Morphological Characteristics and Gene Mapping of Purple Apiculus Formation in Rice

Plant Molecular Biology Reporter - Apiculus color of grain is an important trait which is used as a morphological marker in rice (Oryza sativa. L). In the present study, the purple apiculus mutant...     

Effect of steroids and the estrous cycle on uterine neuromedin U receptor expression

We investigated the role of estrogen and progesterone on rat uterine NmU receptor expression in both intact and ovariectomized animals and examined receptor expression through the estrous cycle. Chronic administration of beta-estradiol 3-benzoate (E2) or progesterone in intact animals was devoid of any effect. RU486 caused a 2-fold up-regulation in NmU receptor density. Ovariectomy caused a 60% decrease in receptor density, but chronic E2 administration to ovariectomized rats significantly increased NmU receptor density. The estrous cycle had no significant effect on NmU receptor density. These results suggest that NmU receptor expression is estrogen-dependent, whereas progesterone or a progestin-induced factor is involved in the modulation of this expression.     

The complex life of simple sphingolipids

The extensive diversity of membrane lipids is rarely appreciated by cell and molecular biologists. Although most researchers are familiar with the three main classes of lipids in animal cell membranes, few realize the enormous combinatorial structural diversity that exists within each lipid class, a diversity that enables functional specialization of lipids. In this brief review, we focus on one class of membrane lipids, the sphingolipids, which until not long ago were thought by many to be little more than structural components of biological membranes. Recent studies have placed sphingolipids-including ceramide, sphingosine and sphingosine-1-phosphate-at the centre of a number of important biological processes, specifically in signal transduction pathways, in which their levels change in a highly regulated temporal and spatial manner. We outline exciting progress in the biochemistry and cell biology of sphingolipids and focus on their functional diversity. This should set the conceptual and experimental framework that will eventually lead to a fully integrated and comprehensive model of the functions of specific sphingolipids in regulating defined aspects of cell physiology.     

Tumour necrosis factor alpha, lipid peroxidation and NO* are increased and associated with decreased free-radical scavenging enzymes in patients with Weill-Marchesani syndrome

AIM: Weill-Marchesani syndrome (WMS) is a rare systemic disorder with both autosomal recessive and dominant inheritances. Accumulation of reactive oxygen species such as O2*-, H2O2 and OH* causes lipid peroxidation (LPO), whereas antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GSHPx)) mediate defence against oxidative stress. Excess tumour necrosis factor (TNF)-alpha and NO* react with O2*- and cause further antioxidant depletion with an increase in mutation frequency by H2O2. This study investigated the levels of SOD, GSHPx, catalase (CAT), TNF-alpha, NO and LPO in patients with WMS. METHODS: A group of 10 WMS patients (four males, six females; age, 26.5+/-19.0 years) and 10 age-matched and sex-matched controls (five males, five females; age, 27.3+/-18.2 years) were included. Serum TNF-alpha levels were determined by a spectrophotometer technique using immulite chemiluminescent immunometric assay. Malondialdehyde (MDA) was determined in plasma; CAT in red blood cells (RBCs), and SOD and GSHPx in both plasma and RBCs. Total serum NO* levels were evaluated by Griess reaction. RESULTS: Mean levels of TNF-alpha (8.3+/-0.6 pg/ml) in WMS patients were significantly (p<0.001) higher than controls (4.3+/-0.2 pg/ml). Plasma MDA levels in patients and controls were 5.4+/-0.8 and 1.8+/-0.6 micromol/l, respectively, and the difference was significant (p=0.0002). SOD and GSHPx activities were significantly lower in both RBCs and plasma of WMS than in controls (RBC-SOD, 3981.9+/-626.6 versus 5261.6+/-523.0 U/g haemoglobin (Hb), p=0.0005; plasma-SOD, 529.4+/-49.3 versus 713.4+/-55.7 U/g protein, p=0.0002; RBC-GSHPx, 682.7+/-42.0 versus 756.5+/-47.6 U/g Hb, p=0.0011; plasma-GSHPx, 107.3+/-15.0 versus 131.4+/-19.7 U/g protein, p=0.0113). In addition, serum NO (NO*-2 + NO*-3) levels were also significantly (p = 0.0002) increased in WMS patients (54.4+/-5.7 versus 26.9+/-6.7 micromol/l). RBC-CAT levels were similar between groups (125.6+/-21.3 versus 131.0+/-21.5 k/g Hb, p = 0.8798). CONCLUSIONS: The elevated LPO, TNF-alpha and NO* with decreased antioxidant enzyme activities indicated impaired antioxidative defence mechanisms with an oxidative injury and cell toxicity in WMS patients. The use of multiple antioxidants and free radical scavengers might be helpful in this genetic disorder.     

