Almost pure I(alpha) cellulose in the cell wall of Glaucocystis.

Crystalline features of cellulose microfibrils in the cell walls of Glaucocystis (Glaucophyta) were studied by combined spectroscopy and diffraction techniques, and the results were compared with those of Oocystis (Chlorophyta). Although these algae are grouped into two different classes, by the composition of their chloroplasts for instance, their cell walls are quite similar in size and morphology. The most striking features of their cellulose crystallites are that they have the highest cellulose I(alpha) contents reported to date. In particular, the I(alpha) fraction of cellulose from Glaucocystis was found to be as high as 90% from (13)C NMR analysis. The mode of preferential orientation of cellulose crystallites in their cell walls is also interesting; equatorial 0.53-nm lattice planes were oriented parallel to the cell surface in the case of Glaucocystis, while the 0.62-nm planes were parallel to the Oocystis cell surface. Such a structural variation provides another link to the evolution of cellulose structure, biosynthesis, and its biocrystallization mechanism.     

In-situ Raman microprobe studies of plant cell walls: Macromolecular organization and compositional variability in the secondary wall of Picea mariana (Mill.) B.S.P.

Native-state organization and distribution of cell-wall components in the secondary wall of woody tissue from P. mariana (Black Spruce) have been investigated using polarized Raman microspectroscopy. Evidence for orientation is detected through Raman intensity variations resulting from rotations of the exciting electric vector with respect to cell-wall geometry. Spectral features associated with cellulose and lignin were studied. The changes in cellulose bands indicate that the pyranose rings of the anhydroglucose repeat units are in planes perpendicular to the cross section, while methine C–H bonds are in planes parallel to the cross section. Changes in bands associated with lignin indicate that the aromatic rings of the phenyl-propane units are most often in the plane of the cell-wall surface. However, regions where lignin orientation departs from this pattern also occur. These results represent direct evidence of molecular organization with respect to cellular morphological features in woody tissue, and indicate that cell-wall components are more highly organized than had been recognized. Studies carried out in order to establish the usefulness and sensitivity of the Raman technique to differences of composition within the cell walls provide evidence of variations in the distribution of cellulose and lignin. Such compositional differences were more prominent between the walls of different cells than within a particular cell wall.     

Atrial proarrhythmia due to increased inward rectifier current (IK1) arising from KCNJ2 mutation – A simulation study

Atrial fibrillation (AF) has been linked to increased inward rectifier potassium current, I_K1, either due to AF-induced electrical remodelling, or from functional changes due to the Kir2.1 V93I mutation. The aim of this simulation study was to identify at cell and tissue levels' mechanisms by which increased I_K1 facilitates and perpetuates AF. The Courtemanche et al. human atrial cell action potential (AP) model was modified to incorporate reported changes in I_K1 induced by the Kir2.1 V93I mutation in both heterozygous (Het) and homozygous (Hom) mutant forms. The modified models for wild type (WT), Het and Hom conditions were incorporated into homogeneous 1D, 2D and 3D tissue models. Restitution curves of AP duration (APD), effective refractory period (ERP) and conduction velocity (CV) were computed and both the temporal and the spatial vulnerability of atrial tissue to re-entry were measured. The lifespan and tip meandering pattern of re-entry were also characterised. For comparison, parallel simulations were performed by incorporating into the Courtmanche et al. model a linear increase in maximal I_K1 conductance. It was found that the gain-in-function of V93I ‘mutant’ I_K1 led to abbreviated atrial APs and flattened APD, ERP and CV restitution curves. It also hyperpolarised atrial resting membrane potential and slowed down intra-atrial conduction. V93I ‘mutant’ I_K1 reduced the tissue's temporal vulnerability but increased spatial vulnerability to initiate and sustain re-entry, resulting in an increased overall susceptibility of atrial tissue to arrhythmogenesis. In the 2D model, spiral waves self-terminated for WT (lifespan < 3.3 s) tissue, but persisted in Het and Hom tissues for the whole simulation period (lifespan > 10 s). The tip of the spiral wave meandered more in WT tissue than in Het and Hom tissues. Increased I_K1 due to augmented maximal conductance produced similar results to those of Het and Hom Kir2.1 V93I mutant conditions. In the 3D model the dynamic behaviour of scroll waves was stabilized by increased I_K1. In conclusion, increased I_K1 current, either by the Kir2.1 V93I mutation or by augmented maximal conductance, increases atrial susceptibility to arrhythmia by increasing the lifespan of re-entrant spiral waves and the stability of scroll waves in 3D tissue, thereby facilitating initiation and maintenance of re-entrant circuits.     

