Operation Everest II: plasma lipid and hormonal responses during a simulated ascent of Mt. Everest

To examine the effect of hypobaric hypoxia on plasma lipid profiles, fasting blood samples were collected from six men (21-31 yr) at 760 Torr and periodically during a 40-day exposure to decreasing barometric pressure culminating in a final ambient pressure of 282 Torr. Preascent plasma total cholesterol concentration ([TC]) was decreased by 25% after the 40-day exposure (P less than 0.01). High-density lipoprotein concentrations ([HDL-C]) decreased 32% (P less than 0.001) with no alteration in the TC-to-HDL-C weight ratio. Plasma triglyceride concentration increased twofold during this period (P less than 0.01). There were no significant differences in fasting plasma free fatty acid concentrations or free fatty acid-to-albumin molar ratio throughout the study. Fasting plasma insulin levels were increased approximately twofold with no significant changes in glucagon concentration or the insulin-to-glucagon molar ratio. Plasma norepinephrine concentrations were increased threefold on reaching 282 Torr (P less than 0.01), with no significant changes in plasma epinephrine concentrations. Mean energy intake (kcal/day) decreased 42%, whereas mean body weights decreased by 8.9 +/- 0.8% (P less than 0.01) with exposure. Increased concentrations of insulin may lead to increased hepatic production of triglyceride-rich lipoproteins, thus eliciting metabolic changes independent of weight loss and dietary intake.     

Single crystals of bacteriophage T7 RNA polymerase

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.     

Is fatty acid uptake in cardiomyocytes determined by physicochemical fatty acid partition between albumin and membranes?

Summary Palmitate uptake by isolated, calcium-resistant cardiomyocytes was measured by using a stimulation chamber in which cell contraction can be evoked electrically. Experiments were performed in a medium containing physiological interstitial concentration of albumin (2%) and palmitate/albumin (P/A) ratios ranging from 0.03 to 2.5, and were compared to experiments with fixed P/A ratio (– 1).Initial rate of uptake (V_i) was calculated from fitted uptake vs. time curves as measured by accumulation of radioactivity in the cells from ¹⁴C-labelled palmitate. V_i-vs.-concentration curves exhibited a saturable component, if albumin concentration was kept constant. Almost no change in V_i was observed in experiments performed at constant P/A. This is in contrast to the albumin receptor hypothesis.The ¹⁴C-palmitate content of the myocytes as estimated by thin-layer-chromatography did reach a plateau at 30 s and had the same value at 30 min after administration. The cellular content of labelled palmitate could be attributed to the membrane compartment as calculated from partition coefficient (Kc) of fatty acids (FA) between albumin and membranes. With electrical stimulation V_i-vs.-palmitate concentration kinetics showed a shift in apparent Km from 62 µM (P/A – 0.22) to 23 µM (P/A = 0.08), and presence of 2,4-dinitrophenol increases V_i.Our results suggest that FA-transfer across the sarcolemmal membranes is determined by a physicochemical equilibrium between the compartments of extracellular FA-albumin complex, the membrane lipid phase, intracellular FA binding proteins and the respective aqueous phases. Consequently in cell suspensions the rate of palmitate uptake is controlled by a step of fatty acid metabolism possibly the formation of Fa CoA by the enzyme FA acyl CoA synthetase which is localized in membranes of endoplasmatic reticulum and mitochondria. This step is influenced by the metabolic state of the cells and by FA concentration in membranes.     

Regeneration of ascorbic acid by rat colon

Plants and animals alike use ascorbic acid in a variety of reactions that result in net generation of dehydro-L-ascorbic acid. The ability to reduce dehydro-L-ascorbic acid back to ascorbic acid would conserve "total ascorbate" and would help to maintain the toxic oxidized form of the molecule at a low level. This study evaluated the rate of dehydro-L-ascorbic acid reduction either by following the rate of NADPH consumption or by analysis of the amount of 14C-labeled dehydro-L-ascorbic acid converted to ascorbic acid. A large percentage of the NADPH consumed by a semipurified preparation of rat colonic mucosa in vitro was dependent on the presence of dehydro-L-ascorbic acid. The tissue factor active in regenerating ascorbic acid is intermediate in size between cytochrome c and blue dextran. The present results indicate that the mucosa reduced dehydro-L-ascorbic acid by a cytosolic enzyme that uses NADPH as a hydrogen donor. Subsequent to precipitation by ammonium sulfate, the 55-70% fraction contains most of the reductase activity while consisting of only 17% of the cellular soluble protein.     