Poloxamer 407-induced atherosclerosis in mice appears to be due to lipid derangements and not due to its direct effects on endothelial cells and macrophages

Coronary heart disease secondary to atherosclerosis is still the leading cause of death in the US. Animal models used for elucidating the pathogenesis of this disease primarily involve rabbits and pigs. Previous studies from this laboratory have demonstrated intraperitoneal injections of poloxamer 407 (P-407) in both male and female mice will lead to hyperlipidemia and atherosclerosis, suggesting the use of this polymer to develop a mouse model of atherosclerosis. In order to understand the mechanism of P-407-induced hyperlipidemia and vascular lesion formation, we evaluated the direct effects of P-407 on endothelial cell and macrophage functions in vitro, and its in vivo effects on the oxidation of circulating lipids following long-term (4 month) administration. Our results demonstrated that incubation of P-407 with human umbilical vein endothelial cells in culture did not influence either cell proliferation or interleukin-6 and interleukin-8 production over a concentration range of 0-40 microM. In addition, nitric oxide production by macrophages was not affected by P-407 over a concentration range of 0-20 microM. Finally, we demonstrated that while P-407 could not induce the oxidation of LDL-C in vitro, long-term (4 month) administration of P-407 in mice resulted in elevated levels of oxidized lipids in the plasma. Thus, it is suggested that the formation of atherosclerotic lesions in this mouse model of atherosclerosis does not result from either direct stimulation of endothelial cells or macrophage activation by P-407. Instead, these data would support the premise that oxidation of lipids (perhaps low-density lipoprotein cholesterol) by an indirect mechanism following injection of P-407 may represent one of the mechanisms responsible for atheroma formation.     

Induction of antigen-specific CTL responses using antigens conjugated to short peptide vectors

Linear peptides (SynB vectors) with specific sequence motifs have been identified that are capable of enhancing the transport of a wide range of molecules into cells. These peptide vectors have been used to deliver exogenous peptides and protein Ags across the cell membrane and into the cytoplasm of cells. Specifically, in vitro analysis indicated that these SynB peptides enhanced the uptake of two 9-mer peptide Ags, NP(147-155) and Mtb(250-258) (T cell epitopes of influenza nucleoprotein and Mycobacterium tuberculosis, respectively) and the M. tuberculosis Ag Mtb8.4 protein, into K562 cells when covalently linked to the respective Ags. Furthermore, selected SynB vectors, when conjugated to these same Ags and used as immunogens, resulted in considerably enhanced Ag-specific CTL responses. Several SynB vectors were tested and resulted in varying levels of cellular uptake. The efficiency of uptake correlated with the ability of the SynB construct to deliver each epitope in vivo and induce specific CTL responses in mice. These data suggest that peptide vectors, such as SynB that transport target Ags across the cell membrane in a highly efficient manner, have significant potential for vaccine delivery.     

Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier

Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice [mdr1a(-/-) and mrp1(-/-)], with and without probenecid, digoxin, PSC833 or d-glucose. No difference was found between P-gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co-perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved. The co-perfusion of [14C]M6G with d-glucose revealed a threefold decrease in M6G uptake. In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.     

Studies on the internalization mechanism of cationic cell-penetrating peptides

A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43-58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time- and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperature- and energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43-58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation.     

cPKC-dependent sequestration of membrane-recycling components in a subset of recycling endosomes

In addition to the classical role of protein kinase C (PKC) as a mediator of transmembrane signals initiated at the plasma membrane, there is also significant evidence to suggest that a more sustained PKC activity is necessary for a variety of long term cellular responses. To date, the subcellular localization of PKC during sustained activation has not been extensively studied. We report here that long term activation of PKC (1 h) leads to the selective translocation of classical PKC isoenzymes, alpha and betaII, to a juxtanuclear compartment. Juxtanuclear translocation of PKC required an intact C1 and C2 domain, and occurred in a microtubule-dependent manner. This juxtanuclear compartment was localized close to the Golgi complex but displayed no overlap with Golgi markers, and was resistant to dispersal with Golgi disrupting agents, brefeldin A and nocodazole. Further characterization revealed that PKCalpha and betaII translocated to a compartment that colocalized with the small GTPase, rab11, which is a marker for the subset of recycling endosomes concentrated around the microtubule-organizing center/centrosome. Analysis of the functional consequence of cPKC translocation on membrane recycling demonstrated a cPKC-dependent sequestration of transferrin, a marker of membrane recycling, in the cPKC compartment. These results identify a novel site for cPKC translocation and define a novel function for the sustained activation of PKCalpha and betaII in regulation of recycling components.