"Matrigenin" activity from bovine bone--III. Effects on glycosaminoglycans and proteoglycans of human synovial cells in culture

1. Human synovial fibroblastic cells were cultured in the presence and absence of an extract from bovine bone containing "matrigenin" activity. The rate of incorporation of radioactivity into the glycosaminoglycans of the medium of "matrigenin"-treated cultures increased after 24 hr of incubation, compared to "controls". 2. Higher serum concentrations had a greater effect on the incorporation of radioactivity into hyaluronic acid synthesized by "matrigenin"-treated cultures, than by "controls". 3. Incorporation of radioactive precursors into the proteoglycans isolated from the medium was greater in the "matrigenin"-treated cultures than in "controls". The synthesis of a large mol. wt proteoglycan was specifically stimulated.     

The origin of Taraxacum agamospecies

The genus Taraxacum is readily divided into "primitive" and "advanced" forms, on morphological and cytological criteria. It is thought that the genus arose in the west Himalayas during the Cretaceous and that apomixis arose at an early stage by means of polyploidy, precocious embryony and asynapsis in the female meiosis. Advances of sexual primitive types and "precursor" types west into Europe were paralleled by the spread of arctic-alpine types into many regions of the world. During the Pleistocene the precursor types are thought to have generated the widespread advanced section Ceratophora , which gave rise to many of the advanced species after the last glacial period by hybridizing with primitive and precursor sexuals, thus "fixing" a hybrid swarm as apomicts.     

Periclinal penetration of potassium permanganate into mature cuticular membranes ofAgave andClivia leaves: new implications for plant cuticle development

Staining cuticular membranes ofAgave americana andClivia miniata en bloc with potassium permanganate results in a strong contrast in the interior cuticular layer while the exterior part remains unstained. This is not caused by a selective chemical reaction with the interior part but by the unidirectional penetration of the reagent from the interior side, the outside being protected by the cuticle proper. In transverse cryosections of the cuticular membrane, permanganate penetrates nearly as easily into the exterior cuticular layer as into the interior one giving the same contrast. However, compared with the periclinal penetration into the cuticle proper this penetration is accelerated five-to tenfold by the polysaccharide network within the cuticular layer which serves as a distribution-channel system. Periclinal penetration into the cuticle proper occurs independently in each cutin penetration unit included between two obvious lucent lamellae and further divided into subunits.     

Studies on choline transport enhancement into fibroblasts from normals and Alzheimer's donors

Human dental fibroblasts transport choline actively. This transport is inhibitable by hemicholinium-3. In this paper, choline transport into fibroblasts of normal donors (four cell lines) and into those of Alzheimer victims (four cell lines, age and sex matched to the normals) is accelerated by methylated xanthines, nicotine, and dexamethasone. At a caffeine concentration of 10 M the stimulation of choline transport into normal cells averages 128% and into Alzheimer donor cells, 45%. 1 M Dexamethasone stimulates choline influx by 86% in normal cells and 36% in Alzheimer cells. Nicotine enhances choline transport by 35% in normal cells and by 16% in Alzheimer cells. The implication is that if Alzheimer's disease is a cholinergic disorder, it may be amenable to transport-directed chemotherapies.Special issue dedicated to Dr. Santiago Grisolia     

Effect of proteolysis inhibitors on the incorporation of labelled amino acids into proteins]

Role of peptide bond breaks in the incorporation of amino acids into proteins in a "protein--amino acid" system is investigated. For this purpose the incorporation of labelled amino acids into trypsin under the inhibition of its autolysis by a specific inhibitor from soybean and epsilon-amino-caproic acid is studied. The trypsin inhibitor from soybean is found to suppress considerably the incorporation of 14C-glycine, 14C-lysine and 14C-methionine into crystal trypsin and not to affect the incorporation of labelled amino acids into chomotrypsin, papain and carboxypeptidase. Epsilon-Aminocaproic acid inhibited 14C-glycine incorporation into crystal trypsin by 40% and did not change its incorporation level into serum albumin. The dependency of amino acid incorporation level into trypsin on the activity of autolysis in the "protein--amino acid" system is demonstrated.     

