Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.     

Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.     

Rapid Removal of Tissue Culture Monolayers by Collodion for Subsequent Staining

Removing cultures from roller tubes before staining eliminates the destaining which often occurs when the cells are first stained and then removed by embedding in collodion. The cells are fixed in situ, dehydrated, and covered with collodion (Merk's flexible) for 10 min. The collodion is poured off, the fluid residue lining the tube allowed to dry for 10 min, and the tube is filled with tap water. The collodion cast containing the cells is loosened and removed, cut into strips, placed on slides and blotted into firm contact. The collodion is then dehydrated and dissolved with absolute alcohol followed by a 1:1 mixture of alcohol and ether. The slides can then be rehydrated and stained by conventional methods.     

Regulation of apolipoprotein E synthesis and mRNA by diet and hormones

Rats were fasted or fasted and refed simple purified diets so the effects of individual carbohydrates or fats could be studied. Freshly isolated hepatocytes from these animals were used to measure both apoE synthesis and mRNA levels so any changes in apoE synthesis that might occur without changes in its mRNA could be detected. Some of these experiments were done with both sexes. Both fasting and fasting and refeeding a 60% glucose fat-free diet significantly increased spoE synthesis. However, cyclic AMP is not likely to rapidly mediate the effect of fasting since dibutyryl cAMP slightly lowered (rather than increased) apoE synthesis and mRNA when injected into rats for 4.5 h. Dietary fat had no effect either in the absence of carbohydrate or when consumption of carbohydrate was constant in pair-fed rats. ApoE mRNA levels remained normal for 4 days in primary hepatocytes cultured in medium that had only amino acids as an energy source. Added hormones or fructose had no significant effect. Thus, only fasting and fasting and refeeding glucose were able to significantly change apoE synthesis or mRNA levels. Synthesis of apoE may be regulated to increase when apoE is secreted with very low density lipoprotein or when apoE in secreted high density lipoprotein is needed to acquire cholesteryl esters for the synthesis of bile salts and acids by liver.     

The role of displacement of the mitral annulus in left atrial filling and emptying in the intact dog

The variations in ventricular-atrial mitral annular position during the cardiac cycle and the simultaneous changes in left atrial silhouette area (obtained by angiography after injections of contrast material into the main pulmonary artery) were investigated in six experiments on intact dogs with chronically implanted intracardiac markers. Frame-by-frame measurements of the angiograms (120 frames/s) were used to determine, under various hemodynamic conditions, the duration, magnitude, and average rate of the mitral annular motion and of the simultaneous changes in left atrial area during atrial filling (ventricular systole) and atrial emptying (early in ventricular diastole). The mitral annulus was seen to move towards the ventricular apex during systole and towards the atrium early in diastole with the duration, average rate, and magnitude of displacement (although varying widely) showing good statistical correlations (P less than 0.0005-0.005) with the changes in projected left atrial area. These findings suggest that the duration, rate, and magnitude of atrial filling and emptying may be, in the intact heart, determined by the movements of the atrioventricular junction.     

Sudden interruption of leaflet opening by ventricular contractions: a mechanism of mitral regurgitation.

The motion of both mitral cusps and the presence of valvular regurgitation during ventricular contractions were investigated in seven experiments on dogs in which radiopaque markers had been sutured to the cusps and the valve annulus 1-32 wk before the studies. Cineangiograms of the left ventricle were obtained during ventricular ectopic beats, interposed throughout the cardiac cycle (20-99% of cycle length) and during induced variations in the P-R interval (0-200 ms). Mitral regurgitation was observed only during a) weak, early ectopic beats (peak pressure below 34 mmHg) which were incapable of closing the cusps and b) when ventricular contractions suddenly interrupted normal leaflet motion toward the ventricle, during three well-defined periods of diastole (diastolic valve opening, diastolic rebound, and atrial opening). Valve closure following sudden reversal of cusp opening was slow and the leaflets often did not arrive simultaneously at their closed positions. These findings suggest that sudden interruption of leaflet opening by ventricular contractions is an important mechanism of transient mitral regurgitation in the normal heart.     

mTOR kinase and the regulatory subunit of protein kinase A (PRKAR1A) spatially and functionally interact during autophagosome maturation

The regulatory subunit 1-alpha (RIalpha) of protein kinase A (PKA) and the mTOR kinase are involved in a common pathway regulating mammalian autophagy. RIalpha was found to localize on Rab7-positive late endosomes and on LC3-positive autophagosomal membranes in cultured cells. RIalpha was also shown to physically interact with mTOR kinase and affect its phosphorylation and activity. In this addendum, we further explore the subcellular distribution of mTOR related to RIalpha and LC3. We present experiments showing that mTOR colocalizes with RIalpha-, Rab7- and LC3-positive membranes in cultured cells. Because RIalpha regulates the phosphorylation and activity of mTOR kinase, which we now show localizes on autophagosomal membranes, the possibility emerges that the RIalpha-mTOR complex acts at the level of autophagosome maturation.     

Evaluation of Plasma Trace Elements in Different Stages of Nonalcoholic Fatty Liver Disease

Biological Trace Element Research - Nonalcoholic fatty liver disease (NAFLD) is considered as the hepatic manifestation of metabolic syndrome. Its global prevalence is estimated between 25 and 45%,...