Depressed sliding velocity of isolated cardiac myosin from cardiomyopathic hamsters: Evidence for an alteration in mechanical interaction of actomyosin

We measured the relative sliding velocity of cardiomyopathic hamster cardiac myosin on actin cables by using anin vitro motility assay system. We also investigated the relationship between the velocity and both myosin isozyme content and ATPase activity. Cardiac myosin was obtained from cardiomyopathic hamsters (BIO 14.6;B) aged 3,6,9, and 18 months and age-matched controls (F1B;F). Long well-organized actin cables of an alga,Nitellopsis, wer used for the motility assay. Small latex beads (2 m in diameter) were coated with purified cardiac myosin. When myosin-coated beads were introduced into an algal cell in the presence of Mg-ATP, myosin interacted with actin and dragged the beads. Active movement of the beads along the actin cables was observed under a photomicroscope and the velocity was measured. The velocity was significantly lower in B than in F for each age group (0.47 vs. 0.71 m/s at the age of 3 months, p<0.05; 0.44 vs. 0.88 m/s at 6 months, p<0.01; 0.44 vs. 0.67 m/s at 9 months, p<0.01; 0.35 vs. 0.52 m/s at 18 months, p<0.05). Both Ca²⁺-activated ATPase activity and the percentage of -myosin heavy chain were also lower in B than in F for each age group. When examined for individual specimens, there was a positive correlation between the velocity and both myosin Ca²⁺-activated ATPase activity (r=0.84) and percentage of -myosin heavy chain (r=0.83). These data points of both control and cardiomyopathic hamsters were distributed near the regression line obtained from control and thyroxine-treated rabbits reported previously. The present results indicate that the difference in mechanical properties between control and cardiomyopathic cardiac myosin is attributed to isozyme redistribution and not to a qualitative change in each myosin molecule.     

Development and pigmentation of chlorosomes in Chloroflexus aurantiacus strain Ok-70-fl

The development of chlorosomes and their pigmentation were studied by growing Chloroflexus aurantiacus strain Ok-7o-fl first under conditions under which BChl c-synthesis is low (50°C, 2000 lux and 30°C, 1500 lux) and subsequently under conditions promoting high BChl c-synthesis (50°C, 400 lux). Electron microscopic observations on and chemical analyses of isolated cell components showed that in BChl c-depleted cells chlorosome-like structures (chlorosome bags) are attached to fragments of cytoplasmic membranes. These chlorosome bags exhibit a periodic fine structure caused by the construction of the baseplates of the chlorosomes. The baseplates are closely attached to the cytoplasmic membrane, they are rich in phospholipids and apparently contain a 790 nm-BChl a-complex. Chlorosome bags of BChl c-depleted cells always contain a limited amount of light-harvesting pigment complexes (BChlc, - and -carotene). The light-harvesting system is restored (50°C, 400 lux) by first refilling the existing chlorosome bags before cell division takes place.Abbreviations BChl Bacteriochlorophyll - LH Light-harvesting complex - RC Reaction center     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

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.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Common antigenic determinants in the glycoproteins of plants,molluscs and insects

An antiserum raised against -fructosidase isolated from the cell walls of suspension-cultured carrot cells cross-reacts with many plant proteins and hemocyanin ofHelix pomatia. The shared epitope appears to be a small complex glycan with a (1–2)-linked xylose residue attached to the -linked mannose residue of the core of an asparagine-linked oligosaccharide. There is strong cross-reactivity with the proteins of many seed plants, molluscs and insects, and no cross-reactivity with the proteins of fungi, algae, mosses, ferns, or any of the vertebrates tested. Xylose-containing glycans appear to increase the immunogenicity of the proteins to which they are attached, and we suggest that they may be responsible for some allergic responses of people that are repeatedly exposed to plant or insect proteins.     

Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

Effect of culture media on lymphokine-activated killer effector phenotype and lytic capacity

Summary We compared the ability of murine lymphokine-activated killer (LAK) cells grown in either a serum-supplemented standard medium (MEM plus fetal calf serum) or a serum-free medium (AIM-V) to lyse a range of tumour targets. LAK cells grown in either of the media killed a cultured murine tumour line (YAC-1 lymphoma) well and spared syngeneic self cells (concanavalin-A-stimulated splenocytes). However, a striking difference was noted in the ability of LAK cells grown in MEM plus fetal calf serum (as opposed to AIM-V) to kill modified self cells (trinitrophenol-modified concanavalin A blasts); LAK cells grown in the former always killed modified self cells better than those grown in the latter. This pattern held under a broad range of experimental manipulations and was found to be related to a relative increase in CD3-bearing LAK cells grown in the standard medium. These data suggest that the two media cannot be used interchangeably. This conclusion may have clinical implications for the use of LAK cells, as animal studies have been done using LAK cells generated in serum-containing medium and clinical studies have used LAK cells generated in serum-free medium.     

Immunocytochemical localization of conglutin γ and legumin-like globulin in developing and mature seeds ofLupinus albus L.

