Human vascular to cardiac tissue selectivity of L- and T-type calcium channel antagonists

1 Voltage-operated calcium channel (VOCC) antagonists are effective antihypertensive and antianginal agents but they also depress myocardial contractility. 2 We compared four L-type calcium channel antagonists, felodipine, nifedipine, amlodipine and verapamil and a relatively T-type selective calcium channel antagonist, mibefradil, on human and rat isolated tissue assays to determine their functional vascular to cardiac tissue selectivity (V/C) ratio. 3 The V/C ratio was calculated as the ratio of the IC50 value of the antagonist that reduced (by 50%) submaximally contracted (K+ 62 mM) human small arteries from the aortic vasa vasorum (vascular, V) mounted in a myograph and the IC50 value of the antagonist that reduced (-)-isoprenaline (6 nM) submaximally stimulated human right atrial trabeculae muscle (cardiac, C) mounted in organ chambers. 4 The average pIC(50) Values (-log IC50 M) for the human vascular preparations were felodipine 8.30, nifedipine 7.78, amlodipine 6.64, verapamil 6.26 and mibefradil 6.22. The average pIC(50) values for the cardiac muscle were felodipine 7.21, nifedipine 6.95, verapamil 6.91, amlodipine 5.94, and mibefradil 4.61. 5 The V/C ratio calculated as antilog [pIC(50)V-pIC(50)C] is thus mibefradil 41, felodipine 12, nifedipine 7, amlodipine 5 and verapamil 0.2. 6 In rat small mesenteric arteries the pIC(50) values for the five drugs were similar to the values for human vasa vasorum arteries contracted by K+ 62 mM. However for methoxamine (10 mu M) contraction in the rat arteries the pIC(50) values were lower for felodipine 7.24 and nifedipine 6.23, but similar for verapamil 6.13, amlodipine 6.28 and mibefradil 5.91. 7 In conclusion in the human tissue assays, the putative T-channel antagonist mibefradil shows the highest vascular to cardiac selectivity ratio; some 3 fold higher than the dihydropyridine, felodipine, and some 200 fold more vascular selective than the phenylalkylamine, verapamil. This favourable vascular to cardiac selectivity for mibefradil, from a new chemical class of VOCC antagonist, may be explained by its putative T-channel selectivity.

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-H-3]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin I. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin I using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an cc-helical secondary structure. (C) 1998 Elsevier Science B.V, All rights reserved.

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli. We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gets. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE(22-88) fragment forms hetero-oligomers with MinE(+) when the proteins are co-expressed. In contrast, the MinE(36-88) fragment does not form MinE(+)/MinE(36-88) hetero-oligomers, although MinE36-88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE(+) in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of mini-celling by expression of MinE(36-88) in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE(36-88),nd the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn2+. The EPR spectrum of Mn2+-activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5 (+/- 0.1) Angstrom. In the X-ray absorption spectrum of Mn2+-activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k less than or equal to 12 Angstrom(-1) by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15 Angstrom, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5 Angstrom, is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn2+-activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.

Three-dimensional solution structure of alpha-conotoxin MII by NMR spectroscopy: Effects of solution environment on helicity