Characteristics of the nuclear translocation of progesterone receptor in fetal guinea pig uterus "in vivo", "in vitro" and in organ culture

The translocation of progesterone receptor from the cytosol into the nucleus was studied under "in vivo" and "in vitro" conditions in the uteri of guinea pig fetuses exposed to progesterone or a synthetic progestin, R5020. Progesterone treatment of estrogen-primed fetuses leads to a rapid (before 1h) transfer of cytosol progesterone receptor into the nucleus which is, however, short-lived (less than 3h). A rapid decrease in the retention of the estrogen receptor in the nucleus also occurs. In the "in vitro" incubations of whole fetal uteri, translocation of progesterone receptor is temperature-dependent and specific for progesterone and R5020; estradiol and cortisol have no effect. Putative progesterone receptors can also be induced in explants of fetal guinea pig uteri in organ culture which translocate from the cytosol into the nucleus under the same "in vitro" conditions as in whole uteri. Fetal uterine progesterone receptor, either stimulated "in vivo" by estrogen-priming or induced in organ culture, translocates from the cytosol into the nucleus and this process seems to be accompanied by a decrease in retention of the estrogen receptor in the nucleus which appears to be the mechanism by which progesterone antagonises estrogen action in fetal guinea pig uterus.     

Partial conversion of vasopressinyl-Gly-Lys-Arg into pharmacologically active vasopressin through secretory granule carboxypeptidase E and alpha-amidating processing enzymes.

Vasopressinyl-Gly-Lys-Arg, the first intermediate derived from vasopressin protein precursor, has been converted into mature vasopressin by an "in vitro" two-step reaction through neurohypophysial secretory granule enzymes. Whereas the conversion into vasopressinyl-Gly is virtually complete at pH 5.5 as judged by HPLC, the conversion of vasopressinyl-Gly into vasopressin is weak at pHs 6.0 or 8.0 as judged by HPLC and measure of generated pressor activity. It is suggested that the high conversion yield usually seen in mammalian neurohypophysis, where no intermediate is detected, might be due to additional "in vivo" factors such as particular membrane-association or binding of the intermediate onto a neurophysin carrier.     

Effect of Pseudomonas aeruginosa exotoxin-A on the synthesis and secretion of proteins in isolated rat pancreatic acini

Exposure of isolated rat dispersed pancreatic acini to increasing concentrations (10 to 1000 ng/ml) of purified exotoxin-A from Pseudomonas aeruginosa resulted in a progressive inhibition of 3H-leucine incorporation into "cellular" (those remaining in the cells) and "secretory" (those released into the medium) proteins. With each concentration of exotoxin-A, magnitude of reduction was found to be greater for the "secretory" proteins than that observed for the "cellular" proteins. Thus, in the presence of 250 ng/ml of exotoxin-A, a dose that produced maximal inhibition in protein synthesis, 3H-leucine incorporation into "cellular" and "secretory" proteins was found to be decreased by about 19 and 50%, respectively, when compared with the corresponding basal controls. Release of trypsinogen, chymotrypsinogen and amylase from the isolated pancreatic acini was also inhibited by high doses of exotoxin-A. However, whereas the exotoxin concentration of 1000 ng/ml, caused a near complete inhibition of chymotrypsinogen release, trypsinogen and amylase secretion were decreased by 40 and 50%, respectively. It is concluded that in isolated pancreatic acini, exotoxin-A inhibits the synthesis and secretion of proteins.     

