Alterations in the expression of the β-cytoplasmic and the γ-smooth muscle actins in hypertrophied urinary bladder smooth muscle

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle -actin, shows over 200% increase in the -actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in -actin, since the 2D gel electrophoresis does not distinguish -smooth muscle actin from -cytoplasmic actin. The bladder smooth muscle -actin and the smooth muscle -actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the -cytoplasmic actin (37%) and a concomitant diminution in the -cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.     

The Synthesis and Antiviral Activity of Glycyrrhizic Acid Conjugates with α-D-Glucosamine and Some Glycosylamines

Glycyrrhizic acid and its 30-methyl ester were conjugated with 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy--D-glucopyranose, 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl amine, 2,3,4-tri-O-acetyl--L-arabinopyranosyl amine, 2-acetamido-2-deoxy--D-glucopyranosyl amine, and -D-galactopyranosyl amine using N,N-dicyclohexylcarbodiimide and its mixtures with N-hydroxybenzotriazole. Structures of the conjugates were confirmed by IR, UV, ¹H, and ¹³C NMR spectroscopy. The glycoconjugate with the residues of 2-acetamido-2-deoxy--D-glucopyranosyl amine in the carbohydrate part of its molecule exhibited antiviral activity (ID₅₀ 4 g/ml) toward the herpes simplex type 1 virus (HSV-1) in the VERO cell culture. Two compounds demonstrated anti-HIV-1 activity (50–70% inhibition of p24) in a culture of MT-4 cells at concentrations of 0.5–20 g/ml.     

The mode of secretion of α-amylase in barley aleurone layers

The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl₂ two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[³H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE N-carbobenzoxy-L-tyrosine p-nitrophenylester - Cyt oxidase Cytochrome oxidase - ER endoplasmic reticulum - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IDPase inosine diphosphatase - K⁺-ATPase pH 6.5 K⁺-stimulated adenosine triphosphatase - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - NADH: Cyt c reductase cyanide-insensitive NADH-linked cytochrome-c reductase - RER rough endoplasmic reticulum - Tris tris-(hydroxymethyl)-aminomethane     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid,pTiC58

The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection. Part of this DNA (the `common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain-specific. Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced. We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM. Gene c codes for a new unknown T-DNA protein. Gene a is homologous to the agrocinopine synthase gene. Genes b, c, d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3. Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes. Our studies show that gene 3 (located on the TR-DNA of octopine strains) is also oncogenic. Although the b–e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora. Shoot formation could be restored by genes c and c in co-infection experiments. Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested.     

Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion

The ultrastructural organization of actively secreting barley (Hordeum vulgare L. cv. Himalaya) aleurone cells was examined using ultrarapid-freezing (<-10 000°C s^-1) followed by freeze-fracture and freeze-substitution. Our analysis indicates that much of the evidence supporting a direct pathway from the endoplasmic reticulum (ER) to the plasma membrane (i.e. bypassing the Golgi apparatus) for the secretion of -amylase (EC 3.2.1.1) may not be valid. Cryofixed ER cisternae show no sign of vesiculation during active -amylase secretion in gibberellic acid (GA₃)-treated cells. At the same time, Golgi complexes are abundant and numerous small vesicles are associated with the edges of the cisternae. Vesicles appear to be involved in the delivery of secretory products to the plasma membrane since depressions containing excess membrane material appear there. Treatment with GA₃ also induces changes in the composition of Golgi membranes; most notably, the density of intramembrane particles increases from 2700 m^-2 to 3800 m^-2 because of an increase of particles in the 3–8.5-nm size range. A slight decrease in 9–11-nm particles also occurs. These changes in membrane structure appear to occur as the Golgi complex becomes committed to the processing and packaging of secretory proteins. We suggest that secretory proteins in this tissue are synthesized in the abundant rough ER, packaged in the Golgi apparatus, and transported to the plasma membrane via Golgi-derived secretory vesicles. Mobilization of reserves is also accompanied by dynamic membrane events. Our micrographs show that the surface monolayer of the lipid bodies fuses with the outer leaflet of the bilayer of protein-body membranes during the mobilization of lipid reserves. Following the breakdown of the protein reserves, the protein bodies assume a variety of configurations.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - P protoplasmic - E exoplasmic     

rol genes of Agrobacterium rhizogenes cucumopine strain: sequence,effects and pattern of expression

By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA-which we refer to as rol, and -were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5 non-coding region of rol and rol was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.     