alpha-Conotoxin MII, a 16-residue polypeptide from the venom of the piscivorous cone snail Conus magus, is a potent and highly specific blocker of mammalian neuronal nicotinic acetylcholine receptors composed of alpha 3 beta 2 subunits. The role of this receptor type in the modulation of neurotransmitter release and its relevance to the problems of addiction and psychosis emphasize the importance of a structural understanding of the mode of interaction of MII with the alpha 3 beta 2 interface. Here we describe the three-dimensional solution structure of MIT determined using 2D H-1 NMR spectroscopy. Structural restraints consisting of 376 interproton distances inferred from NOEs and 12 dihedral restraints derived from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. The final set of 20 structures is exceptionally well-defined with mean pairwise rms differences over the whole molecule of 0.07 Angstrom for the backbone atoms and 0.34 Angstrom for all heavy atoms. MII adopts a compact structure incorporating a central segment of alpha-helix and beta-turns at the N- and C-termini. The molecule is stabilized by two disulfide bonds, which provide cross-links between the N-terminus and both the middle and C-terminus of the structure. The susceptibility of the structure to conformational change was examined using several different solvent conditions. While the global fold of MII remains the same, the structure is stabilized in a more hydrophobic environment provided by the addition of acetonitrile or trifluoroethanol to the aqueous solution. The distribution of amino acid side chains in MII creates distinct hydrophobic and polar patches on its surface that may be important for the specific interaction with the alpha 3 beta 2 neuronal nAChR. A comparison of the structure of MII with other neuronal-specific alpha-conotoxins provides insights into their mode of interaction with these receptors.

Dynamical quantum noise in trapped Bose-Einstein condensates

We introduce the study of dynamical quantum noise in Bose-Einstein condensates through numerical simulation of stochastic partial differential equations obtained using phase-space representations. We derive evolution equations for a single trapped condensate in both the positive-P and Wigner representations and perform simulations to compare the predictions of the two methods. The positive-P approach is found to be highly susceptible to the stability problems that have been observed in other strongly nonlinear, weakly damped systems. Using the Wigner representation, we examine the evolution of several quantities of interest using from a variety of choices of initial stare for the condensate and compare results to those for single-mode models. [S1050-2947(98)06612-8].

Factors affecting biosynthesis by Xanthomonas albilineans of albicidin antibiotics and phytotoxins

Albicidins are important factors in systemic pathogenesis by Xanthomonas albilineans, which causes the devastating leaf scald disease of sugar cane. They ale also of substantial interest as antibiotics that selectively block prokaryote DNA replication. Albicidin biosynthesis is highly sensitive to medium composition. An optimized, chemically defined medium (SMG3) yielded 30-fold more albicidin from half the accumulated biomass, relative to sucrose peptone (SP) medium. Phosphate starvation stimulated albicidin production in SMG3 and SP media. Addition of other amino acids, ammonium ions or peptones to the defined medium increased the growth rate of X albilineans XA3, but differentially inhibited albicidin biosynthesis. Knowledge of these factors indicates new approaches to understanding mechanisms of pathogenesis and resistance to sugar cane leaf scald disease, and to strain improvement for production of albicidin antibiotics.

Tongue piercing: Case report and review of current practice

Although oral piercing has been an uncommon practice in the Western world, the insertion of metal objects into intra-oral and peri-oral pierced sites is growing in popularity. Tongue piercing is one such practice whereby a metal barbell is inserted into the tongue after piercing with a 14-16 gauge needle. Pain, swelling and infection are the most serious consequences associated with this procedure. Other adverse outcomes include mucosal or gingival trauma, chipped or fractured teeth, increased salivary flow, calculus build-up, and interference with speech, mastication and swallowing. This article presents a case report on tongue piercing and highlights the procedure involved. Special attention is given to complications and dental implications associated with such an unusual practice.

Evolution of three-dimensional folds for a non-Newtonian plate in a viscous medium

