Cytokinesis in the green alga Eremosphaera viridis: Plasmalemma formation from “open membranes”

Summary Cytokinesis in the unicellular chlorococcalean alga Eremosphaera viridis de Bary has been investigated by electron microscopy of thin sections. The new plasmalemmata of the daughter cells in this organism form centrifugally within a phycoplast. Unlike other cell division systems each new plasmalemma is formed, not by the fusion of vesicles, but rather by the fusion of open membranes which are characteristically heavily stained. Measurements of these open membranes reveal that they are 11 nm thick with a central 4,5 nm unstained portion. The possible origin of these open membranes as burst-open vesicles has been suggested from the presence of intensely straining vesicles in the vicinity of the cell equator. Calculations of vesicle and open membrane surface areas support this contention.     

Dissecting subdomains involved in multiple functions of the CK2β subunit

We have characterized several subdomains of the subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators. Mutational analysis disclosed that glutamic residues lying in the polyacidic region of the CK2 subunit are involved in the interaction with polyamine molecules and allowed the delineation of an autonomous binding domain. Furthermore, this regulatory domain was shown to mediate the association of CK2 with plasma membrane.The C-terminal domain of the CK2 subunit plays a role in the oligomerization of the kinase since it was observed that a truncated form of this subunit lacking its 33-last amino acids was incompetent for the assembly of polymeric forms of CK2. Altogether, our results support the notion that the subunit of CK2 is a modular protein made by the association of interdependent domains that are involved in its multiple functions.     

Effects of polymyxin B on the sarcoplasmic reticulum membrane

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca²⁺-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 M, 130M and 550 M of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca²⁺-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 M polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.     

Two new species of Isospora (Apicomplexa: Eimeriidae) from passeriform birds of South America

Summary Two new species of Isospora (Apicomplexa: Eimeriidae) are described from the faeces of passeriform birds of South America. I. cyanocoracis n. sp. is described from Cyanocorax chrysops (Passeriformes: Corvidae) and I. paroariae n. sp. from Paroaria coronata (Passeriformes: Emberizidae). I. cyanocoracis oocysts are spherical or subspherical, 28.7×26.8 m (25.0–30.5×24.5–29.0), with bi-layered wall about 2.0 m thick. Micropyle and oocyst residuum are absent; large polar granule present. Sporocysts are ovoid, 19.3×11.4 m (17.0–21.0×10.5–12.2), with smooth, single-layered wall about 0.8 m thick. Stieda and substiedal bodies and sporocyst residuum are present. Sporozoites 12.2×4.2 m (10.5–15.0×3.5–4.5), possess spherical anterior and posterior refractile bodies. I. paroariae oocysts are spherical or subspherical, 22.3×21.4 m (19.5–25.5×18.5–24.0), and have bi-layered wall about 1.8 m thick. Micropyle, polar granule, and oocyst residuum are absent. Sporocysts ovoid, 15.2×10.0 m (14.0–16.5×8.0–11.5), possess smooth, single-layered wall about 0.7 m thick. Stieda and substiedal bodies and sporocyst residuum are present. Sporozoites elongate, 11.3×3.4 m (10.0–13.5×3.2–4.0), have single, large, posterior refractile body. ac]19840712     

Sucrose in storage media and cultivar affects post-storage regrowth of <Emphasis Type="Italic">in vitro</Emphasis> Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama Newberry Gold (NBG) and Hosta Striptease were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4weeks. At the end of stage III, cultures were stored, with the remaining media, at 10°C with 5molm^–2s^–1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14week storage. Sucrose loading by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.     

On the retinal basis of visual adaptation

Summary Visual Adaptation poses numerous problems: in some situations bleached pigment and background lights exert equivalent effects on the visual threshold, while in other situations they do not. Adaptation in man spans a range of more that 10 log units, yet receptor polarization—if this be considered as a measure of the response—saturates over 3 or 4 log units in various animals, including primates. To resolve problems such as these, a model is proposed for the responses and interactions of retinal cells, especially receptor, horizontal, and bipolar cells. It is postulated that receptors can give an output either in terms of ionic flux and polarization changes or chemical synaptic activity. These can both contribute to retinal thresholds, which will also depend, however, on the level of bleached pigment in receptors, as well as on processes in the neural network of the retina. The horizontal cells transmit a signal only if the transmission line is opened at each end through synaptic activity. This opening of the line occurs in response to light stimuli, as distinct from those due to bleaching. Bipolar cells integrate the receptor output with the surround inhibition of the horizontal cells. The amacrine cells perform a second difference operation with respect to rates of change. It is argued that the model is consistent with experimental data on cellular and membrane responses in the retina and that various psychophysical data can be explained on this basis. The objective of this paper is to try to understand the responses of an organ in terms of cell responses and interactions. Specifically, the organ here is the eye and the cells are the neurons in the retina.     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator     

