Liquid chromatographic determination of caffeine in biologic fluids

A high-performance liquid chromatographic procedure was developed for the determination of caffeine in various biologic fluids and coffee. A reversed-phase column and UV detection at 254 nm were used to obtain a sensitivity of 0.1 μg/ml caffeine in serum and saliva using a sample volume of 0.1 ml. Caffeine metabolites and commonly ingested xanthines do not interfere with the assay. The within-day coefficients of variation were 9.8 and 9.9% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. The day-to-day coefficients of variation were 6.8 and 6.6% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. Serum and saliva caffeine concentrations were determined following a single oral dose of coffee and an intravenous infusion of caffeine in one subject. Computer estimates of caffeine pharmacokinetic parameters in one subject are in excellent agreement with previously published values.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Circulating ceramides are inversely associated with cardiorespiratory fitness in participants aged 54–96 years from the Baltimore Longitudinal Study of Aging

Cardiorespiratory fitness (VO₂ peak) declines with age and is an independent risk factor for morbidity and mortality in older adults. Identifying biomarkers of low fitness may provide insight for why some individuals experience an accelerated decline of aerobic capacity and may serve as clinically valuable prognostic indicators of cardiovascular health. We investigated the relationship between circulating ceramides and VO₂ peak in 443 men and women (mean age of 69) enrolled in the Baltimore Longitudinal Study of Aging (BLSA). Individual species of ceramide were quantified by HPLC–tandem mass spectrometry. VO₂ peak was measured by a graded treadmill test. We applied multiple regression models to test the associations between ceramide species and VO₂ peak, while adjusting for age, sex, blood pressure, serum LDL, HDL, triglycerides, and other covariates. We found that higher levels of circulating C18:0, C20:0, C24:1 ceramides and C20:0 dihydroceramides were strongly associated with lower aerobic capacity (P < 0.001, P < 0.001, P = 0.018, and P < 0.001, respectively). The associations held true for both sexes (with men having a stronger association than women, P value for sex interaction <0.05) and were unchanged after adjusting for confounders and multiple comparison correction. Interestingly, no significant association was found for C16:0, C22:0, C24:0, C26:0, and C22:1 ceramide species, C24:0 dihydroceramide, or total ceramides. Our analysis reveals that specific long‐chain ceramides strongly associate with low cardiovascular fitness in older adults and may be implicated in the pathogenesis of low fitness with aging. Longitudinal studies are needed to further validate these associations and investigate the relationship between ceramides and health outcomes.     

Ethanol Alters Glutamate but Not Adenosine Uptake in Rat Astrocytes: Evidence for Protein Kinase C Involvement

Glutamate is the primary excitatory neurotransmitter in brain. By stimulating neuronal activity, glutamate increases cellular energy utilization, enhances ATP hydrolysis and promotes the formation of adenosine. Adenosine has receptor-mediated effects that reduce or oppose the excitatory effects of glutamate. As a possible mechanism for ethanol's ability to inhibit excitatory effects of glutamate and enhance inhibitory effects of adenosine, we tested the hypothesis that ethanol promotes [³H]glutamate uptake and inhibits [³H]adenosine uptake. Using primary cultures of rat astrocytes, we found that acute treatment with ethanol (50 mM, 30 min) inhibited [³H]glutamate uptake and reduced protein kinase C (PKC)-induced stimulation of [³H]glutamate uptake. Prolonged treatment (50 mM, 3 day) with ethanol, however, increased both [³H]glutamate uptake and PKC activity. Contrary to other cell types, neither acute or chronic ethanol exposure affected [³H]adenosine uptake in astrocytes. These data indicate that in rat cortical astrocytes ethanol affects [³H]glutamate uptake but not [³H]adenosine uptake by affecting PKC modulation of transporter activity.     

Disruption of neurogenesis by amyloid beta-peptide,and perturbed neural progenitor cell homeostasis,in models of Alzheimer's disease

Neurogenesis occurs in the adult mammalian brain and may play roles in learning and memory processes and recovery from injury, suggesting that abnormalities in neural progenitor cells (NPC) might contribute to the pathogenesis of disorders of learning and memory in humans. The objectives of this study were to determine whether NPC proliferation, survival and neuronal differentiation are impaired in a transgenic mouse model of Alzheimer's disease (AD), and to determine the effects of the pathogenic form of amyloid beta-peptide (Abeta) on the survival and neuronal differentiation of cultured NPC. The proliferation and survival of NPC in the dentate gyrus of the hippocampus was reduced in mice transgenic for a mutated form of amyloid precursor protein that causes early onset familial AD. Abeta impaired the proliferation and neuronal differentiation of cultured human and rodent NPC, and promoted apoptosis of neuron-restricted NPC by a mechanism involving dysregulation of cellular calcium homeostasis and the activation of calpains and caspases. Adverse effects of Abeta on NPC may contribute to the depletion of neurons and cognitive impairment in AD.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Demographic and clinical variables affecting mid‐ to late‐life trajectories of plasma ceramide and dihydroceramide species

It has been increasingly recognized at the basic science level that perturbations in ceramide metabolism are associated with the development and progression of many age‐related diseases. However, the translation of this work to the clinic has lagged behind. Understanding the factors longitudinally associated with plasma ceramides and dihydroceramides (DHCer) at the population level and how these lipid levels change with age, and by sex, is important for the clinical development of future therapeutics and biomarkers focused on ceramide metabolism. We, therefore, examined factors cross‐sectionally and longitudinally associated with plasma concentrations of ceramides and DHCer among Baltimore Longitudinal Study of Aging participants (n = 992; 3960 total samples), aged 55 years and older, with plasma at a mean of 4.1 visits (range 2–6). Quantitative analyses were performed on a high‐performance liquid chromatography‐coupled electrospray ionization tandem mass spectrometer. Linear mixed models were used to assess the relationships between plasma ceramide and DHCer species and demographics, diseases, medications, and lifestyle factors. Women had higher plasma concentrations of most ceramide and DHCer species and showed steeper trajectories of age‐related increases compared to men. Ceramides and DHCer were more associated with waist–hip ratio than body mass index. Plasma cholesterol and triglycerides, prediabetes, and diabetes were associated with ceramides and DHCer, but the relationship showed specificity to the acyl chain length and saturation. These results demonstrate the importance of examining the individual species of ceramides and DHCer, and of establishing whether intra‐individual age‐ and sex‐specific changes occur in synchrony to disease onset and progression.     

Deficiency of a Niemann-Pick, type C1-related protein in toxoplasma is associated with multiple lipidoses and increased pathogenicity

Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved 'sterol-sensing domain' (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma.     

Involvement of Inositol 1,4,5-Trisphosphate-Regulated Stores of Intracellular Calcium in Calcium Dysregulation and Neuron Cell Death Caused by HIV-1 Protein Tat

HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.