Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.     

Osmium tetroxide footprinting of a scaffold attachment region in the maizeAdh1 promoter

Osmium tetroxide (OsO₄) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO₄ hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO₄ will also identify other torsional perturbations of DNA. In this study, OsO₄ was used to footprint an AT-rich region (between –780 and –500) of the maizeAdh1 promoter. Hyperreactive sites were identified bothin vitro andin vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO₄ hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei.     

The Role of Cellular and Extracellular Matrix Adhesion Proteins in Organ Transplantation

The specific adhesion of cells to other cells or to particular tissue microenvirorvments is a basic function of cell migration and recognition, and underlines many biologic processes including embryogenesis, repair and immunity. Leukocytes express an array of surface receptors broadly known as “accessory adhesion molecules.” which mediate most cell -cell interactions, direct lymphocyte traffic between anatomical compartments, and facilitate cellular adhesion to the inflammation or alloantigenic sites (Springer 1990). In addition, adhesion molecules are involved in the process of antigen recognition, and may costimulate cell activation and transformation. These proteins are thought to affect the very early antigen independent events between host leukocytes and vascular endothelium. Because of these activities, the subject of adhesion molecules is gaining interest in the field of organ transplantation, in both conceptualization and development of novel therapeutic strategies (de Sousa et al. 1991, Kupiec-Weglinski et al. 1993a, Heemann et al. 1993).     

Neuronal responses to purinoceptor agonists in the leech central nervous system

Exracellular nucleotides like ATP and its derivatives are possible chemical messengers in vertebrate nervous systems. In invertebrate nervous systems, however, little is known about their role in neurotransmission. We have studied the reponse of identified neurones of the leech Hirudo medicinalis to the purinoceptor agonist ATP, ADP, AMP, and adenosine using conventional intracellular microelectrodes and whole-cell patch-clamp recording. Bath application of the agoinsts depolarized the different neurons, but not neuropil glial cells. The most effective responses (up to 10 mV) were observed with ATP (100 μM) or ADP (100 μM) in the noxious and touch cells. In most neurons the nonhydrolyzable ATP derivative ATP-γ-S (5 μM) induced larger depolarizations that 100 μM ATP, indicating that most of the potency of ATP is lost presumably due to its degradation by ectonucleotidases. In medial noxios cells, ATP (100 μM) induced an inward current of 1.7 ± 1.1 nA at a holding potential of ?60 mV. The ATP-induced current-voltage relationship showed an inward rectification and a reversal potential close to 0 m V. In a Na⁺-free extracellular solution, the ATP-induced inward current decreased and in a Na⁺- and Ca²⁺-free saline only a small residual current persisted. The possible P₂ purinoceptor antagonist suramin did not antagonize the ATP-induced current, but itself evoked an inward current and a conductance increase. We conclude that ATP activates nonselective cation channels in medial noxious cells of the leech with the order of potency of purinoceptor agonists ATP ≥ ADP > AMP. The results suggest that these cells express purinoceptors of the P₂ type. 1994 John Wiley & Sons, Inc.     

Seasonal determinations of extracellular hydrolytic activities in heterotrophic and mixed heterotrophic/autotrophic biofilms from two contrasting rivers

The temporal changes in extracellular enzyme activities in freshwater microbial biofilms were examined in two contrasting river sites in North Wales over a 12 month period. Sites were a first order, unshaded oligotrophic upland stream (Nant Waen) and a fourth order, mildly eutrophic river with riparian tree cover (River Clywedog). When algal populations were low, biofilms of the more eutrophic site supported greater enzyme activities and higher population densities than the oligotrophic site. Composition, concentration and origin of substrates available to the respective biofilm communities influenced extracellular processing patterns. Reduction in algal populations depressed total and extracellular activities in biofilms from the first order site, suggesting that biofilm communities here were maintained by in situ primary production. Biofilms from Nant Waen were often found to contain higher extracellular activities per cell than the more eutrophic River Clywedog biofilms, which might represent the enhanced ability of an oligotrophic biofilm to accumulate extracellular enzymes. In contrast, light and darkgrown River Clywedog biofilms were not enzymatically distinct, inferring a less important role for biofilm phototrophs. Some evidence was found for increased reliance on allochthonous substrates in the River Clywedog for biofilm maintenance.     

Evidence for anextra-cellular function for protein kinase A

In addition to itsintra-cellular functions, cAMP-dependent protein kinase (PKA) may well have anextra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg⁺⁺; (b) In human serum, an endogenous phosphorylation of one protein (p75, Mr 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser³⁷⁸) which, at physiological pH, is buried in its two-chain form (V₆₅₊₁₀) but becomes exposed in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser³⁷⁸ in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1). Physiologically, this functional modulation may be involved in unleashing PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin.Dedicated to Edmond H. Fischer and Edwin G. Krebs, with gratitude for teaching us the right measure of thoroughness and vision in research.     

The differential expression of lamin epitopes during mouse spermatogenesis

The presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti-lamin AC and an anti-lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti-lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti-lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti-lamin B reacts with a ～ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post-meiotic nuclear matrices. Anti-lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti-lamin B, anti-lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti-lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley-Liss, Inc.     

Ranking species based on sensitivity to perturbations under non-equilibrium community dynamics

Managing ecological communities requires fast detection of species that are sensitive to perturbations. Yet, the focus on recovery to equilibrium has prevented us from assessing species responses to perturbations when abundances fluctuate over time. Here, we introduce two data-driven approaches (expected sensitivity and eigenvector rankings) based on the time-varying Jacobian matrix to rank species over time according to their sensitivity to perturbations on abundances. Using several population dynamics models, we demonstrate that we can infer these rankings from time-series data to predict the order of species sensitivities. We find that the most sensitive species are not always the ones with the most rapidly changing or lowest abundance, which are typical criteria used to monitor populations. Finally, using two empirical time series, we show that sensitive species tend to be harder to forecast. Our results suggest that incorporating information on species interactions can improve how we manage communities out of equilibrium.     

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.