Electron Microscope Studies of Nuclear Extrusions in Pancreatic Acinar Cells of the Rat

This paper describes "blebs" protruding from the surface of the nucleus into the cytoplasm. The "blebs" are separated from the cytoplasm by 2 membranes which are continuous with the outer and inner nuclear membranes. The "blebs" contain 3 structurally distinct substances. Two of these substances (β and γ substances) are similar to extranucleolar karyoplasm and nucleolar material. The other substance (α substance) is present in every "bleb," but it cannot be readily compared to a recognizable nuclear structure. Cytoplasmic vesicles are described that are apparently different from the Golgi vesicles or the vesicular component of the ergastoplasm. It is suggested that these vesicles may be of nuclear "bleb" origin. A dark karyoplasmic zone extending from the region of the nucleolus into the nuclear "bleb" is shown. This zone may be similar in some respects to the preformed pathway ("Leitbahn") described by Altmann (3) and Hertl (28) and could reflect movement of nuclear material from the nucleolar region into the cytoplasm. The "blebs" are thought to be homologous to structures described by many light microscopists, but they are considerably larger than the nuclear "blebs" described previously by electron microscopists.     

An Insect-Resistant Transgenic Cabbage Plant with Cowpea Trypsin Inhibitor(CpTI) Gene

Cowpea trypsin inhibitor (CpTI) gene was transformed into Brassica oleracea var. capitara variety "Yingchun" and "Jingfeng" mediated by the Agrobacterium tumefaciens LBA4404(pRCL27). Transgenic plants were obtained from transformed calli or explants. It was shown from the ELISA assay that NPT Ⅱgene was expressed in the transgenic cabbage cells. The integration of the CpTI gene into cabbage genome DNA was confirmed by Southem blotting. Insect-tolerance of the transgenic plants to Pieris rapae L. was observed by bioassays on the transgenic plants in the laboratory.     

The largest convex patches: A boundary-based method for obtaining object parts

The importance of boundaries for shape decomposition into component parts has been discussed from different points of view by Koenderink and van Doorn (1982), and by Hoffman and Richards (1984). The former define part boundaries as parabolic contours, whereas the latter propose that part boundaries should be defined by contours of negative minima (or maxima) of principal curvature. In this article, building on aspects of both approaches, we develop a new method for shape decomposition. This method relies exclusively on global properties of the surface which are fully characterized by local surface properties. We propose that a useful parcellation of shapes into parts can be obtained by decomposing the shape boundary into the largest convex surface patches and the smallest nonconvex surface patches. The essential computational steps of this method are the following: (i) build initial parts from the largest locally convex patches, (ii) consider an initial part as a constituent part if it is essentially convex, and (iii) obtain the remaining constituent parts by merging adjacent initial parts generated by the largest locally convex and the smallest nonconvex patches of nearly the same sizes. The method is illustrated on both smooth and continuous shapes. We show that the decomposition of shapes into the largest convex patches aims to maximize the thingness in an object, and to minimize its non-thingness. The method is conducive to a natural parcellation of shapes into constituent parts useful for recognition and for inferring function.     

Occurrence of cell junctions and microfilaments in osteoblasts

Osteoblasts in the diaphysis of the tibia during endochondral ossification in young rats are attached to one another by nexus, by "adhaerens" junctions, and by simple appositions. "Adhaerens" junctions and nexus also occur between preosteoblasts and osteoblasts. Furthermore, to osteoblasts exhibit a network of microfilament bundles in the cell periphery overlying the osteoid. From this network bundles extend into the cell processes which protrude into the unmineralized matrix. The mean diameter of individual microfilaments is 5.9 +/- 0.06 nm. A possible role of nexus and microfilaments in controlled bone growth and differentiation is discussed.     

Ultrastructural studies on early events in fertilization of the bivalveLaternula limicola

Summary Ultrastructural studies on sperm-egg interaction at the time of fertilization inLaternula limicola were performed. The temporary-acrosome did not change morphologically while the sperm passed through the egg investments. At the onset of sperm entrance into the egg, however, the temporary-acrosome and mitochondria were eliminated from the sperm. Afterwards the sperm was engulfed by the egg surface without membrane fusion of the gametes. After entry the sperm nucleus was surrounded by four membranes: the plasma membranes of the egg and of the sperm, and the membranes of the sperm nuclear envelope. As the sperm nucleus differentiated into the male pronucleus, the plasma membranes of both the sperm and egg were initially vesiculated, then dispersed into the egg cytoplasm. Finally, the sperm nuclear envelope changed into the male pronuclear membrane accompanying sperm chromatin dispersion.     