Zur Cytologie und taxonomischen Einordnung von Glaucocystis

Zusammenfassung Glaucocystis ist eine einzellige Alge mit endosymbiontischen Cyanophyceen, die gewöhnlich für eine apoplastidische Oocystis gehalten wird. Gegen diese taxonomische Einordnung sprechen verschiedene Befunde unserer licht- und elektronenmikroskopischen Untersuchungen, unter anderem der Besitz zweier rudimentärer Geißeln, pulsierender Vacuolen (während der Zellteilung) und die Zellsymmetrie. Wie aus einer tabellarischen Übersicht über die Anordnung des Golgi-Apparates in den verschiedenen Algengruppen hervorgeht, ist es sehr unwahrscheinlich, daß Glaucocystis überhaupt eine Chlorophycee ist, denn ihre Dictyosomen liegen im Gegensatz zu denen der Grünalgen ringförmig um die Geißelbasis, sind also parabasal angeordnet. Weil weitere spezifische Merkmale sowie eigene Pigmente fehlen, scheint uns eine taxonomische Einordnung vorerst nicht möglich zu sein.

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Summary Glaucocystis is an unicellular alga with endosymbiontic blue-green algae and is usually thought to be an apoplastidic Oocystis. However, our light and electron microscopical investigations show, that there is no relationship to Oocystis, since Glaucocystis has two reduced flagella, contractile vacuoles (during the cell division), and a different symmetry of the cell. A survey on the position of the Golgi apparatus in the different groups of algae demonstrates that Glaucocystis is most probably no chlorophycean since its dictyosomes surround the flagellar base and are therefore in contrast to that of green algae in parabasal position. Due to the lack of other specific characteristics as well as own pigments it seems us very difficult to place at present Glaucocystis in the taxonomic system of the algae.

     

Properties of a novel β-glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2

A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60 °C, and the deactivation energy (E_d) for FPG was 88.6 kJ mo1⁻¹. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg₃. The enzyme hydrolyzed the 3-C, β-(1 → 2)-glucoside of ginsenoside Rg₃ to produce ginsenoside Rh₂, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh₂. The K_m and V_max values of FPG for ginsenoside Rg₃ were 2.37 mM and 0.568 μmol (h mg protein)⁻¹, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides.     

Struktur der Zellwände und Cellulosefibrillen Bei Glaucocystis

Zusammenfassung Die Zellwände und Cellulosefibrillen von Glaucocystis wurden elektronenmikroskopisch an Dünnschnitten und mit dem Negativ-Kontrast-Verfahren untersucht. Die Zellwände sind aus mehreren Lamellen zusammengesetzt. Die Lamellen bestehen aus einer Doppellage teilweise miteinander verwobener Mikrofibrillen in Paralleltextur; die beiden Lagen überkreuzen sich ungefähr rechtwinklig. Gegenüber der nächsten Lamelle ist die Streichrichtung meistens um etwa 45° gedreht.In jungen Wänden sind die Celluloseelemente in eine dichte Matrix eingebettet und so markiert. In alten Autosporenmutterzell-Hüllen ist die Struktur aufgelockert; die Cellulose ist teilweise freigelegt und unmittelbar darstellbar. Die Mikrofibrillen sind abgeplattet und nur selten breiter als 20 nm. Ihre Länge wurde indirekt ermittelt; sie beträgt durchschnittlich etwa 10 .Die Mikrofibrillen bestehen aus zwei (oder mehr) mit ihren Schmalseiten nebeneinander liegenden Elementarfibrillen; sie scheinen aber dennoch ein relativ homogenes Ganzes zu bilden. Ihre elastische Dehnbarkeit beträgt etwa 2,3%. Die Elementarfibrillen haben lang auslaufende, spitze Enden (Minimalbreite etwa 3 nm, in der Mitte des Fadens etwa 10 nm).Die Ergebnisse werden mit anderen Angaben über den Bau der Mikro- und Elementarfibrillen und mit Befunden über die Struktur der Zellwände von Chlorococcalen verglichen.