Effect of alpha-adrenergic blockade on the cardiovascular responses to hypoxemia and hypercapnic acidosis in conscious dogs

In order to evaluate the role of the alpha-adrenergic system in the systemic and renal hemodynamic changes of the acute combined blood gas derangement, seven conscious mongrel dogs in careful sodium balance (80 mEq/day for 4 days) were evaluated. Each animal was evaluated during combined acute hypoxemia (PaO2 = 35 +/- 1 mm Hg) and hypercapnic acidosis (PaCO2 = 56 +/- 2 mm Hg; pH = 7.18 +/- 0.01) with (i) vehicle (D5W) alone and (ii) alpha 1-adrenergic blockade with prazosin, 0.1 mg/kg iv. Mean arterial pressure increased during the combined blood gas derangement with vehicle. In contrast, mean arterial pressure fell during combined acute hypoxemia and hypercapnic acidosis with alpha 1-adrenergic blockade. The mechanism for abrogation of the rise in mean arterial pressure during the combined blood gas derangement by alpha 1-adrenergic blockade appeared to be through attenuation of the rise in cardiac output rather than an exaggerated fall in total peripheral resistance. These observations suggest that the alpha-adrenergic system is important in circulatory homeostasis during the combined blood gas derangement.     

Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity

Mouse macrophages and lymphocytes express two distinct isoforms of a single class of Fc receptor for IgG. The macrophage isoform (FcRII-B2) is identical to the lymphocyte isoform (FcRII-B1) except for an inframe insertion in the cytoplasmic tail of FcRII-B1 that increases its length from 47 to 94 amino acids. To determine the functional significance of this cytoplasmic domain variation, presumably the result of alternative mRNA splicing, we expressed both isoforms in receptor-negative fibroblasts. While FcRII-B2 mediated the efficient ligand internalization and delivery to lysosomes, endocytosis via FcRII-B1--and via a tailminus mutant--was relatively inefficient. This difference reflected the inability of FcRII-B1 (and the tailminus mutant) to accumulate in clathrin-coated pits. Thus, the FcRII-B2 cytoplasmic tail contains a domain needed for accumulation in coated pits, and this domain is disrupted by the 47 amino acid insertion in FcRII-B1.     

Short related sequences in the cytoplasmic domains of CD4 and CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase.

We report that the cytoplasmic domains of the T-lymphocyte glycoproteins CD4 and CD8 alpha contain short related amino acid sequences that are involved in binding the amino-terminal domain of the intracellular tyrosine protein kinase, p56lck. Transfer of as few as six amino acid residues from the cytoplasmic domain of the CD8 alpha protein to the cytoplasmic domain of an unrelated protein conferred p56lck binding to the hybrid protein in HeLa cells. The common sequence motif shared by CD4 and CD8 alpha contains two cysteines, and mutation of either cysteine in the CD4 sequence eliminated binding of p56lck.p56lck also contains two cysteine residues within its CD4-CD8 alpha-binding domain, and both are critical to the interaction with CD4 or CD8 alpha. Because the interaction does not involve disulfide bond formation, a metal ion could stabilize the complex.     

Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase

NAD-linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AIDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and beta = 110.5 degrees. Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the kappa = 180 section at psi = 90 degrees and phi = 71 degrees and 341 degrees, indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.     

Protein kinase NII. Interaction with RNA polymerase II and contribution to immunological cross-reactivity of RNA polymerases I and II

The interaction between antibodies directed against RNA polymerase I purified from Morris hepatoma 3924A and homologous RNA polymerase II was investigated. The activity of partially purified polymerase II was inhibited by the antibodies. In contrast, the reaction catalyzed by the purified enzyme was not affected. Partially purified polymerase II preparations contained a protein kinase activity. Sucrose gradient centrifugation in the presence of 0.3 M KCl resulted in complete separation of RNA polymerase II from protein kinase as well as in complete loss of sensitivity to the anti-RNA polymerase I antibodies. The protein kinase possessed reaction characteristics similar to those of the NII protein kinase (Rose, K.M., Bell, L.E., Siefken, D.A. and Jacob, S.T. (1981) J. Biol. Chem. 256, 7468–7477) which is associated with hepatoma RNA polymerase I (Rose, K.M., Stetler, D.A. and Jacob, S.T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2833–2837). The activities of both kinases were inhibited to the same extent by anti-RNA polymerase I antibodies and polypeptides of M_r 42000 and 25000, present in both kinase preparations, formed immune complexes with the antisera. Readdition of protein kinase NII to purified polymerase II resulted in phosphorylation of the polymerase and a concomitant enhancement of RNA synthesis. After addition of the kinase, RNA polymerase II activity was again sensitive to anti-RNA polymerase I antibodies. Upon reacting with protein kinase NII, RNA polymerase II polypeptides could be detected in immune complexes with anti-RNA polymerase I antibodies. These data indicate that protein kinase NII is associated with RNA polymerase II during early stages of purification and is at least partially responsible for the immunological cross-reactivity of RNA polymerases I and II.