A nodulin specifically expressed in senescent nodules of winged bean is a protease inhibitor.

Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. Immunocytochemical study showed that the protease inhibitor is exclusively localized in infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.     

Biochemical changes in French bean pods infected with Colletotrichum lindemuthianum*

Infection of bean pods with Colletotrichum lindemuthianum leads to the appearance in diffusates of a range of fluorescent and phenolic compounds and of at least two inhibitory compounds. These compounds were found to be absent or in reduced concentration in control diffusates, although the inhibitors were frequently observed to appear at low concentrations without infection. Although sugars and amino acids were found to be released into diffusates, variation in the concentration of these stimulants was found to have little effect on spore germination. Evidence from solvent partition, spectrophotometry and chromatography suggests that the two inhibitors can be tentatively identified with inhibitors previously described from French bean, and both appear to be phenolic substances. It is suggested that inhibitor production may be regarded as part of a general change in aromatic biosynthesis following infection.     

Cell wall residues in yeast protoplast preparations

Protoplast preparations made from Saccharomyces cerevisiae by prolonged treatment with snail digestive juice contained fibrils and chitinous bud-scar residues from the original cell wall.     

Field bean protease inhibitor preparations,unlike methotrexate,can completely suppress Yoshida sarcoma tumor in rats

Protease inhibitor preparations (PIP) with antitryptic and antichymotryptic activities, isolated from field bean legume as well as doxorubicin and cyclophosphamide could effectively suppress the growth of Yoshida sarcoma ascites tumor cells transplanted in adult rats and prevent their death. As against this, methotrexate and heat-inactivated PIP were ineffective in such rats at varied doses of treatment tried. The percent survival of animals appeared to be related to the purity, treatment mode and the dose of PIP used. Zymographic analysis of the trypsin activated sarcoma cell homagenate revealed the presence of six protease bands in the molecular weight range of 51kD to 206kD. Prolonged interactions of such zymograms with protease inhibitors such as 20mM EDTA or 5mM diisopropyl fluorophosphate (DIFP) or 400 · μg/ml of PIP in reaction buffer indicated that these are not metalloproteases but serine proteases whose activities are inhibited by PIP and DIFP. Since proteases are involved in cell growth regulation and cell transformation, we hypothesize a positive relationship between the field bean protease inhibitor;s blocking action on tumor cell proteases and its tumor suppressing activity     

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm²/mm³. In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.     

Characteristics of chromatin preparations from herpes simplex virus infected cells

Chromatin isolated from herpes simplex virus type 1-infected baby hamster kidney cells contains a number of tightly associated virus-induced polypeptides. A subset of these proteins bind to immobilized DNA in vitro (Vmw 175, 155, 130, 63, 43, 38/39). Virus-induced polypeptides extractable with acid from infected cell chromatin include Vmw 155, the major capsid protein of herpes simplex virus type 1 virions, and Vmw 63 and 38/39 which are heterogeneous with respect to charge and are phosphorylated. These chromatin preparations, in the presence of deoxynucleoside triphosphates and MgCl2 were capable of synthesizing viral and cell DNA in a reaction which was stimulated by the addition of ATP, riboNTPs and potassium acetate. In vitro synthesized viral DNA co-sedimented with prelabelled parental DNA but the single-stranded product was smaller than parental DNA. Density labelling indicated that extensive synthesis was taking place and all BamHI fragments of viral DNA were represented by the DNA synthesized in vitro.     

Distribution of O2 within infected cells of soybean root nodules: a new simulation