Tissue specific expression of an α-skeletal actin-lacZ fusion gene during development in transgenic mice

Transgenic mice carrying a chimaeric transgene containing 730 bp of the 5-flanking sequences and the entire first intron of the rat -skeletal actin gene fused to thelacZ reporter gene have been produced by microinjection. ThelacZ reporter gene was used to verify the suitability of using the rat -actin promoter elements to target expression of genes of agricultural and therapeutic value exclusively to skeletal and heart muscle cells and fibres of transgenic mice. Expression of the transgene indicates a tightly regulated developmental and muscle specific control of the rat -skeletal actin gene, making it a useful promoter for gene targeting to muscle tissues. The cells destined to form muscle tissues in these transgenic mice are readily visualized in intact embryos by staining for -galactosidase activity, making them a suitable animal model for studying the origin and development of skeletal and cardiac muscle tissues.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Endoplasmic reticulum and crystalline fibrils in the root protophloem of Nymphoides peltata

Endoplasmic reticulum in the root protophloem of Nymphoides peltata (S.G. Gmel.) O. Kuntze changes form as sieve elements differentiate. In immature sieve elements the individual endoplasmic reticulum (ER) cisternae form large irregular aggregates in the cytoplasm. In older immature sieve elements the ER aggregates are more ordered and membranes in them are convoluted. Although convoluted ER predominates in immature sieve elements the ER of the mature sieve elements consists mainly of flattened stacks of ER cisternae. Some of these stacks of ER may be derived from the existing convoluted ER. Crystalline fibrils first appear in the cytoplasm of the sieve element when the ER starts to aggregate. The crystalline fibrils move to the parietal layer of the sieve element along with the aggregates of ER. A possible ontogenetic relationship between ER and crystalline fibrils is discussed.Abbreviation ER endoplasmic reticulum     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline     

β-Mannoside mannohydrolase and the mobilization of the endosperm cell wall of lettuce seeds,cv. Grand Rapids

Mobilization of the endosperm cell-wall reserves of Lactuca sativa L. cv. Grand Rapids requires endo--mannanase and -galactosidase activity. A third enzyme, -mannoside mannohydrolase (EC 3.2.1.25) is also involved. We have determined the optimum extraction and assay conditions for this enzyme, which is soluble only in high-salt (1 M NaCl) buffer. It is located exclusively within the cotyledons, in association with a cellulosic cell-wall fraction. Its substrates are the products of endosperm cell-wall mobilization, mannobiose and mannotriose, which diffuse to the cotyledons and are hydrolyzed extracellularly by the -mannoside mannohydrolase.Abbreviation PNPM p-nitrophenyl--mannopyranoside We dedicate this paper to the memory of our friend and colleague Dr. Jacob D. Duerksen     

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 m thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.     

Characterization of the fibrillar layer at the epithelial-mesenchymal junction in tooth germs

A characteristic layer containing numerous fibrils is associated with the basement membrane of the inner enamel epithelium during the early stages of odontogenesis. However, its nature is not well understood. In this study, the layer was examined with high-resolution electron microscopy and immuno-histochemical staining. Tooth germs of monkeys (Macaca fuscata) were studied and each fibril in the layer was found to be a tubular structure, 8–9 nm in width, resembling a basotubule, the tubular structure previously observed in various basement membranes. The space between the fibrils was filled with a network formed by irregular anastomosing strands with an average thickness of 4 nm; these strands resembled the cords forming the network in the lamina densa of basement membranes. After immunoperoxidase staining, fine threads immunoreactive for laminin staining were seen winding along the strands of the network, and 1.5-nm wide filaments, immunoreactive for type IV collagen, took the form of a network arrangement. The 5-nm-wide ribbon-like structures associated with the strands were identified as heparan sulfate proteoglycan by immunostaining. These results are similar to those obtained for the cord network of the lamina densa. The fibrillar layer therefore represents a highly specialized lamina fibroreticularis of the basement membrane of the inner enamel epithelium, and rich in basotubules.     

