Comparison of the metabolic effects of chylomicrons and their remnants on isolated hepatocytes

A comparison was made between the effects of chylomicrons and chylomicron remnants on metabolic processes of isolated hepatocytes. Since isolated triacylglycerol-rich lipoproteins are contaminated with nonesterified fatty acids, control incubations were conducted with an amount of fatty acid equivalent to the contaminating fatty acids present in the chylomicrons and the remnant preparations, respectively. Chylomicron remnants, produced in vitro by incubation of chylomicrons in postheparin rat plasma, caused marked inhibition of glycolysis, fatty acid synthesis, and cholesterol synthesis, along with marked stimulation of ketogenesis. These effects were traced to the release of nonesterified fatty acids from these remnant particles as a consequence of contamination with lipoprotein lipase, picked up by the particles during the incubation with rat plasma. Fatty acids inhibit glycolysis, cholesterol, and fatty acid synthesis, but enhance ketone body formation by isolated hepatocytes. Chylomicrons and remnants prepared in vivo by the injection of chylomicrons into functionally hepatectomized rats were not contaminated with lipoprotein lipase and did not inhibit glycolysis and cholesterol synthesis nor increase ketone body formation. These lipoprotein particles did, however, cause significant inhibition of fatty acid synthesis, with the chylomicrons being more effective on a protein basis than the remnants produced in vivo. The mechanism responsible for the inhibition of fatty acid synthesis by chylomicrons and remnants prepared in vivo remains to be resolved.     

Effect of γ-glutamyl cycle inhibitors on brain amino acid transport and utilization

Two inhibitors of the -glutamyl cycle, methionine sulfoximine (MSO) and 2-imidazolidone-4-carboxylic acid (ICA) were administered to C57BL/6J mice. Both agents resulted in a reduced rate of transport of tyrosine from blood to brain and a decreased rate of incorporation of tyrosine from plasma into brain protein. MSO administration also diminished the concentrations of brain tyrosine, dopamine, and norepinephrine. MSO decreased the transport rate of valine by brain as well as the rate of its incorporation into protein when expressed in relation to the plasma specific activity. The results demonstrate a significant role for the -glutamyl cycle in the transport of large neutral amino acids from blood to brain.Presented in part in the April 1977 meeting of the American Academy of Neurology, Atlanta, Georgia.     

Motion of both mitral valve leaflets: a cineroentgenographic study in intact dogs.

Motion and position of both mitral leaflets were studied in five normal dogs 1-11 wk after radiopaque markers were sutured on the valve cusps and on the mitral annulus. Cinefluorograms and cineangiograms (100-120 frames/s) of left atrium and left ventricle were used to study cusp motion and intraventricular flow patterns, and to detect mitral regurgitation during sinus rhythm (42-184 beats/min) and during isolated atrial or ventricular contractions. Time-motion of both leaflets was similar throughout diastole with the exception of delayed posterior cusp opening. Peak opening and closing speeds, opening and closing times, and time of complete closure, measured from the Q wave of the ECG, were not significantly affected by the variations in heart rate. Diastolic leaflet closure began immediately after opening, while the ventricular cavity was small, and was caused by flow eddies behind the cusps. Isolated ventricular contractions closed the valve leaflets completely and symmetric valve closure was ensured by the different rates of leaflet edge approximation. In contrast, atrial closure was slow, partial, and of very short duration.     

Distribution of membrane-bound cyclic AMP-dependent protein kinase in plasma membranes of cells of the kidney cortex

Summary Renal cortical plasma membranes were separated by free flow electrophoresis into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. These membranes were found to contain an intrinsic, self-phosphorylating system which consists of a cyclic AMP-dependent protein kinase, a phosphoprotein phosphatase and the substrate(s) of these enzymes. The kinase, but not the phosphatase, was stimulated by cyclic AMP; maximal (1.7-fold) stimulation was effected at a cyclic AMP concentration of 0.1 m. The degree of phosphorylation of the brush borders was six times greater than that of the basal-lateral membranes in the absence of cyclic AMP and 2.3-fold greater in the presence of cyclic AMP. This preferential phosphorylation of the luminal membrane by membrane-associated protein kinase(s) may play a role in the parathyroid hormone-mediated alterations of solute reabsorption in the proximal tubule.     