Summary A simulation model is presented for the distribution and consumption of O₂ in infected cells of soybean root nodule central tissue. It differs from earlier models in closer adherence to observed structure and embodies new morphometric data about the distribution of > 12,000 mitochondria per cell and about the geometry of the gas-filled intercellular spaces near which the mitochondria are located. The model cell is a rhombic dodecahedron and O₂ enters only through interfaces (totalling 26% of the cell surface) with 24 gas-filled intercellular spaces. These spaces are located at the edges of each rhombic face of the cell, forming an interconnected network over the cell suface. Next, O₂ is distributed through the cytoplasm by a leghaemoglobin-facilitated diffusive process, initially between the mitochondria and amyloplasts in the outer layers of the cell and then between > 6,000 symbiosomes (each containing 6 bacteroids) towards the central nucleus. The symbiosomes and mitochondria consume O₂, but impede its diffusion; all O₂ entering symbiosomes is considered to be consumed there. For the calculations, the cell is considered to consist of 24 structural units, each beneath one of the intercellular spaces, and each is divided into 126 layers, 0.2 m thick, in and through which O₂ is consumed and diffused. Rates of consumption of O₂ and of N₂ fixation in each diffusion layer were calculated from previously-established kinetics of respiration by mitochondria and bacteroids isolated from soybean nodules and from established relationships between bacteroid respiration and N₂ fixation. The effects of varying the O₂-supply concentration and the concentration and type of energy-yielding substrates were included in the simulations. When the model cell was supplied with 0.5 mM malate, mitochondria accounted for a minimum of 50% of the respiration of the model cell and this percentage increased with increased concentration of the O₂ supply. Gradients of concentrations of free O₂ dissolved in the cytoplasm were steepest near the cell surface and in this location respiration by mitochondria appeared to exert a marked protective effect for nitrogen fixation in layers deeper within the cell. Estimates of N₂ fixation per nodule, calculated from the model cell, were similar to those calculated from field measurements.Abbreviations Lb leghaemoglobin - LbO₂ oxyleghaemoglobin - [O₂] concentration of free, dissolved O₂ - e.m. electron micrograph Dedicated to the memory of Professor John G. Torrey     

Elicitor-induced prolyl hydroxylase from French bean (Phaseolus vulgaris). Localization, purification and properties.

The enzyme prolyl hydroxylase (proline: 2-oxoglutarate dioxygenase, EC 1.14.11.12), induced in suspension-cultured cells of Phaseolus vulgaris L. (French bean) by treatment with an elicitor preparation from the phytopathogenic fungus Colletotrichum lindemuthianum, has been investigated. The enzyme, which catalyses the hydroxylation of poly-L-proline with the stoichiometric decarboxylation of 2-oxoglutarate, has been shown to be localized mainly in smooth endoplasmic reticulum. After solubilization from microsomal membranes, the hydroxylase was purified by ion-exchange chromatography and affinity chromatography on poly-L-proline-Sepharose 4B. The subunit Mr, as assessed by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis, was 65 000, the subunit apparently being recovered as a doublet: the subunits associate under non-denaturing conditions to give at least a tetramer. The bean hydroxylase has kinetic properties and cofactor requirements similar to those previously reported for the enzyme from other plants. Elicitor treatment of suspension-cultured bean cells leads to a rapid induction of prolyl hydroxylase activity concomitant with induction of a protein: arabinosyl-transferase and increased levels of an arabinosylated hydroxyproline-rich protein.     

Localization of carbonic anhydrase in legume nodules

Extracts of the central infected zone and the surrounding cortex of nodules from Lupinus angustifolius L., Vigna unguiculata L. (Walp), Pisum sativum L., Phaseolus vulgaris L., Vicia faba L. and Medicago sativa L. contained significant activities of carbonic anhydrase (CA). Immunoassay of extracts using antisera to a putative nodule CA (Msca1) cloned from M. sativa also indicated expression in both tissue types. Quantitative confocal microscopy using laser scanning imaging and a fluorescent CA‐specific probe (5‐dimethylaminonaphthalene‐1‐sulfonamide [DNSA]) localized expression to the infected cells in the central zone tissue and a narrow band of 2–3 files of cells in the cortical tissue that corresponded to the inner cortex. In the infected cells, the enzyme activity was distributed evenly in the cytosol, but in the inner cortical cells, it was restricted to the periphery – possibly to the plasma membrane or cell wall. The functions of CA in these two tissues are considered in relation to the carbon metabolism of nodules and the participation of the inner cortex in the regulation of gaseous diffusive resistance.     

Protein kinase activity of polysome-ribosome preparations from poliovirus infected cells.

