Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes

Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance.     

Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: implications for RNA-based therapeutics

RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2'-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2'-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn(2+) cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.     

茄子光系统Ⅱ的热胁迫特性

以耐热性较弱的黑贝一号圆茄和耐热性较强的黑贝二号圆茄为试材,热胁迫处理后采用植物效率仪PEA进行快速叶绿素荧光诱导曲线及其参数测定.结果表明：当温度高于40 ℃,PSⅡ结构受热胁迫影响较为敏感,表现为初始荧光F_o缓慢上升;PSⅡ原初光化学效率F_v/F_m和ΔF/F_m′大幅度下降,且黑贝二号F_v/F_m的半衰时间T₅₀和ΔF/F_m′的半衰温度t₅₀分别大于黑贝一号.较高的热胁迫剂量（48℃处理5 min或44℃处理20～30min）下,快速荧光诱导动力学曲线呈现OKJIP型,在700μs处出现与放氧复合体失活有关的K相.黑贝一号在44 ℃下处理20 min才有K相出现,黑贝二号则晚10 min出现.与35℃相比,在48℃,特别是在52℃的较高剂量热胁迫下,Strasser能量流动模型参数中的DI_o/RC有大幅度地增加,体现了热耗散对PSⅡ的较强保护能力.随着热胁迫温度的升高和热胁迫时间的延长,两品种的无活性中心F_vi/Fv显著增加.     

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from l-serine to tetrahydrofolate (H₄folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH₂-H₄folate). SHMT is crucial for deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. To define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either l-serine or H₄folate. The dissociation constants for the enzyme·l-serine and enzyme·H₄folate complexes were determined as 0.18 ± 0.08 and 0.35 ± 0.06 mm, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the k_cat value (1.09 ± 0.05 s⁻¹) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development.     

Divanadium(V) complexes with 4-R-benzoic acid (1-methyl-3-oxo-butylidene)-hydrazides: Syntheses, structures and properties

In acetonitrile, reactions of bis(acetylacetonato)oxidovanadium(IV) ([VO(acac)₂]) with 4-R-benzoylhydrazine in 1:1 mole ratio provide coordinatively symmetrical complexes (1-5) of the {OV(μ-O)VO}⁴⁺ motif in 40-47% yields. On the other hand, in methanol the same reactants provide complexes (6-10) containing the {OV(μ-OMe)₂VO}⁴⁺ core in 37-50% yields. In both series of complexes, the ligand is the O,N,O-donor deprotonated Schiff base system 4-R-benzoic acid (1-methyl-3-oxo-butylidene)-hydrazide formed by template condensation of acac⁻ with 4-R-benzoylhydrazine (R = H, Cl, OMe, NO₂ and NMe₂). All the complexes have been characterized by elemental analysis, magnetic and spectroscopic (IR, UV-Vis and NMR) measurements. Molecular structures of three representative complexes (4, 6 and 7) have been determined by X-ray crystallography. In each complex, the dianionic planar ligand is coordinated to the metal centre via the enolate-O, the imine-N and the O-atom of the deprotonated amide functionality. Cyclic voltammetric measurements in dichloromethane revealed that complexes 1-5 are redox inactive, while complexes 6-10 display a metal centred reduction in the potential range −0.06 to 0.0.32 V (versus Ag/AgCl).     

乳酸菌的抗冷冻性及耐受机理

乳酸菌发酵剂在工业生产过程中,会受到冷冻的刺激,如真空冷冻干燥及后期的低温保藏,此外,发酵乳制品的保藏和干酪的成熟过程也都在低温中进行。这些均会对乳酸菌发酵剂及发酵乳制品质量产生一定的影响。因此,掌握乳酸菌在冷冻条件下的反应机理有助于优化发酵剂和发酵乳制品在工业生产中的冷冻、发酵和贮藏条件,从而提高产品质量和生产效益。本文对乳酸菌的抗冷冻性及机理进行了分析,并对发酵剂的保护提出具体措施。     

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.     

Distribution and biochemical properties of an M1-family aminopeptidase in Plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism

Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68- and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steady-state kinetic parameters for both aminoacyl-β-naphthylamide and unmodified dipeptide substrates over the pH range 5.0-8.5 reveals that k(cat) is relatively insensitive to pH, whereas K(m) increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0-5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.     

Saprophytic competence of acid tolerant strains ofRhizobium trifolii in acid soil

Summary Laboratory prescreening ofRhizobium trifolii for acid tolerance, based upon the ability of rhizobia to grow in acid media (pH 4.2) containing Al (15 M), was successful for the selection of strains capable of survival in acid soil.Both sterile and non-sterile soils of varying acidity were inoculated with several strains ofR. trifolii.Acid tolerant strains generally had significantly higher populations at every sample period than an acid sensitive strain. Amelioration of soil acidity by liming improved persistence of all strains. Soil sterilization by autoclaving adversely affected survival of all strains at each soil acidity level.Paper Number 8766 of the Journal Series, North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.