method for in vivo modulation of IL-1 activity and investigation of cytokine involvement in respiratory disease pathogenesis.

Structural characterization and chromosomal localization of the mouse cDNA and gene encoding the bone tyrosine phosphatase, mOST-PTP.

Tyrosine kinases and phosphatases are regulators of the steady-state levels of phosphotyrosine proteins and, in this way, are key players in determining the functional state of the cell. As a unique member of the protein tyrosine phosphatase (PTP) superfamily, osteotesticular PTP (OST-PTP) is a receptor protein whose expression is highly regulated during osteoblast differentiation and in response to modulators of bone remodeling such as parathyroid hormone and vitamin D3. To explore the molecular mechanisms and signaling pathways important in the regulation of this gene, we characterized the structural organization of the mouse OST-PTP cDNA and gene and determined its chromosomal localization. The mouse cDNA is approximately 5.5 kb including 5.1 kb of coding sequence, 315 bp 5' UTR and 102 bp 3' UTR. It is expressed as a single approximately 5.8 kb transcript in day 8 differentiated MC3T3 osteoblasts. Although highly homologous to the rat OST-PTP cDNA, the mouse cDNA possesses a 74 bp insert in the 5' UTR which contains several potential transcription factor binding sites such as AP-2 and NFkappaB. The mouse OST-PTP (mOST-PTP) gene is a single copy gene encompassing 35 exons and spanning only 20.65 kb. As such, it is the smallest gene of the characterized receptor PTP genes. This is due to the lack of large introns and the conserved spatial organization of exons which encode specific protein motifs in the mOST-PTP molecule. Sequence analysis of the putative mOST-PTP promoter revealed basal elements as well as many potential cis-acting regulatory elements with relevance to gene regulation in bone. Of particular interest is the single osteoblast specific element known as osteocalcin specific element 2 (OSE2) found at position -1867, as well as numerous VDRE and NFkappaB sites found throughout the promoter and the 5' UTR. Fluorescence in situ hybridization studies have shown that mOST-PTP localizes to mouse chromosome 1, region F-G which is syntenic to the segment of human chromosome 1q32-33. This characterization of the mOST-PTP cDNA and gene will facilitate future experiments exploring the mechanisms of regulation of this phosphatase during osteogenesis.

Interleukin-10 gene therapy-mediated amelioration of bacterial pneumonia.

Respiratory infection by Actinobacillus pleuropneumoniae causes a highly pathogenic necrotizing pleuropneumonia with severe edema, hemorrhage and fever. Acute infection is characterized by expression of inflammatory cytokines, including interleukin-1 (IL-1), IL-6 and IL-8. To determine if high level production of inflammatory cytokines contributed to disease pathogenesis, we investigated if inhibiting macrophage activation with adenovirus type 5-expressed IL-10 (Ad-5/IL-10) reduced the severity of acute disease. Porcine tracheal epithelial cells infected with Ad-5/IL-10 produced bioactive human IL-10. When pigs were intratracheally infected with A. pleuropneumoniae, pigs pretreated with Ad-5/IL-10 showed a significant reduction in the amount of lung damage when compared to adenovirus type 5-expressing beta-galactosidase (Ad-5/beta-Gal)-treated and untreated pigs. In addition, serum zinc levels were unchanged, the lung weight/body weight ratio (an indicator of vascular leakage) was significantly reduced, and lung pathology scores were reduced. Myeloperoxidase activity in lung lavage fluid samples, an indicator of neutrophil invasion, was decreased to levels similar to that seen in pigs not infected with A. pleuropneumoniae. Reduction in inflammatory cytokine levels in lung lavage fluid samples correlated with the clinical observations in that pigs pretreated with Ad-5/IL-10 showed a corresponding reduction of IL-1 and tumor necrosis factor (TNF) compared with untreated and Ad-5/beta-Gal-treated pigs. IL-6 levels were unaffected by pretreatment with Ad-5/IL-10, consistent with observations that IL-6 was not derived from alveolar macrophages. Since inflammatory cytokines are expressed at high levels in acute bacterial pleuropneumonia, these results indicate that macrophage activation, involving overproduction of IL-1 and TNF, is a prime factor in infection-related cases of massive lung injury.

Reduced light-dependent phosphorylation of an analog visual pigment containing 9-demethylretinal as its chromophore.

