Effect of palmitoyl-CoA and beta-oxidation of fatty acids on the kinetics of mitochondrial citrate transporter.

The kinetics of the hepatic mitochondrial citrate transporter were studied using 1,2,3-benzene tricarboxylate and the inhibitor-stop technique at 8 degrees C. The apparent Km for this transporter was 250 muM and the maximum velocity was 2 nmol of citrate transported per minute per milligram of mitochondrial protein. This apparent Km was increased when hepatic mitochondria were preincubated with both L-palmitoylcarnitine and CoA-SH but not with either alone. This rise in apparent Km was accompanied by a rise in the acid insoluble CoA-SH content. Removal of mitochondrial acid insoluble CoA by "defatted albumin" resulted in a parallel decrease in the apparent Km. The apparent Km for the citrate transporter was increased after coupled beta-oxidation of L-palmitoylcarnitine or octanoate without a detectable increase in acid insoluble CoA. Inhibition of beta-oxidation of L-palmitoylcarnitine by the D-derivative prevented the rise in the apparent Km. Preincubation with ATP resulted in an increase in this apparent Km. When L-palmitoylcarnitine oxidation occurred without ATP accumulation (hexokinase, glucose, ADP, and inorganic phosphate) the apparent Km for the citrate transporter increased two- to threefold. Therefore, the apparent Km for the citrate transporter varied directly with the acid insoluble CoA content. In addition, this Km was increased as a result of beta-oxidation of fatty acids but the mechanism was not solely attributable to a rise in acid insoluble CoA or ATP. The physiological implications of these findings are discussed.     

Network resonance can be generated independently at distinct levels of neuronal organization

Resonance is defined as maximal response of a system to periodic inputs in a limited frequency band. Resonance may serve to optimize inter-neuronal communication, and has been observed at multiple levels of neuronal organization. However, it is unknown how neuronal resonance observed at the network level is generated and how network resonance depends on the properties of the network building blocks. Here, we first develop a metric for quantifying spike timing resonance in the presence of background noise, extending the notion of spiking resonance for in vivo experiments. Using conductance-based models, we find that network resonance can be inherited from resonances at other levels of organization, or be intrinsically generated by combining mechanisms across distinct levels. Resonance of membrane potential fluctuations, postsynaptic potentials, and single neuron spiking can each be generated independently of resonance at any other level and be propagated to the network level. At all levels of organization, interactions between processes that give rise to low- and high-pass filters generate the observed resonance. Intrinsic network resonance can be generated by the combination of filters belonging to different levels of organization. Inhibition-induced network resonance can emerge by inheritance from resonance of membrane potential fluctuations, and be sharpened by presynaptic high-pass filtering. Our results demonstrate a multiplicity of qualitatively different mechanisms that can generate resonance in neuronal systems, and provide analysis tools and a conceptual framework for the mechanistic investigation of network resonance in terms of circuit components, across levels of neuronal organization.     

Systematic Dissection of the Sequence Determinants of Gene 3’ End Mediated Expression Control

The 3’end genomic region encodes a wide range of regulatory process including mRNA stability, 3’ end processing and translation. Here, we systematically investigate the sequence determinants of 3’ end mediated expression control by measuring the effect of 13,000 designed 3’ end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3’ end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3’ end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3’ UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3’UTRs, confirming the role for this element in 3’ end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3’ end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3’ end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3’ end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.     

On the mechanism of accumulation of cholestanol in the brain of mice with a disruption of sterol 27-hydroxylase

The rare disease cerebrotendinous xanthomatosis (CTX) is due to a lack of sterol 27-hydroxylase (CYP27A1) and is characterized by cholestanol-containing xanthomas in brain and tendons. Mice with the same defect do not develop xanthomas. The driving force in the development of the xanthomas is likely to be conversion of a bile acid precursor into cholestanol. The mechanism behind the xanthomas in the brain has not been clarified. We demonstrate here that female cyp27a1^−/− mice have an increase of cholestanol of about 2.5- fold in plasma, 6-fold in tendons, and 12-fold in brain. Treatment of cyp27a1^−/− mice with 0.05% cholic acid normalized the cholestanol levels in tendons and plasma and reduced the content in the brain. The above changes occurred in parallel with changes in plasma levels of 7α-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1^−/− mouse with ²H₇-labeled 7α-hydroxy-4-cholesten-3-one resulted in a significant incorporation of ²H₇-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7α-hydroxy-4-cholesten-3-one across the blood-brain barrier in cyp27a1^−/− mice and subsequent formation of cholestanol. It is suggested that the same mechanism is responsible for accumulation of cholestanol in the brain of patients with CTX.     

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.     

Effects of fasting on the control of fatty-acid synthesis in hepatoma 7777 and host liver. Role of long-chain fatty acyl-CoA,, the mitochondrial citrate transporter and pyruvate dehydrogenase activity.

The effects of fasting on the rate of fatty acid synthesis, the properties of the mitochondrial citrate transporter and on pyruvate dehydrogenase activity were investigated in "poorly-differentiated" tmorris hepatoma 7777 and in host liver preparations. The properties of the citrate transporter from hepatoma mitochondria were similar to those of host liver mitochondria, with the exception that the Km for the liver mitochondrial citrate transporter was 248 plus or minus 20 mu M while that in hepatoma mitochondria was less than 75 mu M. The acid-insoluble CoA content was 180 plus or minus 20 pmol/mg protein in the hepatoma and remained essentially unchanged in the fasted state, while the acid-insoluble CoA levels in livers from fed rats was 720 plus or minus 80 pmol/mg protein and were increased to 1050 plus or minus 50 pmol/mg protein during fasting. After a 36-h fast, the rate of lipogenesis and the percentage of pyruvate dehydrogenase present in the active form were each decreased by approximately 80% in host liver preparations. In contrast, the rate of lipogenesis by hepatoma slices did not decrease during fasting, and essentially all pyruvate dehydrogenase present was in the active form of hepatomas obtained from either fed or fasted animals. Implications concerning the identification of possible regulatory sites in the control of lipogenesis were discussed in relation to the above observations.     

A Novel Functional Human Eukaryotic Translation Initiation Factor 4G

Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.