Recombinant interferon γ up-regulates in vivo and down-regulates in vitro monocyte CD14 antigen expression in cancer patients

Summary The expression of the monocyte membrane glycoprotein CD14 was measured and related to the serum interferon (IFN) concentration in thirteen patients with disseminated cancer during treatment with human recombinant interferon (rIFN). The drug was administered by continuous subcutaneous infusion using an escalating dose schedule, starting at 50 µg/day or 100 µg/day and increasing weekly up to 600 µg/day, if tolerated. Treatment was continued at a mean maximal tolerated dose of 200 µg/day for a median duration of 43 days. Serum IFN concentration and monocyte CD14 antigen expression (immunofluorescence with the monoclonal antibody LeuM3 and fluorescence-activated cell sorting analysis) were determined weekly. The serum IFN concentration was positively correlated with the rIFN dose (P <0.05). Therapy induced a dose-dependant enhancement of CD14 antigen expression. The increase in mean CD14 fluorescence intensity was on average 60% after 3 weeks of treatment at a mean dose of 220 µg rIFN/day and was reversed after withdrawal of therapy. Patients with a rapidly rising serum IFN concentration (starting dose 100 µg/day) showed a smaller increment in CD14 fluorescence intensity than those with slowly rising serum IFN levels (starting dose 50 µg/day). Since rIFN is known to down-regulate CD14 antigen expression in vitro, monocytes from patients off therapy and from healthy volunteers were cultured with this cytokine. A similar decrease of CD14 fluorescence was observed in both groups. In patients several factors, such as IFN concentration, duration of drug effect and type of serum, were evaluated and could not explain the discrepant in vivo and in vitro findings. In conclusion, the monocyte marker CD14 was found to be differentially regulated by rIFN in vivo and in vitro. In vivo, secondary mediators, induced by rIFN and acting on a constantly renewed cell population, may contribute to the enhanced CD14 expression.     

Lipid vesicle-mediated alterations of membrane cholesterol levels: Effects on Na+ and K+ currents in squid axon

Summary We demonstrate that cholesterol can exchange from sonicated lipid vesicles to a perfused squid giant axon membrane and that vesicles with varying cholesterol/phospholipid (C/P) mole ratios can be used to achieve either net loading or net depletion of axon membrane cholesterol. Two types of evidence were obtained which show that net loading or depletion of cholesterol was achieved: (i) changes in the cholesterol/phospholipid (C/P) mole ratios of axons, and (ii) visualization of cholesterol depleted from the preparation by cholesterol-free vesicles by thin-layer chromatography. The C/P mole ratios indicate that cholesterol levels in the preparation were increased or decreased by 30–40%. Increasing or decreasing membrane cholesterol levels were ineffective in altering the Na⁺ or K⁺ occurrents in voltage-clamped axons. In addition, we determined that cholesterol flip-flop across the axonal membrane occurred with at ^1/2 of 7.3 to 15.3 min.     

The high-conductance channels of yeast mitochondrial outer membranes: A planar bilayer study

The high-conductance channels present in the outer membranes of wild-type and porin-less yeast mitochondria have been characterized electrophysiologically after incorporation in planar bilayer membranes. The most prominent activity was ascribed to a voltage-dependent, substaterich, cationic channel which generally inactivated at potentials positive in thecis compartment, in agreement with the observations from patch-clamp experiments on porin-less mitoplasts. This channel has been identified as the so-called peptide-sensitive channel (PSC). We also observed similar channels displaying either no inactivation, or inactivation at both positive and negative potentials. These latter properties match those already described for mammalian and yeast PSC, respectively. These different behaviors are tentatively explained as arising from the presence, or lack of, peptides bound to the PSC. Very high conductances, apparently due to cooperative gating, were frequently displayed. In wild-type membranes, activity ascribable to the porin was also observed.The first two authors contributed equally to the work described in this paper.     

Some observations on the fine structure of the giant synapse in the stellate ganglk of the squid,Doryteuphis bleekeri

Summary The fine structure of the synapse between the second-order giant fibre and the third order-giant fibre of the squid Doryteuphis bleekeri was studied by means of electron microscope. In the synaptic region, the two giant fibres are arranged side by side. Many small processes from the third-order giant fibre penetrate the common sheath which separats the adjacent giant axons making synaptic contact with the second order giant axon.The contact surface consists of opposing two plasma membranes of adjacent axons separated by a narrow space of 20–30 m in width. The synaptic membranes are more electron dense and thicker than the other part of the axon membrane. The synaptic vesicles are concentrated exclusively in the presynaptic axon.The fine structural differences between giant synapse in the stellate ganglion of the squid and the giant-to-motor giant synapse of the crayfish were discussed.This work was supported by Grant Number B-3348 from the National Institutes of Health, United States Public Health Service, Department of Health, Education and Welfare.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

