Effect of L-propranolol on the binding,internalization and degradation of 125I-low density lipoproteins by human skin fibroblasts

The effect of L-propranolol on the receptor mediated uptake of human LDL by fibroblasts was investigated. When L-propranolol was tested between 10^?6 and 10^?4 M at a ¹²⁵I LDL concentration ranging from 6.25 to 198 μg/ml, an increase in binding, internalization and degradation of the labelled lipoproteins was demonstrated with a maximal effect respectively of 52%, 680% and 105% observed with L-propranolol at 10^?4 M and a lipoprotein concentration of 37.5 μg/ml. The effect occurs rapidly and is seen 2 hrs after the addition of the drug. Lower drug concentrations, although less effective, still produced significant increase in lipoprotein uptake and degradation. Procaine, a local anesthetic, used at 10^?4 M has no effect on ¹²⁵I-LDL binding and internalization. D-propranolol, another local anesthetic and L-propranolol enantiomer, is also without effect. This, excluding a drug induced stabilization of the membrane as the responsible mechanism of action, indicates that stereospecificity is important.     

Relationship between the Accumulation of Putrescine and the Tolerance to Oxygen-Deficit Stress in Gramineae Seedlings

The accumulation of putrescine under anoxia was studied in seedlingsof 6 Gramineae species showing tolerance to the stress in thefollowing order: rice, barnyard grass>maize>rye, barleyand wheat. The accumulation of putrescine in shoots and rootsduring 6 h of oxygen deprivation was different among the speciesand correlated with their tolerance to anaerobic conditions.In both tissues, rice and barnyard grass accumulated more than0.6 µmol/g fresh weight. The lower increase in the putrescinetiter was observed in wheat tissues with less than 0.1 µmol/gfresh weight. Feeding experiments with [^l4C]arginine showedthat rice tissues have a greater capacity than wheat tissuesto synthesize putrescine under anoxia. The better tolerant speciesto anaerobic conditions showed high arginine decarboxylase activityin shoots and roots than the sensitive species. The presenceof exogenous Put partially increased the survival of wheat rootsafter 7.5 h of oxygen-deficit stress. The role of putrescinein determining differences in the tolerance to anoxia in Gramineaeseedlings is discussed. (Received December 20, 1989; Accepted March 12, 1990)     

Detrimental role of IL-33/ST2 pathway sustaining a chronic eosinophil-dependent Th2 inflammatory response,tissue damage and parasite burden during Toxocara canis infection in mice

Toxocariasis is a neglected disease that affects people around the world. Humans become infected by accidental ingestion of eggs containing Toxocara canis infective larvae, which upon reaching the intestine, hatch, penetrate the mucosa and migrate to various tissues such as liver, lungs and brain. Studies have indicated that Th2 response is the main immune defense mechanism against toxocariasis, however, there are still few studies related to this response, mainly the IL-33/ST2 pathway. Some studies have reported an increase in IL-33 during helminth infections, including T. canis. By binding to its ST2 receptor, IL-33 stimulating the Th2 polarized immune cell and cytokine responses. Thus, we aimed to investigate the role of the IL-33/ST2 pathway in the context of T. canis larval migration and the immunological and pathophysiological aspects of the infection in the liver, lungs and brain from Wild-Type (WT) BALB/c background and genetically deficient mice for the ST2 receptor (ST2^-/-). The most important findings revealed that the IL-33/ST2 pathway is involved in eosinophilia, hepatic and cerebral parasitic burden, and induces the formation of granulomas related to tissue damage and pulmonary dysfunction. However, ST2^-/- mice, the immune response was skewed to Th1/Th17 type than Th2, that enhanced the control of parasite burden related to IgG2a levels, tissue macrophages infiltration and reduced lung dysfunction. Collectively, our results demonstrate that the Th2 immune response triggered by IL-33/ST2 pathway mediates susceptibility to T. canis, related to parasitic burden, eosinophilia and granuloma formation in which consequently contributes to tissue inflammation and injury.     

Intraocular pressure profile during the modified diurnal tension curve using Goldman applanation tonometry and dynamic contour tonometry

The aim of this study was to compare the intraocular pressure (IOP) profile during the modified diurnal tension curve (mDTC) using Goldman applanation tonometry (GAT) and dynamic contour tonometry (DCT) in treated glaucomatous eyes. Eligible subjects were submitted to the mDTC using GAT and DCT in this sequence. IOP measurements were performed at 8 a.m., 10 a.m., 2 p.m., and 4 p.m.. Central corneal thickness was measured using ultrasound pachymetry in the morning. Statistical analysis was performed using paired Student’s t test and Bland–Altman plot. The mean difference between DCT and GAT measurements was 0.9 mmHg. The mean ± SD IOP measurements during the mDTC were 19.68 ± 4.68, 17.63 ± 4.44, 17.25 ± 5.41, and 17.32 ± 4.25 mmHg using GAT and 19.97 ± 4.75, 18.79 ± 4.61, 19.53 ± 5.30, and 19.43 ± 5.45 mmHg using DCT. IOP measurements were higher in the morning (8 a.m.) and decreased throughout the day using both tonometers. The difference between IOP measurements using GAT and DCT was smaller in the morning and increased throughout the day. The IOP variability using GAT was higher than using DCT. Corneal biomechanical properties might help explain our findings.     