9-Demethyl rhodopsin (9dR), an analog of vertebrate rhodopsin, consists of opsin and a covalently attached chromophore of 11-cis 9-demethylretinal. Electrophysiological evidence that photoactivated 9dR (9dR*) undergoes abnormally slow deactivation in salamander rods (Corson, D. W., Cornwall, M. C., and Pepperberg, D. R. (1994) Visual Neurosci. 11, 91-98) raises the possibility that opsin phosphorylation, a reaction involved in visual pigment deactivation, operates abnormally on 9dR*. This possibility was tested by measuring the light-dependent phosphorylation of 9dR in preparations obtained from bovine rod outer segments. Outer segment membranes containing 9dR or regenerated rhodopsin were flash-illuminated in the presence of [gamma-32P]ATP and rhodopsin kinase, further incubated in darkness, and then analyzed for opsin-bound [32P]Pi. [32P]Pi incorporation by 9dR* increased with both incubation period and bleaching extent but, under all conditions tested, was less than that measured in rhodopsin controls. Results obtained with 30-s incubation periods indicated that the maximal initial rate of incorporation by 9dR* is about 25% of that by photoactivated rhodopsin. The results imply that the low incorporation of Pi by 9dR* results from a reduced rate of phosphorylation by rhodopsin kinase and are consistent with the prolonged lifetime of 9dR* determined electrophysiologically.

Depalmitoylation of rhodopsin with hydroxylamine.

Intrinsic membrane proteins that to date have been investigated with respect to the function of palmitoylation are the beta-adrenergic receptor, rhodopsin, the alpha 2A-adrenergic receptor, and the influenza virus spike glycoprotein. As described above, the studies have led to differing conclusions with respect to the influence of palmitoylation on physiological activity. The basis of the differences remains unclear, but it may relate at least in part to the membrane environment of the protein during these studies, that is, the presence of a native membrane, the membrane composition of the expression cell line (in the case of mutant proteins), or the absence of membrane (in the case of detergent-purified proteins). For example, in the case of rhodopsin, the composition of the ROS disk membrane differs from that of the rod plasma membrane, and presumably also from the plasma membranes of cell lines in which mutant rhodopsins are expressed. Variation in membrane composition is known to have marked effects on the ability of rhodopsin to mediate the photic activation of PDE. Thus, although Karnik et al. clearly demonstrated the absence of an absolute requirement for palmitate in activating transducin, the influence of detergent on tertiary protein structure may have masked the full effect of the elimination of palmitate on the transducin-activating property of rhodopsin. Alternatively, the differing results obtained in the studies of rhodopsin could be a consequence of differences in amino acid sequences of the proteins studied. The precise functional role of the palmitate groups of rhodopsin remains an important question for further research. It was suggested by Ovchinnikov et al. that the hydrolysis of covalently bound palmitate might occur during the process of rhodopsin bleaching, but more recent data argue against this hypothesis. Experiments using synthetic peptides (representing cytoplasmic loop regions of rhodopsin) to identify the sites of interaction of R* and transducin provide support for an alternative possibility, namely, that palmitoylation and the resulting cytoplasmic loop play a role in the coupling of rhodopsin with transducin. The finding that the binding of transducin to R* occurs independently of the presence of palmitate argues against an essential requirement of palmitoylation on the binding step itself. However, available data indicate an enhancement, by depalmitoylation, of light-dependent GTPase activity in ROS preparations, although not in assays of unpalmitoylated, purified mutant rhodopsins (see above).(ABSTRACT TRUNCATED AT 400 WORDS)

Activity decreases as percentage overweight increases.

The purpose of the present research was to clarify the empirical relationship between percentage overweight and degree of ambulatory activity. Two week activity measurements were obtained, over two studies, using pedometers, from 127 women aged 19 to 55 years ranging from 14% underweight to 99% overweight. Regression analysis indicated that activity decreased by 7.6457 x 10(-4) mph for every 1% increase in percentage overweight from 0.25279 mph associated with zero percentage overweight. These data are in good agreement with data published by Chirico and Stunkard in 1960.16 Implications for small activity increases on obesity and general health are discussed.

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin.

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).

Interaction of the gamma-subunit of retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides as functional probes.

