Isolation of genotype- and chromosome-specific DNA markers in a biotype of Colletotrichum gloeosporioides using random amplified polymorphic DNA analysis

Genetic markers that distinguish fungal genotypes are important tools for genetic analysis of heterokaryosis and parasexual recombination in fungi. Random amplified polymorphic DNA (RAPD) markers that distinguish two races of biotype B of Colletotrichum gloeosporioides infecting the legume Stylosanthes guianensis were sought. Eighty-five arbitrary oligonucleotide primers were used to generate 895 RAPD bands but only two bands were found to be specifically amplified from DNA of the race 3 isolate. These two RAPD bands were used as DNA probes and hybridised only to DNA of the race 3 isolate. Both RAPD bands hybridised to a dispensable 1.2 Mb chromosome of the race 3 isolate. No other genotype-specific chromosomes or DNA sequences were identified in either the race 2 or race 3 isolates. The RAPD markers hybridised to a 2 Mb chromosome in all races of the genetically distinct biotype A pathogen which infects other species of Stylosanthes as well as S. guianensis. The experiments indicate that RAPD analysis is a potentially useful tool for obtaining genotype-and chromosome-specific DNA probes in closely related isolates of one biotype of this fungal pathogen.

A study of the stability of subcycling algorithms in structural dynamics

Algorithms for explicit integration of structural dynamics problems with multiple time steps (subcycling) are investigated. Only one such algorithm, due to Smolinski and Sleith has proved to be stable in a classical sense. A simplified version of this algorithm that retains its stability is presented. However, as with the original version, it can be shown to sacrifice accuracy to achieve stability. Another algorithm in use is shown to be only statistically stable, in that a probability of stability can be assigned if appropriate time step limits are observed. This probability improves rapidly with the number of degrees of freedom in a finite element model. The stability problems are shown to be a property of the central difference method itself, which is modified to give the subcycling algorithm. A related problem is shown to arise when a constraint equation in time is introduced into a time-continuous space-time finite element model. (C) 1998 Elsevier Science S.A.

Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: A model for the role of disulfide bonds in structural stability

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by H-1 NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 Angstrom for a family of 35 structures, and comprises primarily a distorted 3(10),, helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13) < GI(2-13;3-7) < GI(2-3;7-13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2-3;7-13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms. A model for the binding of native GI(2-7;3-13) to the mammalian nAChR is proposed, with an alpha-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the alpha and delta subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2-7;3-13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity. (C) 1998 Academic Press Limited.

Thickness and stability of adsorbed film in cylindrical mesopores

The adsorbed film in small cylindrical mesopores is studied by using MCM-41 samples of uniform cylindrical channels as model systems. It is found that at a given relative pressure, the smaller the pore radius, the thicker the adsorbed film is, as postulated by Broekhoff and De Beer. Thermodynamics analysis established that the stability of the adsorbed film is determined by interface curvature and the potential of interaction between adsorbate and adsorbent. A semiempirical equation is proposed to describe the state of stable adsorbed films in cylindrical mesopores. It is also shown to be useful in calculations of pore size distributions of mesoporous solids.

Analysis of small signal stability margins using genetic optimization

Power system small signal stability analysis aims to explore different small signal stability conditions and controls, namely: (1) exploring the power system security domains and boundaries in the space of power system parameters of interest, including load flow feasibility, saddle node and Hopf bifurcation ones; (2) finding the maximum and minimum damping conditions; and (3) determining control actions to provide and increase small signal stability. These problems are presented in this paper as different modifications of a general optimization to a minimum/maximum, depending on the initial guesses of variables and numerical methods used. In the considered problems, all the extreme points are of interest. Additionally, there are difficulties with finding the derivatives of the objective functions with respect to parameters. Numerical computations of derivatives in traditional optimization procedures are time consuming. In this paper, we propose a new black-box genetic optimization technique for comprehensive small signal stability analysis, which can effectively cope with highly nonlinear objective functions with multiple minima and maxima, and derivatives that can not be expressed analytically. The optimization result can then be used to provide such important information such as system optimal control decision making, assessment of the maximum network's transmission capacity, etc. (C) 1998 Elsevier Science S.A. All rights reserved.

The Modified Card Sorting Test: Test-retest stability and relationships with demographic variables in a healthy older adult sample

Objectives. To investigate the test-retest stability of a standardized version of Nelson's (1976) Modified Card Sorting Test (MCST) and its relationships with demographic variables in a sample of healthy older adults. Design. A standard card order and administration were devised for the MCST and administered to participants at an initial assessment, and again at a second session conducted a minimum of six months later in order to examine its test-retest stability. Participants were also administered the WAIS-R at initial assessment in order to provide a measure of psychometric intelligence. Methods. Thirty-six (24 female, 12 male) healthy older adults aged 52 to 77 years with mean education 12.42 years (SD = 3.53) completed the MCST on two occasions approximately 7.5 months (SD = 1.61) apart. Stability coefficients and test-retest differences were calculated for the range of scores. The effect of gender on MCST performance was examined. Correlations between MCST scores and age, education and WAIS-R IQs were also determined. Results. Stability coefficients ranged from .26 for the percent perseverative errors measure to .49 for the failure to maintain set measure. Several measures were significantly correlated with age, education and WAIS-R IQs, although no effect of gender on MCST performance was found. Conclusions. None of the stability coefficients reached the level required for clinical decision making. The results indicate that participants' age, education, and intelligence need to be considered when interpreting MCST performance. Normative studies of MCST performance as well as further studies with patients with executive dysfunction are needed.