High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His(6)-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (approximately 35 mg) and truncated hTK1 (approximately 23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP-MgCl(2) incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (T(m)) of 71.7 and 73.4 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K(M) of 0.51 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM and k(cat) of 1.65 s(-1), thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.     

Asymmetric growth and development of the Xenopus laevis retina during metamorphosis is controlled by type III deiodinase

During the metamorphosis of the Xenopus laevis retina, thyroid hormone (TH) preferentially induces ventral ciliary marginal zone (CMZ) cells to both increase their proliferation and give rise to ipsilaterally projecting ganglion cells. Here we show that dorsal CMZ cells express type III deiodinase (D3), an enzyme that inactivates TH. The dorsal CMZ cells can be induced to proliferate if deiodinase activity is inhibited. D3 or dominant-negative thyroid hormone receptor transgenes inhibit both TH-induced proliferation of the ventral CMZ cells and the formation of the ipsilateral projection. Thus, the localized expression of D3 in the dorsal CMZ cells accounts for the asymmetric growth of the frog retina.     

Synthesis and opioid activity of N,N-dimethyl-Dmt-Tic-NH-CH(R)-R' analogues: acquisition of potent delta antagonism

N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R′ series of compounds produced no significant affect on the high δ-opioid receptor affinity (K_i=0.035–0.454 nM), but dramatically decreased that for the μ-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R′): phenyl (Ph) (5′–8′) elicited a greater reduction in μ-affinity (40–70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss of δ-agonism coupled with the appearance of potent δ antagonism (4′–7′) (pA₂=8.14–9.47), while 1 exhibited only a 160-fold decreased δ agonism (1′) and the δ antagonism of 8 enhanced >10-fold (pA₂=10.62, 8′); and (ii) a consistent loss of μ-affinity resulted in enhanced δ-opioid receptor selectivity. With the exception of compound 1′, the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent δ-selective antagonists.     

Blockade of nociceptin/orphanin FQ transmission in rat substantia nigra reverses haloperidol-induced akinesia and normalizes nigral glutamate release

We recently showed that pharmacological blockade of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors located in the substantia nigra stimulates the nigrostriatal dopaminergic pathway and motor behavior (Marti et al. J. Neurosci. 2004, 24, 6659-6666). To investigate whether such motor-stimulating action was dependent on functional dopaminergic transmission, the selective NOP receptor peptide antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) was microinjected into the substantia nigra reticulata of rats made cataleptic by systemic haloperidol administration. UFP-101 reduced haloperidol-induced akinesia as measured by immobility time in the bar test. UFP-101 also induced contralateral turning in cataleptic rats. To investigate the mechanisms involved in the anti-akinetic action of UFP-101, nigral glutamate release was monitored by microdialysis technique. The anti-akinetic action of UFP-101 correlated with normalization of nigral glutamate release, previously elevated by haloperidol injection. We conclude that endogenous N/OFQ in the substantia nigra sustains akinesia generated by impaired DA transmission and subthalamic nucleus overactivation. NOP receptor antagonists may be beneficial in the symptomatic therapy of parkinsonism, via normalization of subthalamonigral glutamatergic transmission.     

Anti-protozoal and plasmodial FabI enzyme inhibiting metabolites of Scrophularia lepidota roots

The ethanolic root extract of Scrophularia lepidota, an endemic plant of the Turkish flora, has been investigated for its anti-protozoal and inhibitory effect towards plasmodial enoyl-ACP reductase (FabI), a key enzyme of fatty acid biosynthesis in Plasmodium falciparum. Chromatographic separation of the extract yielded 10 iridoids (1-10), two of which are new, and a known phenylethanoid glycoside (11). The structures of the new compounds were determined as 3,4-dihydro-methylcatalpol (8) and 6-O-[4'-O-trans-(3,4-dimethoxycinnamoyl)-alpha-L-rhamnopyranosyl]aucubin (scrolepidoside, 9) by spectroscopic means. The remaining metabolites were characterized as catalpol (1), 6-O-methylcatalpol (2), aucubin (3), 6-O-alpha-L-rhamnopyranosyl-aucubin (sinuatol, 4), 6-O-beta-D-xylopyranosylaucubin (5), ajugol (6), ajugoside (7), an iridoid-related aglycone (10) and angoroside C (11). Nine isolates were active against Leishmania donovani, with the new compound 9 being most potent (IC50 6.1 microg/ml). Except for 4, all pure compounds revealed some trypanocidal potential against Trypanosoma brucei rhodesiense (IC50 values 29.3-73.0 microg/ml). Only compound 10 showed moderate anti-plasmodial (IC50 40.6 microg/ml) and FabI enzyme inhibitory activity (IC50 100 microg/ml). 10 is the second natural product inhibiting the fatty acid biosynthesis of Plasmodium falciparum.