Increased extracellular recovery of recombinant thermostable α-amylase from protein-excreting Escherichia coli cells by a simple water-wash procedure

Extracellular recovery of a recombinant, thermostable -amylase produced by Escherichia coli was increased up to three-fold simply by washing the harvested cells with distilled water. However this phenomenon was confined to this E. coli strain which excretes the same enzyme into the culture fluid. It was demonstrated that the release of -amylase into the resulting water-wash fraction was not caused by cell lysis but weak osmotic shock. © Rapid Science Ltd. 1998     

A study of passive Ca2+ flow through the sarcoplasmic reticulum of skeletal muscles. II. Stimulation of passive Ca2+ influx with monovalent cations and anions

The initial rate of passive Ca2+ influx into "heavy" and "light" fractions of sarcoplasmic reticulum (SR) vesicles increases in the presence of univalent cation chlorides. Stimulation of passive Ca2+ influx decreases in the following order: KCl + valinomycin-KSCN- + valinomycin greater than KSI = NaCl greater than choline chloride. K-gluconate + valinomycin and K-gluconate have no effect on the passive Ca2+ influx into SR vesicles. It is supposed that KCl-stimulation of passive Ca2+ influx into SR vesicles under conditions used may be caused by depolarization of the SR membrane.     

Multiplicity of dog kidney high-Km aldose reductase and conversion mechanism into aldose reductase

• 1.1. High-K_m, aldose reductase purified from dog kidney inner medulla was easily converted into aldose reductase by incubation in the neutral buffer solution.
• 2.2. High-K_m, aldose reductase was found to be in multiple forms, and was separated into three kinds of species designated as a-, b- and c-forms by HPLC.
• 3.3. The a-form observed as a single peak by HPLC was assumed to be present in three forms (al-, a2- and a3-forms), one was aldose reductase (a 1-form) and the others were the precursors of aldose reductase (a2- and a3-form).
• 4.4. The b-form was rapidly converted into the a3-form, followed slowly by the a2-form and finally into the a 1-form.
• 5.5. The c-form was either directly converted into the al-form, or indirectly into the a2-form followed by the al-form.
• 6.6. Four kinds of species (a2-, a3-, b- and c-forms) of high-Ap, aldose reductase were finally converted into aldose reductase (al-form).

     

Organellar DNA synthesis in permeabilized soybean cells

Summary Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L--lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.     

Uptake of dimannoside clusters and oligomannosides by human dendritic cells

Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides. The reducing saccharides were converted into glycosynthons, i.e., into glyco-amino acids. Fluorescein derivatives were obtained by coupling the free carboxyl group of oligosaccharyl-pyroglutamate to the -amino group of -fluoresceinyl-thiocarbamyl lysine methyl ester. It has been shown by others that glycosylated linear oligolysines containing up to six -D-mannopyranosylphenylthiocarbamyl units have a high affinity for the human mannose receptor. In order to obtain fully biodegradable clusters and to improve both the specificity and the selectivity, disaccharides transformed into glycosynthons were coupled to pentalysine carriers (Lys₅-Ala-Cys-NH₂). Glycosylated pentalysyl cysteine conjugates were made fluorescent upon substitution of the cysteine thiol group with fluorescein iodoacetamide. As shown by flow cytofluorimetry, both the dimannoside clusters and yeast oligomannosides were very efficiently taken up by DC, conversely lactoside clusters were not.     

