(-)-CGP 12177 increases contractile force and hastens relaxation of human myocardial preparations through a propranolol-resistant state of the beta1-adrenoceptor

Two forms of the activated beta(1)-adrenoceptor exist, one that is stabilized by (-)-noradrenaline and is sensitive to blockade by (-)-propranolol and another which is stabilized by partial agonists such as (-)-pindolol and (-)-CGP 12177 but is relatively insensitive to (-)-propranolol. We investigated the effects of stimulation of the propranolol-resistant PI-adrenoceptor in the human heart. Myocardium from non-failing and failing human hearts were set up to contract at 1 Hz. In right atrium from non-ailing hearts in the presence of 200 nM (-)-propranolol, (-)-CGP 12177 caused concentration-dependent increases in contractile force (-logEC(50)[M] 7.3+/-0.1, E-max 23+/-1% relative to maximal (-)-isoprenaline stimulation of beta(1)- and beta(2)-adrenoceptors, n=86 patients), shortening of the time to reach peak force (-logEC(50)[M] 7.4+/-0.1, E-max 37+/-5%, n=61 patients) and shortening of the time to reach 50% relaxation (t(50%), -logEC(50)[M] 7.3+/-0.1, E-max 33+/-2%, n=61 patients). The potency and maxima of the positive inotropic effects were independent of Ser49Gly- and Gly389Arg-beta(1)-adrenoceptor polymorphisms but were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (-logEC(50)[M] 7.7+/-0.1, E-max 68+/-6%, n=6 patients, P

Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC 4.1.3.18) catalyzes the first step in branchedchain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides. These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated. Here we report the 2.8 Angstrom resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl. The inhibitor, which has a K-i of 3.3 nM blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance. The structure provides a starting point for the rational design of further herbicidal compounds.

Reciprocal changes in forebrain contents of glycogen and of glutamate/glutamine during early memory consolidation in the day-old chick

Passive avoidance learning is with advantage studied in day-old chicks trained to distinguish between beads of two different colors, of which one at training was associated with aversive taste. During the first 30-min post-training, two periods of glutamate release occur in the forebrain. One period is immediately after the aversive experience, when glutamate release is confined to the left hemisphere. A second release, 30 min later, may be bilateral, perhaps with preponderance of the right hemisphere. The present study showed increased pool sizes of glutamate and glutamine, specifically in the left hemisphere, at the time when the first glutamate release occurs, indicating de novo synthesis of glutamate/glutamine from glucose or glycogen, which are the only possible substrates. Behavioral evidence that memory is extinguished by intracranial administration at this time of iodoacetate, an inhibitor of glycolysis and glycogenolysis, and that the extinction of memory is counteracted by injection of glutamine, supports this concept. A decrease in forebrain glycogen of similar magnitude and coinciding with the increase in glutamate and glutamine suggests that glycogen rather than glucose is the main source of newly synthesized glutamate/glutamine. The second activation of glutamatergic activity 30 min after training, when memory is consolidated into stable, long-term memory, is associated with a bilateral increase in pool size of glutamate/glutamine. No glycogenolysis was observed at this time, but again there is a temporal correlation with sensitivity to inhibition by iodoacetate and rescue by glutamine, indicating the importance of de novo synthesis of glutamate/glutamine from glucose or glycogen. (C) 2003 Elsevier B.V All rights reserved.

Effects of additional sequences directly downstream from the AUG on the expression of GFP gene

We have studied the expression of the green fluorescent protein (GFP) gene to gain more understanding of the effects of additional nucleotide triplets (codons) downstream from the initiation codon on the translation of the GFP mRNA in CHO and Cos1 cells. A leader sequence of six consecutive identical codons (GUG, CUC, AGU or UCA) was introduced into a humanized GFP (hm gfp) gene downstream from the AUG to produce four GFP gene variants. Northern blot and RT-PCR analysis indicated that mRNA transcription from the GFP gene was not significantly affected by any of the additional sequences. However, immunoblotting and FACS analysis revealed that AGU and UCA GFP variants produced GFP at a mean level per cell 3.5-fold higher than the other two GFP variants and the hm gfp gene. [35S]-Methionine labeling and immunoprecipitation demonstrate that GFP synthesis was very active in UCA variant transfected-cells, but not in GUG variant and hm gfp transfected-cells. Moreover, proteasome inhibitor MG-132 treatment indicated that the GFPs encoded by each of the GFP variants and the hm gfp were equally stable, and this together with the comparable mRNA levels observed for each construct suggested that the different steady-state GFP concentrations observed reflected different translation efficiencies of the various GFP genes. In addition, the CUC GFP variant, when transiently transfected into CHO or COS-1 cells, did not produce any GFP expressing cells (fully green cells), and the GUG variant produced GFP expressing cells less than 10%, while AGU and UCA GFP variants up to 30–35% in a time course study from 8 to 36 h posttransfection. Analysis of the potential secondary structure of the GFP variant mRNAs especially in the translation initiation region suggested that the secondary structure of the GFP mRNAs was unlikely to explain the different translation efficiencies of the GFP variants. The present findings indicate that a change of the initiation context of the GFP gene by addition of extra coding sequence can alter the translation efficiency of GFP mRNA, providing a means of more efficient expression of GFP in eukaryotic cells.

Matrix metalloproteinase-2 and-9 involvement in canine tumors

Matrix metalloproteinases (MMPs) are a family of enzymes implicated in the degradation and remodeling of extracellular matrix and in vascularization. They are also involved in pathologic processes such as tumor invasion and metastasis in experimental cancer models and in human malignancies. We used gelatin zymography and inummohistochemistry to determine whether MMP-2 and MMP-9 are present in canine tumors and normal tissues and whether MMP production correlates with clinicopathologic parameters of prognostic importance. High levels of pro-MMP-9, pro-MMP-2, and active MMP-2 were detected in most canine tumors. Significantly higher MMP levels were measured in canine tumors than in nontumors, malignancies had higher MMP levels than benign tumors, and sarcomas had higher active MMP-2 than carcinomas. Cartilaginous tumors produced higher MMP levels than did nonsarcomatous malignancies, benign tumors, and normal tissues, and significantly greater MMP-2 than osteosarcomas and fibrosarcomas. Pro-MMP-9 production correlated with the histologic grade of osteosarcomas. The 62-kd form of active MMP-2 was detected only in high-grade, p53-positive, metastatic malignancies. Zymography proved to be a sensitive and quantitative technique for the assessment of MMP presence but has the limitation of requiring fresh tissue; inummohistochemistry is qualitative and comparatively insensitive but could be of value in archival studies. MMP presence was shown in a range of canine tumors, and their link to tumor type and grade was demonstrated for the first time. This study will allow a substantially improved evaluation of veterinary cancer patients and provides baseline information necessary for the design of clinical trials targeting MMPs.

New fluorescence parameters for monitoring photosynthesis in plants - 1. The effect of illumination on the fluorescence parameters of the JIP-test

Chlorophyll fluorescence measurements have a wide range of applications from basic understanding of photosynthesis functioning to plant environmental stress responses and direct assessments of plant health. The measured signal is the fluorescence intensity (expressed in relative units) and the most meaningful data are derived from the time dependent increase in fluorescence intensity achieved upon application of continuous bright light to a previously dark adapted sample. The fluorescence response changes over time and is termed the Kautsky curve or chlorophyll fluorescence transient. Recently, Strasser and Strasser (1995) formulated a group of fluorescence parameters, called the JIP-test, that quantify the stepwise flow of energy through Photosystem II, using input data from the fluorescence transient. The purpose of this study was to establish relationships between the biochemical reactions occurring in PS II and specific JIP-test parameters. This was approached using isolated systems that facilitated the addition of modifying agents, a PS II electron transport inhibitor, an electron acceptor and an uncoupler, whose effects on PS II activity are well documented in the literature. The alteration to PS II activity caused by each of these compounds could then be monitored through the JIP-test parameters and compared and contrasted with the literature. The known alteration in PS II activity of Chenopodium album atrazine resistant and sensitive biotypes was also used to gauge the effectiveness and sensitivity of the JIP-test. The information gained from the in vitro study was successfully applied to an in situ study. This is the first in a series of four papers. It shows that the trapping parameters of the JIP-test were most affected by illumination and that the reduction in trapping had a run-on effect to inhibit electron transport. When irradiance exposure proceeded to photoinhibition, the electron transport probability parameter was greatly reduced and dissipation significantly increased. These results illustrate the advantage of monitoring a number of fluorescence parameters over the use of just one, which is often the case when the F-V/F-M ratio is used.

Comparison of the in vitro neuromuscular activity of venom from three Australian snakes (Hoplocephalus stephensi, Austrelaps superbus and Notechis scutatus): Efficacy of tiger snake antivenom

1. Tiger snake antivenom, raised against Notechis scutatus venom, is indicated not only for the treatment of envenomation by this snake, but also that of the copperhead (Austrelaps superbus ) and Stephen's banded snake (Hoplocephalus stephensi ). The present study compared the neuromuscular pharmacology of venom from these snakes and the in vitro efficacy of tiger snake antivenom. 2. In chick biventer cervicis muscle and mouse phrenic nerve diaphragm preparations, all venoms (3-10 mug/mL) produced inhibition of indirect twitches. In the biventer muscle, venoms (10 mug/mL) inhibited responses to acetylcholine (1 mmol/L) and carbachol (20 mumol/L), but not KCl (40 mmol/L). The prior (10 min) administration of 1 unit/mL antivenom markedly attenuated the neurotoxic effects of A. superbus and N. scutatus venoms (10 mug/mL), but was less effective against H. stephensi venom (10 mug/mL); 5 units/mL antivenom attenuated the neurotoxic activity of all venoms. 3. Administration of 5 units/mL antivenom at t(90) partially reversed, over a period of 3 h, the inhibition of twitches produced by N. scutatus (10 mug/mL; 41% recovery), A. superbus (10 mug/mL; 25% recovery) and H. stephensi (10 mug/mL; 50% recovery) venoms. All venoms (10-100 mug/mL) also displayed signs of in vitro myotoxicity. 4. The results of the present study indicate that all three venoms contain neurotoxic activity that is effectively attenuated by tiger snake antivenom.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and

Generation of aldehyde-derived protein modifications in ethanol-exposed heart

Background: Although excessive ethanol consumption is known to lead to a variety of adverse effects in the heart, the molecular mechanisms of such effects have remained poorly defined. We hypothesized that posttranslational covalent binding of reactive molecular species to proteins occurs in the heart in response to acute ethanol exposure. Methods: The generation of protein adducts with several aldehydic species was examined by using monospecific antibodies against adducts with malondialdehyde (MDA), acetaldehyde (AA), MDA-AA hybrids, and hydroxyethyl radicals. Specimens of heart tissue were obtained from rats after intraperitoneal injections with alcohol (75 mmol/kg body weight) with or without pretreatment with cyanamide (0.05 mmol/kg body weight), an aldehyde dehydrogenase inhibitor. Results: The amounts of MDA and unreduced AA adducts were found to be significantly increased in the heart of the rats treated with ethanol, cyanamide, or both, whereas no other adducts were detected in statistically significant quantities. Immunohistochemical studies for characterization of adduct distribution revealed sarcolemmal adducts of both MDA and AA in the rats treated with ethanol and cyanamide in addition to intracellular adducts, which were also present in the group treated with ethanol alone. Conclusions: These findings support the role of enhanced lipid peroxidation and the generation of protein-aldehyde condensates in vivo as a result of excessive ethanol intake. These findings may have implications in the molecular mechanisms of cardiac dysfunction in alcoholics.