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Structure of cell walls and cellulose fibrils in Glaucocystis

Summary Glaucocystis is an apoplastidic alga related to Oocystis (Chlorococcales) containing endosymbiontic blue-green algae. The cell walls and cellulose fibrils of two species were studied with the electron microscope in thin sections and by means of the negative staining technique. The walls are composed of several lamellae. Each lamella consists of two layers of cellulose microfibrils. In young walls the fibrils are masked by a dense matrix. In older envelopes of autospore mother cells the matrix is partly disintegrated; the fibrils are loosened and can therefore be seen directly. The microfibrils in one layer are oriented in a parallel pattern. They are partly interwoven with the microfibrils of the other layer and cross them at an angle of about 90°. In successive lamellae the direction of the micelles mostly changes by about 45°. The microfibrils are flat bands which are seldom broader than 20 nm, and their average length is calculated to be about 10 . Although they are composed of two or more elementary fibrils, they nevertheless seem to be relatively homogenous structures. Their elastic extensibility is about 2,3%. The elementary fibrils are tapered; the ends are about 3 nm broad, while the middle part measures up to 10 nm.The results are discussed in relation to other observations on cellulose fibrils, and on the cell wall structure of other Chlorococcales.

     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I _Na) and calcium currents (I _Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I _Ca density was unchanged whereas I _Na density after stepping from –80 to –30 mV was decreased by 30% (–9.0 ± 1.16 pA pF^–1 in control and –6.31 ± 0.67 pA pF^–1 in hypertrophy, p < 0.05, n= 6). Steady-state activation/inactivation variables of I _Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I _Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca²⁺ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I _Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na⁺ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I _Na density so that net Na⁺ influx remains unaltered. Changes in the fast I _Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle. (Mol Cell Biochem 261: 217–226, 2004)     

The role of integrin alpha D beta2 (CD11d/CD18) in monocyte/macrophage migration

Integrin α_Dβ₂ (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin α_Mβ₂ (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of α_Dβ₂ in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of α_Dβ₂ and examined their migration to various extracellular matrix proteins. When expressed at a low density, α_Dβ₂ on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with β₁/β₃ integrins to support cell migration. However, its increased expression on the α_Dβ₂-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of α_Dβ₂ with anti-α_D blocking antibodies restores β₁/β₃-driven cell migration by removing the excess α_Dβ₂-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-α_D antibody. These results demonstrate that the density of α_Dβ₂ is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of α_Dβ₂ contribute to monocyte migration while α_Dβ₂ upregulation on differentiated macrophages may facilitate their retention at sites of inflammation.     

On the endothelial cell ISOC

Ca²⁺ store depletion activates both Ca²⁺ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca²⁺ selective current, I_SOC. Indeed, I_SOC is a relatively small inward Ca²⁺ current that exhibits an approximate +40 mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I_SOC, although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I_SOC requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca²⁺ store depletion and channel activation. Thus, at present findings indicate a TRPC4–protein 4.1 physical linkage regulates I_SOC activation following Ca²⁺ store depletion.     