Polysome-ribosome preparations from poliovirus infected HeLa cells contain 10–15 fold more protein kinase activity $\text{in vitro}$ than similar preparations from uninfected cells. Five peptides of mol. wt. 135K, 85K, 65K, 28K and 14K are phosphorylated by polysome-ribosome preparations from infected cells. Extracts obtained from cells infected in the presence of guanidine phosphorylate the same proteins whereas polysome-ribosome extracts obtained from uninfected cells treated with cycloheximide phosphorylate the 135K peptide and two others with mol. wt. values of 77K and 60K. Over 90% of the 135K peptide and over 70% of the other peptides can be washed off ribosomes with 0.5 M KCl.     

Cytopathology of soybean tissue culture cells infected with southern bean mosaic virus

Summary Virus distribution patterns and ultrastructural changes in soybean callus cells after infection with the type, or bean strain, of southern bean mosaic virus (SBMV) were observed. Calli grown in liquid Linsmaier and Skoog (LS) medium were inoculated with SBMV and incubated in fresh LS medium. Calli were sampled at 5, 10, 15, and 20 d after inoculation and examined by transmission electron microscopy. Five days after inoculation, viruslike particles (VLP) measuring 22 to 27 nm in d were observed in the cytoplasm. The particles formed loose aggregates with some tendency to associate in regular patterns. By the 10th d after infection, particles were observed in the vacuoles in similar loose arrangements. Viruslike particles were readily identified in vacuoles because of the absence of ribosomes. Crystalline aggregations of VLP were found from Day 10 to Day 20 in the cytoplasm only. Five days after inoculation particles similar to the VLP observed in the cytoplasm also were present in nuclei. Other cytopathic effects were noted, particularly several types of inclusion bodies. These observations differ considerably from reports of the type strain in intact bean plant tissues in the frequent occurrence of VLP in vacuoles and virus crystals in cytoplasm of soybean callus infected with the same strain of virus. Michigan Agricultural Experiment Station Journal Article Number 10020. We thank Dr. Karen Baker for useful suggestions.     

Co-localisation of membranes and actin in growing infected cells of Glycine max root nodules

The root nodule of Glycine max (L.) Merr. is almost spherical at maturity, and its central tissue consists of infected cells filled with numerous symbiosomes containing bacteroids, interspersed with uninfected cells. During the growth of the nodule, the volume of each infected cell and the number of bacteroids per cell increases, and thus abundant membranes are required for the proliferation of symbiosomes. In expanding infected cells, there are areas adjacent to the nucleus that are devoid of bacteroids, but these areas are filled with numerous membranes and actin filaments, surrounded by endoplasmic reticulum membranes, indicating a perinuclear reservoir of newly formed membranes and a role for actin in delivering membranes to proliferating symbiosomes.     

Localization of a new serine protease, ingobsin, in goblet cells in rat, pig and man

Summary A serine protease, ingobsin, that cleaves Lys-x and Arg-x, has been purified from rat duodenal tissue. By immunohistochemical methods, the enzyme was localized in goblet cells in the small intestine of rat, pig, and man. The immunoreactive cells were most numerous in the proximal part of the intestine. In the electron microscope, the immunoreaction was localized mainly to the rough endoplasmic reticulum of the goblet cells and to the secretion being extruded from the cells.     

Localization of viral-specific 21 kDa protein in nucleoli of herpes simplex infected cells

Viral-encoded 21 kDa protein has been localized in herpes simplex virus type 1 (HSV-1) infected cells by immunocytochemical techniques using peroxidase and colloidal gold as markers. During early infection, 21 kDa protein was shown to be in cytoplasmic areas rich in ribosomes located near the nucleus and in the viral DNA-containing fibrillo-granular material of the virus-specific electron-translucent region in the nucleus. Late in infection, an additional marked accumulation occurred in both fibrillar and granular components of the nucleolus. Host chromatin and the nuclear dense bodies remained unlabeled. No immunolabeling was obtained in the absence of DNA replication. In contrast, inhibition of RNA synthesis did not modify the distribution of the protein. On the other hand, persistence of cytoplasmic and nuclear immunolabeling following the inhibition of protein synthesis performed late in infection indicated that the distribution of 21 kDa protein represented, at least in part, sites of accumulation and retention of preexisting molecules.