We derive analytical solutions for the three-dimensional time-dependent buckling of a non-Newtonian viscous plate in a less viscous medium. For the plate we assume a power-law rheology. The principal, axes of the stretching D-ij in the homogeneously deformed ground state are parallel and orthogonal to the bounding surfaces of the plate in the flat state. In the model formulation the action of the less viscous medium is replaced by equivalent reaction forces. The reaction forces are assumed to be parallel to the normal vector of the deformed plate surfaces. As a consequence, the buckling process is driven by the differences between the in-plane stresses and out of plane stress, and not by the in-plane stresses alone as assumed in previous models. The governing differential equation is essentially an orthotropic plate equation for rate dependent material, under biaxial pre-stress, supported by a viscous medium. The differential problem is solved by means of Fourier transformation and largest growth coefficients and corresponding wavenumbers are evaluated. We discuss in detail fold evolutions for isotropic in-plane stretching (D-11 = D-22), uniaxial plane straining (D-22 = 0) and in-plane flattening (D-11 = -2D(22)). Three-dimensional plots illustrate the stages of fold evolution for random initial perturbations or initial embryonic folds with axes non-parallel to the maximum compression axis. For all situations, one dominant set of folds develops normal to D-11, although the dominant wavelength differs from the Biot dominant wavelength except when the plate has a purely Newtonian viscosity. However, in the direction parallel to D-22, there exist infinitely many modes in the vicinity of the dominant wavelength which grow only marginally slower than the one corresponding to the dominant wavelength. This means that, except for very special initial conditions, the appearance of a three-dimensional fold will always be governed by at least two wavelengths. The wavelength in the direction parallel to D-11 is the dominant wavelength, and the wavelength(s) in the direction parallel to D-22 is determined essentially by the statistics of the initial state. A comparable sensitivity to the initial geometry does not exist in the classic two-dimensional folding models. In conformity with tradition we have applied Kirchhoff's hypothesis to constrain the cross-sectional rotations of the plate. We investigate the validity of this hypothesis within the framework of Reissner's plate theory. We also include a discussion of the effects of adding elasticity into the constitutive relations and show that there exist critical ratios of the relaxation times of the plate and the embedding medium for which two dominant wavelengths develop, one at ca. 2.5 of the classical Biot dominant wavelength and the other at ca. 0.45 of this wavelength. We propose that herein lies the origin of parasitic folds well known in natural examples.

Cerebrospinal fluid and plasma concentrations of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in patients before and after initiation of intracerebroventricular morphine for cancer pain management

Twenty-three patients treated with intracerebroventricular (ICV) morphine in this study not only obtained excellent pain relief without rapid increases in dose, but also experienced a reduction in morphine-related side effects. By 24 h after initiation of ICV morphine, the mean trough cerebrospinal fluid (CSF) morphine concentration (approximately 20 mu M) was 50-fold higher than the baseline concentration (approximately 0.4 mu M), and the CSF concentration of morphine-6-glucuronide (M6G) was undetectable (

mRNA differential display of 2-amino-1-methyl-6-phenylinlidazo[4,5-b]pyridine-induced rat mammary gland tumors

The mRNA differential display technique was used to compare mRNAs between normal mammary gland and turner-derived epithelial cells from female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a high-fat diet (23.5% corn oil). Two genes, beta-casein and transferrin, were identified as differentially expressed. The expression of these genes was examined across a bank of rat mammary gland tumors derived from animals on a low-fat diet (5% corn oil) or the high-fat diet. Carcinomas had over a 10- and 50-fold lower expression of beta-casein and transferrin, respectively than normal mammary gland. In addition, carcinomas from animals on the high-fat diet showed on average a 5-fold higher expression of beta-casein, and transferrin than carcinomas from animals on the low-fat diet. The results indicate the process of mammary gland tumorigenesis alters the expression of certain genes in the mammary gland, and that the level of dietary fat further modulates the expression of these genes.

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal. growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its H-1 chemical shifts suggested that its structure was also very similar to native.