Centrin- and α-actinin-like immunoreactivity in the ciliary rootlets of insect sensilla

Summary Long ciliary rootlets are a characteristic feature of the dendritic inner segments of the sensory cells in insect sensilla. These rootlets are composed of highly ordered filaments and are regularly cross-striated. Collagenase digestion and immunohistochemistry reveal that the rootlets are probably not composed of collagen fibers. However, double-labeling experiments with phalloidin and anti--actinins show that antibodies to -actinin react with the ciliary rootlets of the sensilla, but do not stain the scolopale, which is composed of actin filaments as visualized by phalloidin. Antibodies to centrin, a contractile protein isolated from flagellar rootlets of green algae, also stain the ciliary rootlets. Within the ciliary rootlets of insect sensilla, -actinin may be associated with filaments other than actin filaments. The immunohistochemical localization of a centrin-like protein suggests that contractions probably occur within the rootlets. The centrin-like protein may play a role during the mechanical transduction or adaptation of the sensilla.     

Effects of temperature on cytomorphogenesis and ultrastructure ofMicrasterias denticulata Bréb

Summary Exposure of growingMicrasterias cells to high (32°–36°C) and low (3°–10°C) temperatures produces changes in morphology that are accompanied by several ultrastructural alterations. Whereas low temperatures essentially cause simplification of cell ornamentation, a variety of cell malformations result from high temperature treatment. These are the loss of cell symmetry leading to markedly aberrant cell shapes and an increase of main lobes with reduced degree of differentiation. Preliminary studies indicate that a shift in the distribution of membrane-associated Ca²⁺ by elevated temperatures probably underlies these abnormal cytomorphogenetic events. Both, low and high temperature cause a reduction in size of the young half cell and affect cytoplasmic streaming. Moreover, nuclear migration is retarded and chloroplast arrangement is influenced by temperature treatment at both ranges. Growth velocity of primary wall responsible for cell shaping is increased at high and slowed down at low temperatures compared to cells grown at 20°C.The main ultrastructural alterations induced by high temperatures are an increase in amount and length of ER cisternae, the appearance of heat shock granule aggregations localized in the cytoplasm, a reduced number of ribosomes and polysomes, the presence of oil bodies in growing cells and a varying thickness of the primary wall. Influences of low temperatures on ultrastructure are less pronounced. They are manifested in the formation of large aggregations of ER cisternae slightly differing from those found in untreated cells, a disturbed arrangement of the microtubule system surrounding the nucleus and a decrease of the number of cell wall forming cytoplasmic vesicles.It is thought that most of the temperature effects are due to an influence on membranes probably an alteration of ionic currents and, in addition, a modulation of normal protein synthesis.Dedicated to my teacher Professor Oswald Kiermayer in deep gratitude     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.     

The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) II. A cytochemical analysis

Summary Cytochemical analysis of the endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), reveals distinct compartmentalization. Phosphatase localization shows that: IDPase is located throughout all cisternae of the dictyosome and vesicles associated with the contractile vacuole. Other alkaline phosphatases like TPPase, ATPase and ITPase were localized within the trans-face cisternae and vesicles of the contractile vacuole. IMPase was localized at the plasmamembrane and not within the endomembrane system. Acid phosphatases, incl. CMPase, NADPase and -glycerophosphatase, were localized in vesicles emerging from the central terminus of the trans-face of the dictyosome and in the peripheral vacuolar network. Silver proteinate labeling was noted in the dictyosome, contractile vacuole and on the anterior plasmamembrane. A summary of endomembrane compartmentalization and a putative interpretation of membrane flow and economy are presented.Abbreviations ER endoplasmic reticulum - IDPase inosine 5-diphosphatase - ITPase inosine 5-triphosphatase - ATPase adenosine 5-triphosphatase - TPPase thiamine pyrophosphatase - CMPase cytidine 5-monophosphatase - NADPase -nicotinamide adenine diphosphatase - AcPase acid phosphatase