Glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction

Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of homotypic CCI between “Os Fr.B” having 5–6 GlcNAc termini, vs. absence of such homotypic CCI between “Os Fr.1” having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3 ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using BAP conjugates of glycosyl epitopes.     

Varied and repeated atropine dosages and exercise-heat stress

Comparisons of physiological responses to 0, 0.5, 1, and 2 mg atropine (IM) were made in seven males (X +/- SD: age, 24 +/- 3 years; ht, 174 +/- 12 cm; wt, 76 +/- 3 kg) while they exercised (approximately 390 W) in a hot-dry (40 degrees C, 20% rh) environment. Responses to 4 mg, as well as repeatability of responses to 2 mg, were studied in two and six of these subjects, respectively. On 8 test days an intramuscular injection of atropine or saline control was administered 20 min before subjects walked on a treadmill for two 50-min bouts. Heart rate (HR) during exercise did not change in the control trial but by min 50 increased during all atropine trials (P less than 0.01). Rectal temperature (Tre) increased (P less than 0.01) in all trials by min 50 and continued increasing (P less than 0.01) in the 2-mg trial during the second exercise bout. For the two subjects tested with all dosages (0.5 - 4 mg atropine), the change in HR and Tre between the atropine and control trials at 50 min of exercise was regressed against the various atropine dosages. The relationship (r = 0.92) for HR was curvilinear while the relationship (r = 0.99) for Tre was linear. Mean weighted skin temperature (Tsk) was relatively constant during exercise and was warmer (P less than 0.05) with increasing atropine dosage. In a repeat 2 mg trial, HR was 6 bt . min-1 lower (P less than 0.05) on the second exposure but Tre was the same (P greater than 0.05) on both days. For subjects walking in the heat, three new observations were: 1) 0.5 mg of atropine resulted in increased HR and Tsk compared to control values; 2) HR was elevated but the magnitude of change decreased with increasing dosage, while the elevation in Tre was consistent with increasing dosage; and 3) rectal temperatures (in trials with and without atropine) were unaffected by previous days of atropine administration.     

Using diffusion distances for flexible molecular shape comparison

Background  

Many molecules are flexible and undergo significant shape deformation as part of their function, and yet most existing molecular shape comparison (MSC) methods treat them as rigid bodies, which may lead to incorrect shape recognition.     

Interactions between GABAergic and cholinergic mechanisms involved in monocular optokinetic nystagmus in frogs]

The systemic administration of atropine, a muscarinic cholinergic antagonist, was found to suppress the Nasal-Temporal (N-T) component of the frog monocular optokinetic nystagmus (OKN), which had appeared following a prior injection of bicuculline and which does not exist in the normal animal. On the contrary, the administration of a nicotinic cholinergic antagonist (D-TC, alpha-BGT, Hexamethonium) following that of bicuculline has prolonged the duration of the induced N-T component. Thus, ACh was shown to attenuate or to reinforce the GABAergic inhibition of the N-T component through muscarinic receptors or nicotinic receptors respectively. These data point to the existence of strong interactions between these two neurotransmission systems involved in frog monocular OKN.     

Selective proteolysis of ovine lutropin or its beta subunit by endoproteinase Arg-C. Properties of the Arg beta 43 cleaved hormone

Ovine lutropin (oLH) and its beta subunit (oLH beta) were nicked by short-term incubations with endoproteinase Arg-C. Isolated oLH beta was rapidly nicked and converted from an Mr 18,000 band on sodium dodecyl sulfate-polyacrylamide gels to an Mr 13,000 band. Partial nicking of only the beta subunit in intact oLH was also observed as indicated by the appearance of small amounts of the Mr 13,000 band detected in Arg-C-treated oLH samples. The alpha subunit was protected by association with the beta subunit, but free alpha subunit was rapidly degraded. Sequence analysis of nicked oLH beta indicated that one of the peptide bonds on either side of Arg43 was cleaved by the protease, with a slight preference for the amino side of this residue. Nicked oLH beta was reassociated with oLH alpha, and the resulting dimer was separated from unrecombined subunits. The biologic activity of nicked oLH beta + oLH alpha in an LH radioligand assay was only 2% that of intact oLH.     

