Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Tumor necrosis factor induces acute phase proteins in rats

Inoculation of WAG rats with recombinant mouse tumor necrosis factor results in a rapid and marked increase in several acute phase proteins in the serum (haptoglobin, alpha 1 acid glycoprotein, alpha 2 macroglobulin) and in the plasma (fibrinogen). We conclude that TNF may play an important role in the inflammatory response in vivo and possibly in the pathogenesis of inflammatory disorders.     

Hepatocellular Carcinoma in Hepatitis B Surface Antigen Carriers in Eight Institutions

We reviewed records of all persons dying between 1979 and 1986 in eight California institutions for the mentally retarded. Autopsies had been done in 71% of the 1,181 deaths. Nine deaths were due to hepatocellular carcinoma, which invariably developed in carriers of hepatitis B surface antigen (HBsAg) and was fatal within four months of diagnosis. The mean age at death was 32.7 years. The incidence of hepatocellular carcinoma in HBsAg carriers was 140 times greater than in the US population. Persistent hepatitis B infection was probably etiologically related to hepatocellular carcinoma in this population, which is relatively free of exposure to other hepatocarcinogens.     

A new experimental approach for the study of cardiopulmonary physiology during early development

In this report, an experimental approach and newly designed apparatus for liquid ventilation of preterm animals are described. Findings of age-related changes in cardiopulmonary function of this animal preparation are presented. Thirty-one lambs, 102-137 days gestation (term 147 +/- 3 days), were studied. The carotid artery, jugular vein, and trachea of the exteriorized fetus were cannulated under local anesthesia. Immediately after cesarean section delivery, ventilation commenced; warmed (39 degrees C) and oxygenated (PIO2 greater than 500 Torr) liquid fluorocarbon (RIMAR 101) was delivered to the lung by a mechanically assisted liquid ventilation system. Skeletal muscle paralysis, low-dose exogenous buffering, and thermal support were maintained during the 3-h experiment. Pulmonary gas exchange, acid-base status, and cardiopulmonary and metabolic function were assessed. By utilizing these techniques, effective arterial oxygenation, CO2 elimination, acid-base status, and cardiovascular stability were supported independent of gestational age. The results demonstrate a developmental increase in specific lung compliance and mean arterial pressure and decrease in heart rate and systemic O2 consumption per kilogram with advancing gestational age. These findings demonstrate that liquid ventilation negates the dependency of effective pulmonary gas exchange on surfactant development, thereby extending the limits of viability of the immature extrauterine lamb. As such this new experimental approach is useful for the study of physiological development over an age range previously limited to fetal animal preparations and, therefore, may provide insight regarding adaptation of the premature to the extrauterine environment.     

Autonomous DNA replication in human cells is affected by the size and the source of the DNA.

We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.     

Behaviors of southern red oak hemicelluloses and lignin in a mild sulfuric acid hydrolysis

Removal and modification of southern red oak hemicelluloses and lignin in a 0.05%(w/v) sulfuric acid hydrolysis were investigated. The hydrolysis profile was to raise the reaction from room temperature to 150 degrees C for in 38 min and to extend the hydrolysis at 150 degrees C for 1 h. At the end of the hydrolysis, 25.5% of red oak components were dissolved, of which 58% was xylose and 17% lignin. As the hydrolysis proceeded from room temperature to 150 degrees C, a part of red oak xylan was removed to yield an oligomer fraction having maximal yield and average molecular weight of 3460 at 150 degrees C. This fraction and the bulk xylan extracted during the first 30 min at 150 degrees C were further degraded to give a lower molecular weight oligomer fraction, of which the yield and average molecular weight (2610) were highest at the end of the bulk removal of xylan. Red oak lignin, syringyl and guaiacyl units in particular, was increasingly removed with the progress of the hydrolysis. Lignin derivatives and a part of red oak extractives soluble in the hydrolysate were identified.     

Antithrombin III Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity

Antithrombin III Basel is a hereditary abnormal antithrombin with normal progressive inhibition activity (normal reactive site) and reduced heparin cofactor activity (impaired heparin binding site). Structures of antithrombin III Basel and normal antithrombin III isolated from the same patient were compared by peptide mapping using the dimethylaminoazobenzene isothiocyanate precolumn derivatization technique. Of the approximately 50 tryptic peptides of normal and abnormal antithrombin III, one peptide comprising residues 40-46 had a different retention time in reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide from antithrombin III Basel had a single substitution of Pro (normal) by Leu (abnormal) at position 41. This substitution is close to an Arg (residue 47) and a Trp (residue 49) which have previously been shown to be critical for heparin binding by antithrombin III. Although additional amino acid substitutions in antithrombin III Basel cannot be ruled out, this Pro-Leu replacement could cause a conformational change by increasing both the helical structure and the hydrophobicity around residue 41. These data suggest that: (i) the heparin binding site of antithrombin III encompasses the region containing residues 41, 47, and 49; and (ii) the impaired heparin cofactor activity of antithrombin III Basel is likely due to a conformational change of the heparin binding site induced by the Pro-Leu substitution at position 41.     

Studies of the fluorescence from tryptophan in melittin

The fluorescence lifetime and rotational correlation time of the tryptophan residue in melittin, as both a monomer and tetramer, have been measured between pH 6 and 11. The fluorescence decays are non-exponential and give lifetimes of 0.7±0.1 ns and 3.1±0.1 ns. This emission is consistent with a model in which the tryptophan residue is in slightly different environments in the protein. In a dilute solution of monomer the mean fluorescence lifetime is 2.3±0.1 ns, below pH 10, but falls to 1.7 ns at higher pH. In contrast, the melittin tetramer has a mean fluorescence lifetime of only 2.2 ns at pH 6, which falls to 1.9 ns by pH 8, and falls again above pH 10 to the same value as in monomeric melittin. The behaviour between pH 6 and 8 is explained as the quenching of the Trp residue by lysine groups, which are near to the Trp in the tetramer but in the monomer, are too distant to quench. Fluorescence anisotropy decays show that the Trp residue has considerable freedom of motion and the range of wobbling motion is 35±10° in the tetramer     

Prostaglandin synthesis by the cochlea of the guinea pig. Influence of aspirin, gentamicin, and acoustic stimulation

This study describes the synthesis of prostaglandins (PGs) by the vascular structures of the inner ear (lateral wall = stria vascularis and spiral ligament) in vitro. The main PGs produced were PGI2, PGF2 alpha and PGE2. PGI2 and PGF2 alpha were also found in the perilymph. A 350 mg/kg ip injection of aspirin decreased PG synthesis by the lateral wall and PG levels in perilymph. This effect was reversed after 3 days. Gentamicin (10(-9) to 10(-5) M) decreased significantly and reversibly PG synthesis in vitro, as did 100 mg/kg ip injection. Acoustic stimulation increased ex vivo PGI2 and PGE2 synthesis without modifying PG levels in perilymph. Results suggest that PGs could be one humoral mediator of the cochlear microcirculation homeostasis, and, possibly, of the circulatory disturbances reported after acoustic stimulation. The decreased PG synthesis after gentamicin treatment could account for the angiotoxic component observed in aminoglycoside ototoxicity.