Isolation and derivatization of plasma taurine for stable isotope analysis by gas chromatography-mass spectrometry

A method for the isolation and derivatization of plasma taurine is described that allows stable isotope determinations of taurine to be made by gas chromatography-mass spectrometry (gc-ms). The isolation procedure can be applied to 0.1 ml of plasma: the recovery of plasma taurine was 70–80%. For gc separation, taurine was converted to its dimethylaminomethylene methyl ester derivative which could not be detected by hydrogen flame ionization, but could be monitored readily by NH₃ chemical ionization mass spectrometry. The derivatization reaction occurred partially on-column and required optimization of injection conditions. Using stable isotope ratiometry multiple ion detection, $\text{[M + 2 + H]}{}^{\mathrm{+}}\text{[M + H]}{}^{\mathrm{+}}$ ion ratio of natural abundance taurine was determined with a standard deviation of less than ±0.07% of the ratio. The [1,2-¹³C]taurine/taurine mole ratios of standard mixtures could be accurately determined to 0.001. This stable isotope gc-ms method is suitable for studying the plasma kinetics of [1,2-¹³C]taurine in infants who are at risk with respect to taurine depletion.     

Purification and characterization of a latent precursor of a double-stranded RNA dependent protein kinase from reticulocyte lysates

Double-stranded RNA (dsRNA) activates a cyclic 3′: 5′-AMP independent protein kinase (dsI) in reticulocyte lysates which inhibits protein synthesis by phosphorylating the 38, 000 dalton (38K) subunit of the initiation factor eIF-2 (eIF-2α). A latent precursor form of dsI (latent dsI) has been partially purified (1000–2000 fold) from lysates. Activation of dsI at all stages in the purification of latent dsI requires ATP and low levels of dsRNA (1–20 ng/ml), and is accompanied by the phosphorylation of a broad 67,000 dalton (67K) band. However, as purification proceeds the 67K band is resolved into two phosphorylated polypeptides of 68,500 and 67,000 daltons ($\text{68.5K}\text{67K}$). Although latent dsI and activated dsI have distinctly different chromatographic properties, both forms have similar molecular weights (～120,000) and similar sedimentation coefficients (～3.8S) in glycerol gradients. The data support the view that one or both components of the $\text{68.5K}\text{67K}$ doublet are associated with the dsRNA-dependent protein kinase activity.     

β-Galactosidase from Termination and Deletion Mutant Strains

β-Galactosidase fragments were isolated from strains of Escherichia coli with mutations in the lacZ gene. The polypeptide obtained from a termination mutant (lacZNG125) appeared to be the intact gene product, containing the first half of the β-galactosidase amino acid sequence. From an internal deletion mutant strain (lacZU163), an aggregate was obtained of several partially degraded polypeptides. Each of these was smaller than predicted from genetic data for the fragment. Introduction of the lacZU163 mutation into a protein degradation-deficient strain (Deg⁻) resulted in the protection of the amino-terminal region of the protein. Some of the BrCN peptides from the U163 polypeptides were separated and identified. From such experiments it was shown that in both Deg⁻ and Deg⁺ strains the COOH-terminal region is rapidly degraded. This indicates that the complete gene product of lacZU163 has not been detected. The use of genetically defined enzyme fragments in studying structure-function relationships and in determination of primary structure is discussed.     

Resonance Raman spectroscopy of iron-oxygen vibrations in hemerythrin

Resonance Raman spectra are reported for oxyhemerythrin and 15 anionic adducts of methemerythrin. All methemerythrin derivatives except sulfidomethemerythrin contain a Raman band near 510 cm^?1 which is assigned to an iron-oxygen stretching vibration. The effect of H₂¹⁸O on the frequency of this vibration was studied extensively. On the basis of the exchange results, the vibration is assigned to OH^?, H₂O, or a μ-oxo bridge between the irons.