Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Oscillations in calcium-cyclic AMP control loops form the basis of pacemaker activity and other high frequency biological rhythms.

In this paper theoretical and experimental evidence is presented which indicates that oscillations in internal calcium and cyclic AMP concentrations due to an instability in their common control loops are possible and indeed may be widespread. Further, it is demonstrated that fluctuations in various cellular properties, in particular membrane potential, are a direct consequence of these second messenger oscillations. Given the central importance of calcium and cyclic AMP to the regulation of metabolism, these oscillations would influence most metabolic processes especially rhythmic behaviour. We propose that these oscillations form the basis of several biological rhythms including, potential oscillations in cardiac pacemaker cells, neurones and insulin secreting β-cells, the minute rhythm in smooth muscle, cyclic AMP pulses in Dictyostelium, rhythmical cytoplasmic streaming in Physarum and transepitheliel potential oscillations in Calliphora salivary gland. This model makes possible an explanation of the frequency and amplitude effects of hormones.     

Phosphatidylinositol-hydrolysing enzymes in blowfly salivary glands.

The salivary glands of adult blowflies (Calliphora erythrocephala) contain enzymes that hydrolyse phosphatidylinositol, predominantly by a Ca2+-independent deacylation, though a Ca2+-dependent phosphodiesterase (phospholipase C) activity could be detected. The deacylating enzymes could also hydrolyse phosphatidylcholine and phosphatidylethanolamine, and were secreted in the saliva. Homogenization of salivary glands prelabelled with [3H]inositol resulted in a rapid deacylation of the endogenous 3H-labelled phosphatidylinositol; this hydrolysis was unaffected by addition of 5-hydroxytryptamine to the homogenate.     

Method development in liquid chromatography with a charged cyclodextrin additive for chiral resolution of rac-amlodipine utilising a central composite design

A negatively charged derivative of β-cyclodextrin, sulphobutyl ether-β-cyclodextrin (SBE-β-CD), was examined as a chiral mobile phase additive in reversed-phase high-performance liquid chromatography for the enantiomeric resolution of the calcium channel blocker rac-amlodipine. Theoretical and practical aspects are discussed for setting up a central composite design applicable to any analytical method. These include the correct location of factor points for maintaining orthogonality within the design and the augmentation of centrepoint experiments to allow a larger factor space by increasing the distance of axial star points. Optimised separation was achieved using a reverse-phase column with eluent comprising: acetonitrile (ACN)—potassium dihydrogen phosphate (pH 3.93) containing 2.66 mM SBE-bgr;-CD (26.5:73.5% v/v) at a flow rate of 1.0 ml/min. This yielded a Kaiser peak separation index, P_i = 0.96, at t_R2 = 52 min with satisfactory reproducibility, relative standard deviation values: t_R1, 0.39%; t_R2, 0.47% (n = 5). These experimental results were in excellent agreement with those predicted by the SAS software package for a chromatographic response function model. Multiple regression analysis in four dimensions, with three response models based on R_s, P_i, and a function of P_i, produced response surfaces which revealed zones of optimum robustness and illustrated the interactions involved between the key chromatographic factors. Putative proposals for a mechanism involving the interaction of each of the positively charged enantiomers with the negatively charged cyclodextrin are also discussed. These examine the possibility of ion-pairing and inclusion phenomena to account for the excellent resolution observed. © 1996 Wiley-Liss, Inc.     

Giemsa-based cytological assessment of area,shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 μm, the shortest dimension averaged 14.4 ± 1.8 μm and the nucleus averaged 6.3 ± 0.8 μm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 μm² (average 193 ± 50 μm²). The area could be predicted reliably from the longest and shortest dimensions (r² = 0.903). The areas of goblet cell nuclei were 15–58 μm² (average 33 ± μm²) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.     

Multiple proteins with single activities or a single protein with multiple activities: the conundrum of cell surface NADH oxidoreductases

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.     

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.     

Induction of apoptosis by the marine sponge (Mycale) metabolites, mycalamide A and pateamine

The marine sponge metabolites mycalamide A (myca-lamide) and pateamine are extremely cytotoxic. While mycalamide has been shown to inhibit protein synthesis, the mechanism by which these compounds induce cell death is unknown. Using DNA laddering, Annexin-V staining, and morphological analysis, we demonstrate that both metabolites induce apoptosis in several different cell lines. Furthermore, both mycalamide and pateamine were more potent inducers of apoptosis in the 32D myeloid cell line after transformation with either the ras or bcr-abl oncogenes. This increased sensitivity was also observed in response to the protein synthesis inhibitors cycloheximide and puromycin, and cytosine--D-arabinofurano-side (Ara-C), an inducer of DNA damage. We propose, therefore, that in 32D cells where Ras signalling has been altered either by constitutive expression of oncogenic ras or by Bcr/abl-mediated perturbation of upstream signalling events, increased susceptibility to apoptosis by a range of stimuli is conferred.     

Fine structural organization of the rectum in the blowfly, Calliphora erythrocephala (Meig.) with special reference to connective tissue, tracheae and neurosecretory innervation in the rectal papillae

The rectal epithelium of Calliphora is made up of three quite distinct cell types: rectal, cortical and junctional cells. The thin wall of the rectal pouch is made up of rectal cells which are relatively simple and unspecialized; their general structure does not suggest any direct participation in ion transport. A function of ion and water transport can probably be ascribed to the cortical cells, which are arranged in the form of four cones which project into the rectal lumen. The cavity of each cone is filled up with tracheae, nerve and neurosecretory terminals, and connective tissue to form medulla. The medulla and cortex are separated from each other by deeply staining bridges or trabeculae to form an infundibular space. The most conspicuous feature of the cortex is the presence of an extensive intercellular sinus formed by complex infoldings of the lateral plasma-membranes. It is postulated that fluid, which is absorbed from the rectal lumen, is transported into the intercellular sinus and finally reaches the haemolymph via the infundibular space. The actual site of ion transport is probably the stacks of lateral plasma-membrane which are closely associated with mitochondria. The junctional cells, which are packed with microtubules, form a collar around the base of the papillae at the point of their insertion into the rectal wall. It is suggested that the neurosecretory terminals present in the medulla might release a hormone which controls rate of ion and water reabsorption by the papillae cells.