Ultrastructure of leaves in C4 Cyperus iria and C3 Carex siderosticta

The ultrastructural aspects ofCyperus iria leaves showing the C₄ syndrome and the typical C₃ species,Carex siderosticta, in the Cyperaceae family were examined.C. iria exhibited the chlorocyperoid type, showing an unusual Kranz structure with vascular bundles completely surrounded by two bundle sheaths. The cellular components of the inner Kranz bundle sheath cells were similar to those found in the NADP-ME C₄ subtype, having centrifugally arranged chloroplasts with greatly reduced grana and numerous starch grains. Their chloroplasts contained convoluted thyla-koids and a weakly-developed peripheral reticulum, although it was extensive mostly in mesophyll cell chloroplasts. The outer mestome bundle sheath layer was sclerenchymatous and generally devoid of organelles, but had unevenly thickened walls. Suberized lamellae were present on its cell walls, and they became polylamellate when traversed by plasmodesmata. Mesophyll cell chloroplasts showed well-stacked grana with small starch grains. InC. siderosticta, vascular bundles were surrounded by the inner mestome sheath and the outer parenchymatous bundle sheath with intercellular spaces. The mestome sheath cells degraded in their early development and remained in a collapsed state, although the suberized lamellae retained polylamellate features. Plastids with a crystalline structure, sometimes membrane-bounded, were found in the epidermal cells. The close interveinal distance was 35–50 μm inC. iria, whereas it was 157–218 μm inC. siderosticta. These ultrastructural characteristics were discussed in relation to their photosynthetic functions.     

Simulation analysis of intracellular Na and Cl homeostasis during β1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

The Influences of I h on Temporal Summation in Hippocampal CA1 Pyramidal Neurons: A Modeling Study

Recent experimental and theoretical studies have found that active dendritic ionic currents can compensate for the effects of electrotonic attenuation. In particular, temporal summation, the percentage increase in peak somatic voltage responses invoked by a synaptic input train, is independent of location of the synaptic input in hippocampal CA1 pyramidal neurons under normal conditions. This independence, known as normalization of temporal summation, is destroyed when the hyperpolarization-activated current, I _h, is blocked [Magee JC (1999a), Nature Neurosci. 2: 508–514]. Using a compartmental model derived from morphological recordings of hippocampal CA1 pyramidal neurons, we examined the hypothesis that I _h was primarily responsible for normalization of temporal summation. We concluded that this hypothesis was incomplete. With a model that included I _h, the persistent Na⁺ current (I _NaP), and the transient A-type K⁺ current (I _A), however, we observed normalization of temporal summation across a wide range of synaptic input frequencies, in keeping with experimental observations.     

Formation and isolation of nanocrystal complexes between dextrins and n-butanol

Starch dextrins of different molecular sizes (DP_n 311, 142 and 39) were prepared by hydrolyzing a high amylose maize starch in acidic alcohol solutions. The dextrins were dissolved in an aqueous dimethyl sulfoxide solution (90% DMSO), and then the solution was allowed to migrate down into n-butanol separated by a membrane filter. The complex was gradually formed between the dextrin and butanol, and precipitated in the butanol layer. The dextrin–butanol complex yielded V6-I type crystals with broad reflections (d-spacings 1.123, 0.657 and 0.429 nm) under X-ray diffractograms. Platelets of average length less than 100 nm, interspersed in amorphous matrices, were observed in complexes of DP_n 311 and 142, but that of DP_n 39 showed different morphology, and the formation of complexes was limited. By hydrolyzing the complex of DP_n 311 with α-amylase, amorphous matrices were selectively removed, and crystallites of 23–72 nm showing a V6-I X-ray diffraction pattern were obtained. However, crystallites in complexes of DP_n 142 and 39 were eroded by amylolysis, forming large aggregates.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

The vicissitudes of the pacemaker current <Emphasis Type="Italic">I</Emphasis><Subscript>Kdd</Subscript> of cardiac purkinje fibers

Summary The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I _Kdd. An alternative proposal is that the hyperpolarization-activated current I _f underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K⁺ depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I _K1. In turn, this decay would be responsible for a “fake” reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba²⁺ was used to block the decay of I _K1. In the presence of Ba²⁺ the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as −120 mV. In this regard, the distinct possibility needs to be considered that Ba²⁺ had blocked I _Kdd (and not only I _K1). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba²⁺. In the absence of Ba²⁺, I _Kdd was present in the pacemaker potential range and reversed at E _K. In the presence of Ba²⁺, I _Kdd was blocked and I _f appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I _Kdd but not with I _f. The fact that I _f is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I _Kdd (but not of I _f) in Purkinje fibers.     

The sequence and phylogenesis of the α-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion)

In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked ^Iα and ^IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the ^Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.     