There is considerable evidence which suggests that the gamma-subunit of cGMP phosphodiesterase (PDE gamma) is a multifunctional protein which may interact directly with both the catalytic subunits of PDE (PDE alpha beta) and the alpha-subunit of transducin (T alpha) (Whalen, M., and Bitensky, M. (1989) Biochem. J. 259, 13-19; Griswold-Prenner, I., Young, J. H., Yamane, H. K., and Fung, B. K.-K. (1988) Invest. Ophthalmol. & Visual Sci. 29, (Suppl.) 218). To determine the region of interaction between the multifunctional PDE gamma and T alpha, and to determine the significance of this interaction, peptides corresponding to various regions of PDE gamma were synthesized and tested for their ability to inhibit the GTPase activity of T alpha. One of these peptides, PDE gamma-3 (bovine amino acid residues 31-45), inhibited the GTPase activity of T alpha with an I50 of 450 microM. The peptide (PDE gamma-3) was found to inhibit the GTPase activity of T alpha by inducing the binding of transducin to the rod outer segment membrane and by altering the GTP/GDP exchange. Analogs of PDE gamma-3 were synthesized to determine the required structure of the PDE gamma-3 region needed for the interaction of PDE gamma with T alpha. The results of these studies indicated that the removal of the positively charged amino acids or any of the potential hydrogen-bonding amino acids increased the I50 for the inhibition of the GTPase activity of T alpha Substitution of the hydrophobic amino acids had no effect. These results indicate the hydrophilic interactions may be essential for the binding of PDE gamma to T alpha and for the inhibition of the GTPase activity of T alpha by PDE gamma. The observed effects of PDE gamma-3 on T alpha and on PDE suggest that PDE gamma is a multifunctional protein which may play more than one role in the deactivation of the retinal transduction cascade.

The human atrial strength-interval relation. Influence of cycle length and procainamide.

We defined the atrial strength-interval relation in 23 patients at cycle lengths of 600, 450, and 300 msec before and after procainamide. The atrial diastolic threshold was similar at cycle lengths of 600 and 450 msec, but the threshold at 300 msec was significantly higher than that determined at 600 and 450 msec both before and after procainamide. Procainamide significantly increased the diastolic threshold only at a cycle length of 300 msec. The strength-interval relation was nonlinear, showing progressively decreasing decrements in the measured refractory period as the stimulating current was increased. Progressive decreases in the drive cycle length from 600 to 450 to 300 msec caused similar decreases in refractory periods. The shape of the curves was similar at cycle lengths of 600 and 450 msex. However, at low current strengths, the slope of the curve determined at 300 msex was significantly more vertical than the slopes of the curves at the longer drive cycle lengths. Procainamide caused similar increases in apparent refractory periods at each paced cycle length. Procainamide did not alter the shape of the curves at any paced cycle length. These observations confirm the importance of stimulation frequency on atrial excitability. They suggest that the effects of procainamide on the effective refractory period of the atrium are not cycle length dependent, although the drug effects on threshold are dependent on the drive cycle length.

Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase gamma-subunit.

Synthetic peptides corresponding to various regions of the light-activated guanosine 3',5'-cyclic monophosphate phosphodiesterase (PDE) gamma-subunit (PDE gamma) from bovine retinal rod outer segments were synthesized and tested for their ability to inhibit PDE activity, and GTPase activity of transducin. One of these peptides, corresponding to PDE gamma residues 31-45, inhibited PDE activity and GTPase activity in a dose-dependent manner. The GTPase activity was inhibited by PDE gamma-3 non-competitively. This region of the PDE gamma subunit may be involved in the direct interaction of transducin and PDE alpha beta with PDE gamma.

Effects of nitroprusside and dopamine on pulmonary arterial vasculature in children after cardiac surgery.

The hemodynamic effects of nitroprusside and dopamine were studied in 28 children early after intracardiac repair. Children were placed in six groups, five according to their anatomic lesion and one made up of those who had postoperative pulmonary artery hypertension, to evaluate the possible differences in response of the pulmonary arterial vasculature to the drugs. Seven children had repair of an atrial septal defect; six, repair of tetralogy of Fallot; four, repair of ventricular septal defect; five, surgery for pulmonary stenosis; one, closure of a left ventricular to right atrial tunnel; and five, postoperative pulmonary artery hypertension. Dopamine was infused at 8 microgram/kg/min, and nitroprusside at 3 microgram/kg/min. With dopamine, the heart rate increased an average of 10% and the cardiac index 11%; both increases were statistically significant. Changes in systemic and pulmonary vascular resistance, however, were not. With nitroprusside, the heart rate increased an average of 9% and the cardiac index 5%, while there was a significant decrease in both systemic (-20%) and pulmonary (-27%) vascular resistance. With the combination of dopamine and nitroprusside, both the cardiac index (+13%) and heart rate (+20%) increased significantly while systemic vascular resistance fell an average of 23% from control, and the pulmonary vascular resistance decreased 21%. Drug response among all five anatomic subgroups tended to be similar. We conclude that an afterload-reducing agent, such as nitroprusside and an inotropic drug such as dopamine, may have potential clinical advantages when used together in children providing heart rate does not become excessive.