Architecture and morphogenesis of grain sorghum, Sorghum bicolor (L.) Moench

Plant architecture has been neglected in most studies of biomass allocation in crops. To help redress this situation for grain sorghum (Sorghum bicolor (L.) Moench), we used a 3D digitiser to measure the dimensions and orientations of vegetative and reproductive structures and derived thermal time-based functions for architectural changes during morphogenesis. Our plants, which were grown in a greenhouse, controlled environment cabinets and the field, covered a large, three-fold, size range when mature. This allowed us to detect some general architectural relationships and to fit morphogenetic functions common across the size range we observed. For example, the relationship between the lengths of successive fully-expanded leaves within a plant was nearly constant for all plants. The lengths of existing leaf blades were accurate predictors of the lengths of up to six subsequently-formed blades in our plants. Similar constant relationships were detected for internode lengths in the panicle and for heights above ground of the collars of successive leaves, even though these traits varied a lot between growth conditions. We suggest that such architectural relationships may be used to link the effect of previous growth conditions to future growth potential, and in that way to predict future partitioning. Our results provide the basis for a preliminary model of sorghum morphogenesis which could eventually become useful in conjunction with crop models by allowing resource acquisition to be related to changes in plant architecture during development. (C) 1999 Elsevier Science B.V. All rights reserved.

Hepatocellular carcinoma

Hepatitis C infection is associated with the development of hepatocellular carcinoma, and progress has been made in a number of areas. Transgenic mice lines expressing the hepatitis C core protein develop hepatic steatosis, adenomas, and hepatocellular carcinomas, with no significant hepatitis or fibrosis. This implies that hepatitis C can lead directly to malignant transformation, A new lesion, irregular regeneration, has been described in chronic hepatitis C infection and is associated with a 15-fold increase in the relative risk of developing hepatocellular carcinoma. A minority of patients with hepatitis C-related hepatocellular carcinoma have intense lymphocytic infiltration of the cancer, a feature associated with a better prognosis, Several studies have confirmed the association between large cell dysplasia and hepatocellular carcinoma, However, large cell dysplasia may not be a premalignant lesion and instead may be a marker for premalignant alterations elsewhere in the liver. Oral contraceptives previously have been linked to an increased risk of hepatocellular carcinoma. A large multicenter European case-control study has shown minimal increased risk of hepatocellular carcinoma with use of steroidal contraception. Tamoxifen had shown promise in the management of advanced hepatocellular carcinoma. However, a randomized placebo-controlled study of 477 patients with hepatocellular carcinoma found no benefit from tamoxifen, In a preliminary study, however, octreotide has shown improved survival and quality of life in patients with advanced hepatocellular carcinoma, Finally, interferon treatment continues to be linked to a reduced risk of hepatocellular carcinoma in patients with hepatitis C, These studies generally are not randomized, and a randomized prospective study is required to address this issue. (C) 1999 Lippincott Williams & Wilkins, Inc.

Glycine metabolism by plant roots and its occurrence in Australian plant communities

Soluble organic nitrogen, including protein and amino acids, was found to be a ubiquitous form of soil N in diverse Australian environments. Fine roots of species representative of these environments were found to be active in the metabolism of glycine. The ability to incorporate [N-15]glycine was widespread among plant species from subantarctic to tropical communities. In species from subantarctic herbfield, subtropical coral cay, subtropical rainforest and wet heathland, [N-15]glycine incorporation ranged from 26 to 45% of (NH4+)-N-15 incorporation and was 2- to 3-fold greater than (NO3-)-N-15 incorporation. Most semiarid mulga and tropical savanna woodland species incorporated [N-15]glycine and (NO3-)-N-15 in similar amounts, 18-26% of (NH4+)-N-15 incorporation. We conclude that the potential to utilise amino acids as N sources is of widespread occurrence in plant communities and is not restricted to those from low temperature regimes or where N mineralisation is limited. Seedlings of Hakea (Proteaceae) were shown to metabolise glycine, with a rapid transfer of N-15 from glycine to serine and other amino compounds. The ability to take up and metabolise glycine was unaffected by the presence of equimolar concentrations of NO3- and NH4+. Isonicotinic acid hydrazide (INH) did not inhibit the transfer of N-15-label from glycine to serine indicating that serine hydroxymethyltransferase was not active in glycine catabolism. In contrast aminooxyacetate (AOA) strongly inhibited transfer of N-15 from glycine to serine and labelling of other amino compounds, suggesting that glycine is metabolised in roots and cluster roots of Hakea via an aminotransferase.