Food quality effects on life history traits and fitness in the generalist herbivore Daphnia

Summary Life table experiments were conducted on the generalist suspension feeder Daphnia galeata, using as food the two green algae (Chlorophyta) Scenedesmus acutus and Oocystis lacustris. Oocystis was hypothesized to be a lower quality food because it is convered with a thick sheath, believed to reduce digestibility. Results showed that Oocystis is a lower quality food for Daphnia, but only at relatively low food concentrations (0.15 mg C/L) and not at higher concentrations (1.0 mg C/L). At 0.15 mg C/L, Daphnia intrinsic rate of increase (r) when grown on Oocystis was only half that when grown on Scenedesmus. Daphnia r was similar at 0.15 mg C/L Oocystis and 0.075 mg C/L Scenedesmus, indicating that Daphnia requires twice as much Oocystis as Scenedesmus to achieve the same fitness. Intrinsic rate of increase was lower on Oocystis mainly because age at first reproduction was greatly delayed compared to that on Scenedesmus (13.6 vs 7.3 d). In addition, juvenile growth and survivorship were reduced on Oocystis compared with Scenedesmus. Clutch sizes were similar on the two foods, indicating that once individuals reached adulthood, the two foods were similar in quality. In contrast, at high food concentrations (1.0 mg C/L), the two algae were similar in quality for both juveniles and adults, and r was not significantly different on the two foods. Ingestion and assimilation rate experiments whowed that Daphnia consumes the two algae at identical rates, and that adults assimilate the two algae at similar rates. However, juveniles assimilate Oocystis at much lower rates than Scenedesmus, possibly accounting for reduced juvenile growth and delay in age at maturity at low concentrations. Thus, Daphnia exhibits an ontogenetic shift in its ability to utilize Oocystis, and this can result in juvenile bottlenecks in which survival and growth of young age classes are of critical importance in determining population dynamics. Because food quality effects were manifested primarily in juveniles and at low concentrations, food quality effects in nature will depend on phytoplankton abundance and age-structure of Daphnia populations.     

Different photoreceptors within the same retina express unique combinations of potassium channels

Single electrode current and voltage clamp recordings in Calliphora, and whole-cell voltage clamp recordings in Drosophila were used to characterise the voltage-gated K channels in both major classes of photoreceptors, R7/8 (long visual fibres, LVFs) and R1-6 (short visual fibres, SVFs). R7/8 were identified by their unique spectral properties, ca. 3–4 fold higher input resistances and 3–4 fold lower cell capacitance. In Calliphora SVFs possess both fast and slow activating delayed rectifier potassium conductances. Drosophila SVFs possess a slowly inactivating delayed rectifier (I_Ks), a very rapidly inactivating A channel encoded by the Shaker gene (I_A), and, in a minority of cells, a third K conductance with intermediate kinetics (I_Kf). In both specs the LVFs lack the slowest component, but exhibit the faster K conductance(s) with properties indistinguishable from those in the SVFs. These findings add to established evidence demonstrating the significant role played by potassium channels in tuning the photoreceptor membrane. The results also suggest that R1-6 photoreceptors and R7/8 form inputs to visual subsystems tuned to different temporal frequencies.Abbreviations LVF long visual fibre - SVF short visual fibre - R1-6 retinular cells 1 to 6 inclusive - R7/8 retinular cell 7 and 8 - I _A rapidly inactivating A type potassium conductance; channel coded by Shaker gene - I _Kf rapidly activating, slowly inactivating delayed rectifier-like potassium conductance - I _Ks slowly activating, slowly inactivating delayed rectifier-like potassium conductance - I _KDs slowly activating delayed rectifier potassium conductance - I _KDf rapidly activating delayed rectifier potassium conductance     

The Cytochrome b5 Tail Anchors and Stabilizes Subdomains of Human DNA Topoisomerase IIα in the Cytoplasm of Retrovirally Infected Mammalian Cells

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIα are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIα and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b₅ anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1–266) is weakly and diffusely expressed, addition of the cytb₅ anchor (1–266-b₅) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1–426). A C-terminal domain (aa 1030–1504) of human topo IIα containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb₅ anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.