Polyhalogenobenzimidazoles: synthesis and their inhibitory activity against casein kinases

A series of novel polyhalogenated benzimidazoles have been prepared by exhaustive bromination of a variety of 2-substituted benzimidazoles. The efficacy of both new compounds and a number of their previously described cognates as inhibitors of casein kinases CK1, CK2 and G-CK was investigated. The type of N-1 alkyl substituent as well as introduction of a polyfluoroalkyl moiety at position 2 did not markedly influence the inhibitory efficacy toward CK2 of the respective 4,5,6,7-tetrabromobenzimidazole derivatives which conversely were almost ineffective toward CK1 and G-CK. However, 4,5,6,7-tetrabromobenzimidazoles substituted at position 2 with either chlorine, bromine or sulfur atom, while manifesting a still considerable inhibitory activity against CK2 (IC(50) in the 0.49-0.93 microM range) proved to be potentially powerful inhibitors also against CK1 (IC(50) in the 18.4-2.2 microM range).     

Hepatic overexpression of sterol carrier protein-2 inhibits VLDL production and reciprocally enhances biliary lipid secretion

We examined in vivo a role for sterol carrier protein-2 (SCP-2) in the regulation of lipid secretion across the hepatic sinusoidal and canalicular membranes. Recombinant adenovirus Ad.rSCP2 was used to overexpress SCP-2 in livers of mice. We determined plasma, hepatic, and biliary lipid concentrations; hepatic fatty acid (FA) and cholesterol synthesis; hepatic and biliary phosphatidylcholine (PC) molecular species; and VLDL triglyceride production. In Ad.rSCP2 mice, there was marked inhibition of hepatic fatty acids and cholesterol synthesis to <62% of control mice. Hepatic triglyceride contents were decreased, while cholesterol and phospholipids concentrations were elevated in Ad.rSCP2 mice. Hepatic VLDL triglyceride production fell in Ad.rSCP2 mice to 39% of control values. As expected, biliary cholesterol, phospholipids, bile acids outputs, and biliary PC hydrophobic index were significantly increased in Ad.rSCP2 mice. These studies indicate that SCP-2 overexpression in the liver markedly inhibits lipid synthesis as well as VLDL production, and alters hepatic lipid contents. In contrast, SCP-2 increased biliary lipid secretion and the proportion of hydrophobic PC molecular species in bile. These effects suggest a key regulatory role for SCP-2 in hepatic lipid metabolism and the existence of a reciprocal relationship between the fluxes of lipids across the sinusoidal and canalicular membranes.     

The control of the proventriculus in the honeybee (Apis mellifera carnica L.) II. Feedback mechanisms

The mechanisms underlying the control of solution transport rates through the proventriculus in foraging honeybees were investigated in individuals trained to collect defined amounts of sugar solutions. Following feeding, bees were injected either with metabolisable (glucose, fructose, trehalose), or non-metabolisable (sorbose) sugars, in order to distinguish between haemolymph osmolarity and haemolymph sugar levels as factors controlling the solution transport rates through the proventriculus. After a fixed period, workers were dissected in order to measure crop content and haemolymph sugar titers. Between feeding and dissection, the metabolic rate of every investigated forager was measured using open-flow respirometry. Bees injected with metabolisable sugars 15 min after feeding were observed to reduce their solution transport rates through the proventriculus, but injection of non-metabolisable sugars had no influence on them. This suggests that the solution transport rate through the proventriculus is controlled by the concentration of metabolisable compounds in the haemolymph, and not by the haemolymph osmolarity. A period of 10 min after injection of metabolisable sugars was enough to observe reduced solution transport rates. However, if bees were injected only 5 min after feeding, no reduced solution transport rates were observed 10 min after injection.     

Kynurenine 3-mono-oxygenase inhibitors attenuate post-ischemic neuronal death in organotypic hippocampal slice cultures

Kynurenine 3-mono-oxygenase (KMO) inhibitors reduce 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) neosynthesis and facilitate kynurenine metabolism towards kynurenic acid (KYNA) formation. They also reduce tissue damage in models of focal or transient global cerebral ischemia in vivo. We used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) to investigate KMO mechanism(s) of neuroprotective activity. Exposure of the slices to 30 min of OGD caused CA1 pyramidal cell death and significantly decreased the amount of KYNA released in the incubation medium. The KMO inhibitors (m-nitrobenzoyl)-alanine (30-100 micro m) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (1-10 micro m) reduced post-ischemic neuronal death and increased KYNA concentrations in slice incubation media. The maximal concentration of KYNA detected in the incubation media of slices treated with KMO inhibitors was approximately 50 nm and was too low to efficiently interact with alpha7 nicotinic acetylcholine receptors or with the glycineb site of N-methyl-d-aspartate (NMDA) receptors. On the other hand, the addition of either 3-HK or QUIN (1-10 micro m) to OGD-exposed hippocampal slices prevented the neuroprotective activity of KMO inhibitors. Our results suggest that KMO inhibitors reduce the neuronal death found in the CA1 region of organotypic hippocampal slices exposed to 30 min of OGD by decreasing the local synthesis of 3-HK and QUIN.     

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.     

Metabotropic Glutamate Receptors Stimulate Phospholipase D via Different Pathways in the Adult and Neonate Rat Hippocampus

In order to characterize the ontogenetic profile of metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) we examined the effects of selected mGlu agents on PLD activity in immature and adult rat hippocampus. The group I mGlu receptor agonist 3,5-dihydroxyphenylglycine stimulated PLD in immature tissue, but reduced the PLD response evoked by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] in adult hippocampus. (2R,1S,2R,3S)-2-(2-Carboxy-3-phenylcyclopropyl)glycine (PCCG-13), a recently characterized selective antagonist of PLD-coupled mGlu receptors, displayed a much greater activity in reducing the PLD response to (1S,3R)-ACPD in adult than in neonate hippocampus. Our results lend support to the hypothesis that glutamatergic activation of PLD in the rat hippocampus is developmentally regulated.     

Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells

Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells.