A common inhibitory binding site for zinc and odorants at the voltage-gated K+ channel of rat olfactory receptor neurons

This study compared the effects of zinc and odorants on the voltage-gated K+ channel of rat olfactory neurons. Zinc reduced current magnitude, depolarized the voltage activation curve and slowed activation kinetics without affecting inactivation or deactivation kinetics. Zinc inhibition was potentiated by the NO compound, S-nitroso-cysteine. The pH- and diethylpyrocarbonate-dependence of zinc inhibition suggested that zinc acted by binding to histidine residues. Cysteine residues were eliminated as contributing to the zinc-binding site. The odorants, acetophenone and amyl acetate, also reduced current magnitude, depolarized the voltage activation curve and selectively slowed activation kinetics. Furthermore, the diethylpyrocarbonate- and pH-dependence of odorant inhibition implied that the odorants also bind to histidine residues. Zinc inhibitory potency was dramatically diminished in the presence of odorants, implying competition for a common binding site. These observations indicate that the odorants and zinc share a common inhibitory binding site on the external surface of the voltage-gated K+ channel.

An ATP-sensitive K+ conductance in dissociated neurones from adult rat intracardiac ganglia

1. An ATP-sensitive K+ (K-ATP) conductance has been identified using the perforated patch recording configuration in a population (52%) of dissociated neurones from adult rat intracardiac ganglia. The presence of the sulphonylurea receptor in approximately half of the intracardiac neurones was confirmed by labelling with fluorescent glibenclamide-BODIPY. 2. Under current clamp conditions in physiological solutions, leveromakalim (10 muM) evoked a hyperpolarization, which was inhibited by the sulphonylurea drugs glibenclamide and tolbutamide. 3. Under voltage clamp conditions in symmetrical (140 mM) K+ solutions, hath application of levcromakalim evoked an inward current with a density of similar to8 pA pF(-1) at -50 mV and a slope conductance of similar to9 nS, which reversed close to the potassium equilibrium potential (E-K). Cell dialysis with an ATP-free intracellular solution also evoked an inward current, which was inhibited by tolbutamide. 4. Bath application of either glibenclamide (10 muM) or tolbutamide (100 muM) depolarized adult intracardiac neurones by 3-5 mV, suggesting that a K-ATP conductance is activated under resting conditions and contributes to the resting membrane potential. 5. Activation of a membrane current by levcromakalim leas concentration dependent, with an EC50 of 1.6 muM. Inhibition of the levcromakalim-activated current by glibenclamide leas also concentration dependent, with an IC50 of 55 nM. 6. Metabolic inhibition with 2,4-dinitrophenol and iodoacetic acid or superfusion with hypoxic solution (P-O2 similar to 16 mmHg) also activated a membrane current. These currents exhibited similar I-P characteristics to the levcroinakalim-induced current and were inhibited by glibenclamide. 7. Activation of K-ATP channels in mammalian intracardiac neurones may contribute to changes in neural regulation of the mature heart and. cardiac function during ischaemia-reperfusion.

Developmental changes in hyperpolarization-activated currents I-h and I-K(IR) in isolated rat intracardiac neurons

The hyperpolarization-activated nonselective cation current, I-h, was investigated in neonatal and adult rat intracardiac neurons. I-h was observed in all neurons studied and displayed slow time-dependent rectification. I-h was isolated by blockade with external Cs+ (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I-h was approximately twofold greater in neurons from neonatal (-4.1 pA/pF at -130 mV) as compared with adult (-2.3 pA/pF) rats; however, the reversal potential and activation parameters were unchanged. The reversal potential and amplitude of I-h was sensitive to changes in external Na+ and K+ concentrations. An inwardly rectifying K+ current, I-K(IR), was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba2+. I-K(IR) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of -0.6 pA/pF at -130 mV. I-K(IR) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K+ rectifier described in other mammalian autonomic neurons. I-K(IR) was sensitive to changes in external K+, whereby raising the external K+ concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na+ had no effect on the reversal potential or amplitude of I-K(IR). I-K(IR) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I-h may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.

Effect of restricted feed intake on early reproductive development in Large White gilts

Forty-five Large White gilts were used to study the effect of energy intake from 28 to 176 d of age on body composition and reproductive development. From 28 to 60 d, the gilts were fed ad libitum a 16.6 MJ DE/kg, 24% crude protein and 1.3% total lysine diet. From 61 d of age three dietary treatments were used; 1) ad libitum access to feed (15.6 MJ DE/kg, 21% crude protein and 1.07% total lysine) (H), 2) feed offered at 75% (M) of the previous days intake of H, and 3) feed offered at 60% (L) of the previous days intake of H. ADG from 61 to 176 d of age was (p <0.05) affected by treatment. Although live weight at 176 d of age did not differ (p >0.1) the H gilts had higher (p <0.08) carcass weights than the M or L gilts. Back fat depths were similar (p >0.1) for all treatments at 115 d of age, however by 176 d of age M and H gilts were fatter (p <0.1) than L gilts. The mean lipid deposition (LD) from 115 to 176 d of age for L gilts (78.9 g/d) was less (p <0.05) than for M gilts (143.6 g/d) and H gilts (135.6 g/d). There were no differences between treatments for protein deposition (PD) over the same period. More (p <0.05) H gilts (n=8) attained puberty (first observed estrus) than either M gilts or L gilts (n=4 for both). Follicle numbers were similar (p >0.1) across treatments. For gilts that attained puberty, H gilts had fewer (p <0.05) follicles (13.5) than M gilts (19.7) and L gilts (21.3). For gilts with follicular development, H gilts had the heaviest (458.7 g) reproductive tract weight (RTW). However, for those that attained puberty, L gilts had the heaviest RTW. RTW were lowest for those with no follicular development. Energy restriction had a negative impact on puberty attainment, i.e. it took longer to reach puberty. However, for gilts that attained puberty, the number of follicles was greater for those on lower feed intakes. It would appear that rate of fat deposition, but not necessarily the total amount of fat, plays an important role in puberty attainment.

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

dEndocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and enclocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Free field and parafermionic realizations of twisted su(3)(k)((2)) current algebra

Free field and twisted parafermionic representations of twisted su(3)(k)((2)) current algebra are obtained. The corresponding twisted Sugawara energy-momentum tensor is given in terms of three (beta, gamma) pairs and two scalar fields and also in terms of twisted parafermionic currents and one scalar field. Two screening currents of the first kind are presented in terms of the free fields.

Oral disease in animals: The Australian perspective. Isolation and characterisation of black-pigmented bacteria from the oral cavity of marsupials

A wide range of animals suffer from periodontal disease. However, there is very little reported on disease and oral micro-biota of Australian animals. Therefore, the oral cavity of 90 marsupials was examined for oral health status. Plaque samples were collected from the subgingival margins using curettes; or swabs. Plaque samples were plated onto. non-selective trypticase soy agar plates, selective trypticase soy agar, non-selective and selective Wilkens Chalgrens, Agar. Plates were incubated in an anaerobic atmosphere and examined after 7-14 days for the presence of black-brown-pigmented colonies. A combination of morphological and biochemical tests were used (colonial morphology, pigmentation, aerobic growth, Gram reaction, fluorescence under long-wave UV light (360 nm), production of catalase, enzymatic activity with fluorogenic substrates and haemagglutination of sheep red cells) to identify these organisms. Black-pigmented bacteria were cultivated from the plaque of 32 animals including six eastern grey kangaroos, a musky rat kangaroo, a whiptail and a red-necked wallaby, 18 koalas, a bandicoot and five brushtail possums. No black-pigmented colonies were cultivated from squirrel or sugar gliders or quokkas or from marsupial mice. The majority of isolates were identified as Porphyromonas gingivalis-like species with the higher prevalence of isolation from the oral cavity of macropods (the kangaroos and wallabies). Oral diseases, such as gingivitis can be found in native Australian animals with older koalas having an increase in disease indicators and black-pigmented bacteria. Non-selective Wilkens Chalgren Agar was the medium of choice for the isolation of black-pigmented bacteria. (C) 2002 Elsevier Science Ltd. All rights reserved.

Expression of a truncated Brca1 protein delays lactational mammary development in transgenic mice

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.

The metamorphosis of lambda-fold K-3,K-3-designs into lambda-fold 6-cycle systems

In this paper necessary and sufficient conditions are given for the metamorphosis of a lambda-fold K-3,K-3-design of order n into a lambda-fold 6-cycle system of order n, by retaining one 6-cycle subgraph from each copy of K-3,K-3, and then rearranging the set of all the remaining edges, three from each K-3,K-3, into further 6-cycles so that the result is a lambda-fold 6-cycle system.

The radiation chemistry of ultem at 77 K as revealed by ESR spectroscopy

Radical formation in ultem following gamma-radiolysis has been reassessed, and the G(R*) values at different temperatures have been determined by ESR spectroscopy. The radical assignment and radical reactivity have been re-examined by photobleaching and thermal annealing studies. Photobleachable radical anions were found to comprise approximate to40% of the total number of radicals formed on radiolysis at 77 K. Spectral subtraction methods, ESR spectral simulations, measurement of g-values and the hyperfine splitting constants were used to identify the other radical intermediates. The principal chain scission radicals are formed due to scission of the main-chain at (i) the ether linkage, (ii) the isopropylidene group and (iii) the imide ring in the main chain. The side chain methyl groups of the isopropylidine units also lose hydrogen to form methylene radicals. The five-line spectrum observed to decay in the temperature range 370-430 K, which has not been assigned previously, has been identified as being characteristic of a di-substituted benzyl radical. (C) 2002 Elsevier Science Ltd. All rights reserved.

NMR study of the gamma radiolysis of poly(dimethyl siloxane) under vacuum at 303 K

The structural changes which occur on the gamma -radiolysis of poly(dimethyl siloxane) (PDMS) under vacuum at 303 K have been investigated using Si-29 and C-13 NMR. New structural units consistent with main chain scission and crosslinking through both H-linking and Y-linking reactions have been identified. The results obtained at various absorbed doses have been used to calculate the G-values for scission and crosslinking. G-values for scission of G(S) = 1.3 +/- 0.2, for H-linking of G(D-CH2-R) = 0.34 +/- 0.02 and for Y-Linking of G(Y) = 1.70 +/- 0.09 were obtained for radiolysis under vacuum at 303 K. Thus crosslinking predominates over scission for radiolysis of PDMS under these conditions, and, by contrast with previous studies, Y-links have been shown to be the predominant form of crosslinks. (C) 2001 Elsevier Science Ltd. All rights reserved.

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically, important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at similar to2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450,in, it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K-D = 0.7 mum), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.