Resolving sorting mechanisms into exosomes

The complexity of mechanisms driving protein sorting into exosomes is only beginning to emerge. In a paper recently published in Cell Research, Roucourt et al. report that trimming of heparan sulfate side chains of syndecans by endosomal heparanase facilitates sorting into exosomes by the formation of tight syndecan clusters that are recruited by the multivalent adaptor syntenin to the ALIX-ESCRT sorting machinery at endosomes.Multicellular organisms function by virtue of intercellular communication, which can be accomplished through direct cell-cell contact or transfer of secreted molecules. A third mode of communication, which is increasingly recognized, involves extracellular vesicles (EVs)¹. EVs are composed of integrated molecular packages of membrane proteins, cytosolic proteins, lipids, and RNA, and can transmit complex messages from donor to specific target cells. EVs have been implicated in many different physiological processes, including immune regulation, stem cell regulation, tissue morphogenesis, and gamete function, but also in disease such as cancer progression and metastasis, and neurodegeneration. EVs can be released from donor cells by shedding from the plasma membrane, and these are then generally referred to as microvesicles (MVs). In contrast, EVs that are secreted by multivesicular endosomes (MVEs) are called exosomes. The intraluminal vesicles of MVEs are generated by inward budding of the endosomal limiting membrane (Figure 1), and hence exosomes have the same topology as MVs and cells, with their exoplasmic side exposed.Open in a separate windowFigure 1Syndecans are processed at endosomes, first by trimming associated heparin sulfate site chains, followed by proteolytic cleavage. Clustered syndecan can now be recruited by multivalent syntenin, which on its turn is coupled to the ESCRT machinery via ALIX. Similarly, CD63 is recruited by syntenin, and with it presumably other membrane proteins that are associated with tetraspanin webs. Heparanase also stimulates sorting of CD63, indicating that the two pathways driven by syndecan and CD63 are somehow integrated.Although physiological functions of EVs have nowadays been proposed in > 9 000 reports, MVs and exosomes are usually not discerned. This is mainly due to lack of knowledge on molecular mechanisms that drive or regulate the sorting of molecules into MVs or exosomes, and this also hampers the design of experiments to demonstrate in vivo relevance of EVs in intercellular communication. Molecular mechanisms for exosome formation at endosomes involve the endosomal-sorting complex required for transport (ESCRT) machinery. ESCRT was originally identified to drive the sorting of ubiquitin-conjugated membrane proteins into vesicles that bud into the lumen of a distinct set of MVEs that ultimately fuse with lysosomes rather than with the plasma membrane, resulting in the degradation of their vesicular contents. ESCRT is composed of 4 multiprotein sub-complexes, of which ESCRT-0, -I and -II recognize and sequester ubiquitinated membrane proteins at the endosomal delimiting membrane, while ESCRT-III drives membrane budding and actual scission of intraluminal vesicles². Sorting of proteins into exosomes, however, appears to occur independently of cargo ubiquitination, and only a selected number of ESCRT components are involved in exosome formation³. For example, ubiquitination of MHC class II is required for its sorting into intraluminal vesicles of lysosome-targeted MVEs, but not for incorporation into exosomes⁴. Sorting of the transferrin receptor to exosomes, which occurs efficiently only in reticulocytes, is also independent of ubiquitination, but instead relies on direct binding of its cytoplasmic domain to the ESCRT accessory protein ALIX⁵.The research group of Guido David has previously shown that sorting of membrane proteins of the syndecan family into exosomes also involves ALIX, but in that case is linked to ALIX via the cytosolic adaptor syntenin⁶. Syntenin couples to membrane proteins with two PDZ domains and to ALIX with three LYPX_nL motifs, and ALIX binds on its turn to ESCRT-III, the machinery responsible for intraluminal vesicle formation at MVEs. Syndecans form complexes through lateral interactions between their attached heparan sulfate polysaccharide chains. Guido David and co-workers have now shown that heparanase activity in endosomes trims long heparan sulphate chains into shorter ones, allowing clustering of syndecans. Further condensation is achieved as a consequence of removal of the syndecan luminal domain by endosomal proteases, leaving the membrane-embedded C-terminal domain. Heparanase-induced clustering is thought to stimulate the binding of syndecan cytoplasmic domains to the tandem PDZ domains of syntenin, driving ALIX-ESCRT-mediated sorting into exosomes. Interestingly, heparanase activity also facilitated the recruitment of CD63 into exosomes, in a syntenin-dependent manner^6,7. Sorting of many membrane proteins into exosomes coincides with their association with tetraspanin membrane proteins⁸. Webs of interacting tetraspanins and associated proteins are stabilized by protein palmitoylation, and lipids also play an important role in the formation of tetraspanin webs, conceivably explaining the relative enrichment of cholesterol and glycosylceramides in exosomes, as well as the dependency on sphingomyelinase activity for exosome formation⁹. Non-tetraspanin membrane proteins may in this way piggy-back onto tetraspanin webs for their sorting into exosomes⁸. Interestingly, the tetraspanin CD63, which is highly enriched in exosomes and considered to be important for chaperoning cargo into exosomes¹⁰, can also be recruited by syntenin¹¹. Sorting of tetraspanin webs at endosomes into exosomes could thus, similar to syndecans, be driven by the cytoplasmic adaptor syntenin, and the recruitment by syntenin of tetraspanin webs and syndecan clusters are thus integrated processes (Figure 1).All in all, a complex picture is emerging, in which both CD63 and syndecans, and possibly other membrane proteins that associate with endosomal syndecan and/or tetraspanin-enriched microdomains, are sorted into exosomes by a shared syntenin-ALIX-ESCRT machinery.Importantly, not all EV cargoes appear to depend on heparanase-syntenin-ALIX-ESCRT, as exemplified by flotillin, CD9 and CD81^6,7. One possible explanation is that distinct but parallel sorting mechanisms exist that drive cargo into a single population of exosomes. Another possibility is that separate sorting mechanisms drive the formation of distinct EV populations containing different cargo molecules. For example, syntenin-dependent and -independent EVs may be represented by exosomes and plasma membrane-derived MVs, respectively. The origin of EVs, exosomes or MVs, is ill defined in most research. The current study by Roucourt and coworkers⁷ provides molecular tools, supplementation with heparanase to stimulate, or syntenin depletion to interfere with, cargo incorporation into exosomes, that could help researchers to establish the precise origin of EVs, as well as their roles in biological processes.     