Stereoselectivity and regioselectivity of uridine-5'-diphosphoglucuronosyltransferase toward vicinal dihydrodiols of polycyclic aromatic hydrocarbons

The ability of a purified rat liver microsomal uridine-5'-diphosphoglucuronosyltransferase to catalyze the glucuronidation of stereoisomeric trans- and cis-9, 10-dihydroxy-9, 10-dihydrophenanthrenes and 4, 5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes is examined. The enzyme shows the ability to discriminate kinetically between the antipodes of trans-9, 10-dihydroxy-9, 10-dihydrophenanthrene with turnover numbers of 0.070 and 1.4 s-1 and kc/Kmapp values of 4.4 X 10(3) and 1.1 X 10(3) M-1 s-1 for the 9R, 10R and 9S, 10S stereoisomers. Glucuronidation of the nondissymmetric cis-9, 10-dihydroxy-9, 10-dihydrophenanthrene proceeds with a turnover number of 0.037 s-1 and kc/Kmapp of 18 X 10(3) M-1 s-1 to give a 60/40 mixture of the two possible diastereomeric products. Three of the four stereoisomers of 4,5-dihydroxy-4,5-dihydrobenzo[alpha] pyrene are regioselectively glucuronidated by the enzyme with a high degree of kinetic discrimination. Turnover numbers for the 4S,5S, 4R,5R, and 4S,5R stereoisomers are 4.1, 0.37, and 0.23 s-1 with kc/Kmapp values of 23.8 X 10(3), 0.23 X 10(3), and 3.15 X 10(3) M-1 s-1, respectively. The 4R,5S cis isomer is not a substrate. Enzyme-catalyzed reactions of the 4S,5S and 4S,5R isomers give exclusively (greater than or equal to 95%) the 4-glucuronide with the 4R,5R isomer giving the 5-glucuronide. The kinetic and regiochemical results indicate that the enzyme recognizes hydroxyl groups on the beta-face or bottom face of the 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors associated with whole carcass condemnation rates in provincially-inspected abattoirs in Ontario 2001-2007: implications for food animal syndromic surveillance

Background  

Ontario provincial abattoirs have the potential to be important sources of syndromic surveillance data for emerging diseases of concern to animal health, public health and food safety. The objectives of this study were to: (1) describe provincially inspected abattoirs processing cattle in Ontario in terms of the number of abattoirs, the number of weeks abattoirs process cattle, geographical distribution, types of whole carcass condemnations reported, and the distance animals are shipped for slaughter; and (2) identify various seasonal, secular, disease and non-disease factors that might bias the results of quantitative methods, such as cluster detection methods, used for food animal syndromic surveillance.     

Estimation of transport distance of fine particulate organic matter in relation to channel morphology in tailwaters of the Lake Biwa and reservoir dams

Knowledge on transport and deposition of fine particulate organic matter (FPOM) from reservoir dams is increasingly required for habitat management and restoration of dam tailwater ecosystems. Variations in the transport distance of FPOM, however, have never been studied well, particularly in relation to channel morphology, due to channel size restrictions of artificial tracers such as corn pollen when applied to larger river channels. This study aims to show the relations between FPOM retention efficiency and channel morphology in dam tailwaters using lentic plankters as tracers. We estimated the mean transport distance, S _p, by calculating downstream reduction ratios of lentic tracer plankters and calculated the deposition velocity, v _dep. Suspended FPOM samples were collected in tailwaters of two river channels below reservoir dams and two artificial canals below Lake Biwa in the Yodo River system. The longest S _p (19.2 km) and the shortest one (2.2 km) were recorded in the deep canal and shallow canal, respectively, showing a positive correlation with channel hydraulic radius. The values of v _dep were 4.7–6.4 times higher in river channels than in artificial canals. These results indicate that increasing complexity of bed morphology can minimize S _p, whereas bed degradation and armored bed materials may lead to increased S _p. Advantages of lentic plankters as tracers for estimating distance ranges of reservoir dam impact on river ecosystems are also discussed.