Summary The distribution of two seed proteins, namely conglutin and a legumin-like globulin, in developing and mature seeds ofLupinus albus L. has been examined by immunocytochemistry and the concomitant modifications of their constituent polypeptides followed by SDS-PAGE. Both proteins were found within vacuolar protein bodies in various tissues of the cotyledons, although with some differences in the distribution patterns. The legumin-like protein was found to be deposited within the large storage parenchyma cells of the cotyledons in a manner similar to that reported for other storage proteins; little or no immunolabelling was associated with the cotyledonary epidermal and vascular parenchyma cells. In contrast conglutin was present in all cell types.A precursor of the legumin-like protein accumulated transiently in the developing cotyledon, but was subsequently modified by proteolytic cleavage. The onset of such modification was concomitant with a transition in the predominant vacuolar forms within the storage parenchyma cells. No precursor molecules of conglutin have been detected in this study, thus indicating that this protein is deposited in the protein bodies in its mature form.Abbreviations LM light microscopy - EM electron microscopy - DAF days after flowering - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - GAR goat antirabbit antiserum     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole     

Two recessive mutations with maternal effect upon colour and cleavage ofXenopus l. laevis eggs

Summary Pale eggs and partial cleavage are two mutations with a maternal effect that are found in the same family ofXenopus l. laevis. The pale eggs have animal hemispheres of a yellow to beige colour and give rise to normally pigmented tadpoles and frogs. The cells of pale embryos contain fewer melanosomes than those of controls. The partial cleavage eggs are characterized by an abnormality of cleavage visible from the eight-cell stage onwards, by abnormal yolk platelet distribution and abnormal cytological features. Cleaved, syncytial and uncleaved areas are observed in these eggs, which are lethal at the blastula stage.     

Dynamics of the cytoskeleton of epidermal cells in situ and in culture

Summary The cytoskeleton of primary tissue-culture cells from the epidermis of Xenopus laevis tadpoles was investigated by phase-contrast, immunofluorescence, and electron microscopy. The connection between the arrangement of different types of filaments and the mechanical properties of the epidermis is discussed. The bilayered epidermis attains stability from thick bundles of tonofilaments interconnecting the basal desmosomes. Twisting of tonofilaments around each other can explain the occurrence of elastic filamentous curls forming a meshwork braced between rows of small desmosomes in the apical region of the epidermis. Actin is arranged as a diffuse meshwork and sometimes forms bundles intermingling with tonofilament bundles. Surface membranes and rows of small desmosomes are delineated by actin and contain -actinin. Actin raises the tension for rounding and spreading of cells. Microtubules stabilize already well-developed lamellae.     

Regeneration of whole plants from callus culture of diverse genetic lines of Pisum sativum L.

Sixteen genetic lines of peas were screened for their ability to regenerate whole plants from callus cultures. Epicotyl sections from germinating seeds were placed on callus-inducing medium; the resulting callus was subcultured monthly and was tested every other month for its regeneration ability. Six lines were found that would regenerate after 2 months' growth as callus. Four of these continued to regenerate after 4 months and, of these, two after 6 months. The cultivars Frosty and Alaska were among the lines that would not regenerate at all.Michigan Agricultural Experiment Station Journal Article No. 8932     

Effect of 17β-estradiol on cells stained for FSHβ and/or LHβ in the dog pituitary gland

Summary The effect of long-term treatment (52 weeks) with high doses of 17-estradiol (1.28 mg/kg/week intramuscularly) on gonadotrophs was studied in the pituitary gland of the beagle bitch. For immunochemical staining the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH were employed. For control purposes antisera to the following hormones were also used: bovine TSH, canine GH, canine PRL and porcine ACTH¹. In the pars distalis and pars tuberalis of control bitches, in addition to the cells which react solely with antisera to either LH or FSH, most cells were reactive to both antisera. The cells stained for FSH were less numerous than those shown to contain LH. TSH, PRL, GH and ACTH/MSH were localized in distinctly different cell types in the pars distalis of all control animals. In the treated bitches, almost complete regression of cells classically identified as gonadotrophs and stained for LH was observed. On the other hand, using the antiserum to FSH, selective immunochemical staining was localized in cells fitting the morphological characteristics of TSH cells. All these cells were also stained for TSH. However, a few cells were also shown to react solely with the antiserum to TSH. These cells, which seem to contain both TSH and FSH, were further clearly differentiated from PRL, GH and ACTH/MSH cells on the basis of their cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not necessarily apply to the glycoprotein hormones of the dog pituitary gland.Abbreviations of Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Mrs. K. Oertel for carrying out the experimental work on animals, to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Photoactive amino acid derivatives with long alkyl chains

Summary Azobenzene amphiphiles containing-alanine, L-lysine or-homolysine moieties were synthesized and characterised. Stable monomolecular layers at the air/water interface and LB multilayers has been obtained from some of them. One sample of azobenzenes synthesized has been shown to undergo a reversible trans/cis photoisomerization upon light irradiation in both solution and LB multilayer.Abbreviations DCC N,N-dicyclohexyl carbodiimide - HONSu N-hydroxy succinimide - Boc t-butyloxycarbonyl - Bu^t t-butyl - EtOAc ethyl acetate - THF tetrahydrofuran - TEA triethylamine - HPLC high performance liquid chromatography - TLC thin layer chromatography - Ph phenyl - i-PrOH isopropyl alcohol     

The effect of dibutyryl cyclic AMP on the expression of actin isoforms in astroglia

Mammalian cells contain at least 8 actin isoforms. The functional significance and the mechanisms that regulate the expression of each actin isoform are not yet known. Using immunofluorescence staining, it was found that all astroglia in tissue culture express -actin isoform and about 86% of astroglia express -smooth muscle actin isoform. When astroglia were treated with dibutyryl cyclic AMP for 4,7,14 and 21 days, it was found that the number of the cells expressing -smooth muscle actin isoform progressively decreased, whereas, the number of the cells expressing -actin isoform remained constant. The western blot experiment showed that the amount of total -smooth muscle actin isoform (soluble and insoluble) and of the insoluble isoform expressed by astroglia treated with dibutyryl cAMP decreased whereas, the amount of total and insoluble -actin isoform expressed by the same cells did not show any significant changes. The cells treated with the cAMP failed to migrate and to close the area created by the scratch wound in monolayer culture. However, the non-treated cells migrated and closed the area created by the scratch after 3 days. This study shows that the astroglia have different mechanisms in regulating the expression of different actin isoforms and that the -sm actin isoform is important in migration of astroglia.