Origin and Evolution of Plastids and Photosynthesis in Eukaryotes

Recent progress in understanding the origins of plastids from endosymbiotic cyanobacteria is reviewed. Establishing when during geological time the endosymbiosis occurred remains elusive, but progress has been made in defining the cyanobacterial lineage most closely related to plastids, and some mechanistic insight into the possible existence of cryptic endosymbioses perhaps involving Chlamydia-like infections of the host have also been presented. The phylogenetic affinities of the host remain obscure. The existence of a second lineage of primary plastids in euglyphid amoebae has now been confirmed, but the quasipermanent acquisition of plastids by animals has been shown to be more ephemeral than initially suspected. A new understanding of how plastids have been integrated into their hosts by transfer of photosynthate, by endosymbiotic gene transfer and repatriation of gene products back to the endosymbiont, and by regulation of endosymbiont division is presented in context.Photosynthesis is biology’s equivalent of alchemy converting a common substance (CO₂) into a precious one (reduced carbon compounds rich in chemical energy). Freely available light energy is initially converted to precious chemical energy in the form of ATP. This energy, and the reducing power generated by splitting water molecules to release electrons, is used to fix carbon from atmospheric CO₂ and generate reduced carbon compounds that underpin the biosphere. It is estimated that plants and algae convert 258 billion tons of carbon dioxide into biomass by photosynthesis annually (Geider et al. 2001). Microfossils in ancient stromatolites indicate that cyanobacterium-like organisms had invented this process—or an early, perhaps nonoxygenic, version of it—at least 3.5 byo (billions of years old) (Lowe 1980; Walter et al. 1980; Schopf 1993). These photosynthetic prokaryotes substantially predate eukaryotes, which emerged much later (Rasmussen et al. 2008; Koonin 2010). The common ancestor of all eukaryotes entered into an endosymbiotic partnership with an α-proteobacterium that evolved into the mitochondrion, now the site of aerobic respiration in most eukaryotes (Gray 2012); animals and fungi are heterotrophic descendants of this partnership. Another lineage, which eventually produced the plants, entered into a second endosymbiotic partnership, this time with a cyanobacterium, which transplanted photosynthetic alchemy into eukaryotes to create plastids (Gray and Archibald 2012). This review will highlight recent progress in our understanding of the origin and evolution of plastids.