Familial partial epilepsy with variable foci: A new partial epilepsy syndrome with suggestion of linkage to chromosome 2

Familial partial epilepsy with variable foci (FPEVF) joins the recently recognized group of inherited partial epilepsies. We describe an Australian family with 10 individuals with partial seizures over four generations. Detailed electroclinical studies were performed on all affected and 17 clinically unaffected family members. The striking finding was that the clinical features of the seizures and interictal electroencephalographic foci differed among family members and included frontal, temporal, occipital, and centroparietal seizures. Mean age of seizure onset was 13 years (range, 0.75-43 years). Two individuals without seizures had epileptiform abnormalities on electroencephalographic studies. Penetrance of seizures was 62%. A genome-wide search failed to demonstrate definitive linkage, but a suggestion of linkage was found on chromosome 2q with a LOD score of 2.74 at recombination fraction of zero with the marker D2S133. FPEVF differs from the other inherited partial epilepsies where partial seizures in different family members are clinically similar. The inherited nature of this new syndrome may be overlooked because of relatively low penetrance and because of the variability in age at onset and electroclinical features between affected family members.

Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.

A gene (Cargl) that regulates tissue resistance to Candida albicans maps to chromosome 14 of the mouse

Tissue susceptibility and resistance to infection with the yeast Candida albicans is genetically regulated. Analysis of the strain distribution pattern of the C. albicans resistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant for Carg1, Tcra and Rib1. Therefore, Carg1 is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes. (C) 1998 Academic Press.

Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest

DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Tev sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. Ln support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the Rm-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork. (C) 1999 Academic Press.

Complexes of 1-thia-4,7,10-triazacyclododecane ([12]aneN(3)S) - Synthesis, structure and stability constants

The 12-membered macrocyclic ligand 1-thia-4,7, 10-triazacyclododecane ([12]aneN(3)S) has been synthesised, although upon crystallization from acetonitrile a product in which carbon dioxide had added to one secondary amine in the macrocyclic ring (H[12]aneN(3)SCO(2). H2O) was isolated and subsequently characterised by X-ray crystallography. The protonation constants for [12]aneN(3)S and stability constants with Zn(II), Pb(II), Cd(II) and Cu(II) have been determined either potentiometrically or spectrophotometrically in aqueous solution, and compared with those measured or reported for the ligands 1-oxa-4,7,10-triazacyclododecane ([12]aneN(3)O) and 1,4,7,10-tetraazacyclododecane ([12]aneN(4)). The magnitudes of the stability constants are consistent with trends observed previously for macrocyclic ligands as secondary amine donors are replaced with oxygen and thioether donors although the stability constant for the [Hg([12]aneN(4))](2+) complex has been estimated from an NMR experiment to be at least three orders of magnitude larger than reported previously. Zinc(II), mercury(II), lead(II), copper(II) and nickel(II) complexes of [12]aneN(3)S have been isolated and characterised by X-ray crystallography. In the case of copper(II), two complexes [Cu([12]aneN(3)S)(H2O)](ClO4)(2) and [Cu-2([12]aneN(3)S)(2)(OH)(2)](ClO4)(2) were isolated, depending on the conditions employed. Molecular mechanics calculations have been employed to investigate the relative metal ion size preferences of the [3333], asym-[2424] and sym-[2424] conformation isomers. The calculations predict that the asym-[2424] conformer is most stable for M-N bond lengths in the range 2.00-2.25 Angstrom whilst for the larger metal ions the [3333] conformer is dominant. The disorder seen in the structure of the [Zn([12]aneN(3)S)(NO3)](+) complex is also explained by the calculations. (C) 1999 Elsevier Science Ltd. All rights reserved.

Is there a role for transversus abdominis in lumbo-pelvic stability?

There has been considerable interest in the literature regarding the function of transversus abdominis, the deepest of the abdominal muscles, and the clinical approach to training this muscle. With the development of techniques for the investigation of this muscle involving the insertion of fine-wire electromyographic electrodes under the guidance of ultrasound imaging it has been possible to test the hypotheses related to its normal function and function in people with low back pain. The purpose of this review is to provide an appraisal of the current evidence for the role of transversus abdominis in spinal stability, to develop a model of how the contribution of this muscle differs from the other abdominal muscles and to interpret these findings in terms of the consequences of changes in this function.

Extraordinary and extensive karyotypic variation: A 48-fold range in chromosome number in the gall-inducing scale insect Apiomorpha (Hemiptera : Coccoidea : Eriococcidae)

Chromosome number reflects strong constraints on karyotype evolution, unescaped by the majority of animal taxa. Although there is commonly chromosomal polymorphism among closely related taxa, very large differences in chromosome number are rare. This study reports one of the most extensive chromosomal ranges yet reported for an animal genus. Apiomorpha Rubsaamen (Hemiptera: Coccoidea: Eriococcidae), an endemic Australian gall-inducing scale insect genus, exhibits an extraordinary 48-fold variation in chromosome number with diploid numbers ranging from 4 to about 192. Diploid complements of all other eriococcids examined to date range only from 6 to 28. Closely related species of Apiomorpha usually have very different karyotypes, to the extent that the variation within some species- groups is as great as that across the entire genus. There is extensive chromosomal variation among populations within 17 of the morphologically defined species of Apiomorpha indicating the existence of cryptic species-complexes. The extent and pattern of karyotypic variation suggests rapid chromosomal evolution via fissions and (or) fusions. It is hypothesized that chromosomal rearrangements in Apiomorpha species may be associated with these insects' tracking the radiation of their speciose host genus, Eucalyptus.