The ultrastructure of plasma and thylakoid membrane vesicles from the fresh water cyanobacteriumAnacystis nidulans adapted to salinity

Summary Photoautotrophically growing cultures of the fresh water cyanobacteriumAnacystis nidulans adapted to the presence of 0.4–0.5 M NaCl (about sea water level) with a lag phase of two days after which time the growth rate reassumed 80–90% of the control. Plasma and thylakoid membranes were separated from cell-free extracts of French pressure cell treatedAnacystis nidulans by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Identity of the plasma and thylakoid membrane fractions was confirmed by labeling of intact cells with impermeant protein markers prior to breakage and membrane isolation. Electron microscopy revealed that each type of membrane was obtained in the form of closed and perfectly spherical vesicles. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. On the average, diameters of plasma membrane vesicles from salt adapted cells were only one-third of the diameters of corresponding vesicles from control cells. By contrast, the diameters of thylakoid membrane vesicles were the same in both cases.Freeze-etching the cells and counting the number of membrane-intercalating particles on both protoplasmic and exoplasmic fracture faces of plasma and thylakoid membranes indicated a roughly 50% increase of the particle density in plasma membranes during the adaptation process while that in thylakoid membranes was unaffected. Comparison between particle densities on isolated membranes and those on corresponding whole cell membranes permitted an estimate as to the percentage of inside-out and right-side-out vesicles. Stereometric measurement of particle sizes suggested that two distinct sub-populations of the particles in the plasma membranes increased during the adaptation process, tentatively correlated to the cytochrome oxidase and sodium-proton antiporter, respectively. The effects of salt adaptation described in this paper were fully reversed upon withdrawal of the additional NaCl from the growth medium (deadaptation). Moreover, they were not observed when the NaCl was replaced by KCl.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - EF exoplasmic fracture face - PF protoplasmic fracture face - DABS diazobenzosulfonate; Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonate - PMSF phenylmethylsulfonylfluoride Dedicated to the memory of Professor Oswald Kiermayer     

Effect of cell-surface binding on development of Ascidian egg

Summary The early development ofPhallusia mammillata eggs, dechorionated with trypsin and treated with Concanavalin A, was studied. Vital staining with a very dilute solution of acridine orange (0.01 g/ml) helped to visualize the mitochondrial crescent by fluorescence. At high concentrations of Concanavalin A (20–200 g/ml) fertilized eggs did not cleave, but went through early ooplasmic segregation movements (formation of the crescent) and multinuclear syncytia were formed. At lower concentrations of Concanavalin A (less than 10 g/ml), cleavage occurred, but the blastomeres remained rounded, leading to a grapelike embryo. Eggs attached to Concanavalin A treated nylon surfaces either did not cleave or produced grapelike embryos. Attachment of the eggs did not affect ooplasmic segregation. Considering modern theories of membrane structure it was concluded that Concanavalin A prevented cleavage either by immobilizing surface structures connected with microfilaments or by indirectly modifying other membrane structures. These structures could not have been involved in ooplasmic segregation, but their mobility was necessary for further morphogenesis.This work was performed at the Station Zoologique, Villefranche-sur-Mer and at the Station de Biologie Marine et Lagunaire. Sète     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

Investigations on the arrangement and fine structure ofPorphyridium cruentum phycobilisomes

Summary Phycobilisomes ofPorphyridium cruentum were investigated in ultrathin sectionsin situ and after isolation and positive or negative staining. The phycobilisomes have the approximate shape of one half of a compressed rotation ellipsoid with the following dimensions: length 2 a=40±4 nm, width 2 b=19±2 nm, height c=28±3 nm. The phycobilisomes form rows and pillars on the thylakoid membranes. A plug-like structure (foot) which apparently fixes the phycobilisomes to the thylakoid membrane is described.     

Characterization of catecholamine uptake2 in isolated cardiac myocytes

Extraneuronal catecholamine uptake was investigated in isolated quiescent rat myocardial cells. By administration of (³H-)(–)noradrenaline concentration of 22 nmol/l up to 1000 mol/l the following data were obtained: (1) The K_M of the uptake process amounted to 260 mol/l, the V_max to 4.24 nmol/(10 min × mg Protein) corresponding to 179 nmol/(min × g_WWt)(WWT = Wet Weight). (2) The uptake was largely inhibited by the uptake₂-inhibitors corticosterone (100 mol/l), isoprenaline (IC so = 30.6 mol/l), and O-methylisoprenaline (IC₅₀ = 2.1 pmol/l), but not by the uptake₁-inhibitors cocaine (100 mol/l) and desipramine (10 mol/l). (3) The affinity-values K_M and IC₅₀ closely agreed with those already known, but the V_max-value was higher than those obtained in whole rat hearts by a factor of at least 1.79. This is caused presumably by the voltage dependence of the uptake mechanism and the resulting inhibition of uptake 2 during the periods of depolarisation in beating hearts of other studies.