Streptococcus iniae expresses a cell surface non-immune trout immunoglobulin-binding factor when grown in normal trout serum

Three capsulated isolates of S. iniae representing serotype I and II and being arginine dihydrolase positive, negative or variable (AD+ve, AD-ve, AD+-ve) were investigated for their ability to bind rainbow trout serum immunoglobulin by the Fc region. Using a coagglutination assay with bacteria grown in Todd-Hewitt broth (THB), no evidence of non-specific Fc-binding of trout immunoglobulin (Ig) was obtained. However, when grown in normal trout serum, all isolates produced similar protein patterns in SDS-PAGE, but they were markedly different from the patterns of the bacteria grown in THB. Some bands with MW 70 kDa and over 100 kDa were very intense in the profiles of the serum-grown isolates. In Western blots, these bands of all isolates were immunostained with the conjugated goat antiserum to trout Ig, after blocking with normal goat serum, demonstrating that the bacteria had bound the trout Ig during growth in the serum. When the isolates were grown overnight in trout antiserum against Lactococcus garvieae they coagglutinated with L. garvieae cells but S. iniae isolates grown in normal trout serum did not. These data indicate that S. iniae grown in serum express surface factors which can bind trout Ig by the Fc-region.     

Interaction of cblA/adhesin-positive Burkholderia cepacia with squamous epithelium

A highly transmissible strain of Burkholderia cepacia from genomovar III carries the cable pilin gene, expresses the 22 kDa adhesin (cblA +ve/Adh +ve), binds to cytokeratin 13 (CK13) and is invasive. CK13 is expressed abundantly in the airway epithelia of cystic fibrosis (CF) patients. We have now investigated whether binding of cblA +ve/Adh +ve B. cepacia to CK13 potentiates bacterial invasion and epithelial damage using bronchial epithelial cell cultures differentiated into either squamous (CK13-enriched) or mucociliary (CK13-deficient) epithelia. Three different B. cepacia isolates (cblA +ve/Adh +ve, cblA +ve/Adh -ve and cblA -ve/Adh -ve) showed minimal binding to mucociliary cultures, and did not invade or cause cell damage. In contrast, the cblA +ve/Adh +ve isolate, but not others, bound to CK13-expressing cells in squamous cultures, caused cytotoxicity and stimulated IL-8 release within 2 h. By 24 h, this isolate invaded and migrated across the squamous culture, causing moderate to severe epithelial damage. A specific antiadhesin antibody, which blocked the initial binding of the cblA +ve/Adh +ve isolate to CK13, significantly inhibited all the pathologic effects. Transmission electron microscopy of squamous cultures incubated with the cblA +ve/Adh +ve isolate, revealed bacteria on the surface surrounded by filopodia by 2 h, and within the cells in membrane-bound vesicles by 24 h. Bacteria were also observed free in the cytoplasm, surrounded by intermediate filaments containing CK13. These findings suggest that binding of B. cepacia to CK13 is an important initial event and that it promotes bacterial invasion and epithelial damage.     

Surface antigens of Proteus mirabilis revealed by electroblotting from sodium dodecyl sulphate-polyacrylamide gels

Western Blotting of whole cell preparations of three strains of Proteus mirabilis after separation by electrophoresis on SDS-polyacrylamide gels revealed a complex pattern of antigens. Similar antigen profiles were obtained with isolated outer membranes indicating that the majority of cell surface antigens are located in the outer membrane. Major outer membrane proteins were strongly antigenic and cross-reactive. The highly immunogenic flagella were detected in whole cell preparations and visible in isolated outer membranes. Whereas the protein and flagellar antigens were cross-reactive, lipopolysaccharide (LPS) could only be detected as immunoreactive material using homologous antisera for each strain. The LPS appeared as two broad bands (high and low Mr, respectively) in immunoblots of whole cells, isolated outer membranes and purified LPS. However, isolated LPS could be resolved into multiple sharp bands when 4 M urea was included in the gel system. These discrete bands are assumed to represent differing O antigen chain lengths of the LPS as reported for other Gram-negative organisms.     

Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95.     

Localization of fungal fimbriae by immunocytochemistry in pathogenic and nonpathogenic isolates of Venturia inaequalis

Pathogenic and nonpathogenic isolates of Venturia inaequalis were grown in liquid culture. Hyphae were treated with two types of fimbrial antiserum (AU- and AV-1) and examined by immunofluorescent microscopy, in order to establish the distribution of fimbrial epitopes in whole cell mounts. The AV-1 antiserum was specific for the glycoprotein subunits while the AU-antiserum was specific for the protein moieties present on the fimbriae of Mycobotryum violaceum. The use of fimbrial antiserum with immunocytochemistry and transmission electron microscopy demonstrated a clear distinction between pathogenic and nonpathogenic isolates of V. inaequalis, based on the appearance of the fungal cell wall and the distribution of fimbrial epitopes labeled with AV-1 antiserum and immunogold complex. In actively growing hyphae of the pathogenic isolate, characterized by distinct cellular organelles, small vacuoles, and lipid bodies, fimbrial epitopes were concentrated in the fungal cell wall and were present minimally on the outer surface. In contrast, actively growing hyphae of the nonpathogenic isolate of V. inaequalis had extensive fine hair-like protrusions in the fungal cell wall which labeled with the AV-1 antiserum and immunogold. The distribution of fimbrial epitopes in V. inaequalis was highly dependent on the developmental growth stage of the fungal mycelium. Aging mycelia in both the pathogenic and nonpathogenic isolates of V. inaequalis were characterized by a large central vacuole and no label. In the pathogenic and nonpathogenic isolates of V. inaequalis grown in vitro, the distribution of fimbrial glycoprotein epitopes provided a more complex profile than that seen in M. violaceum.     

The influence of dexamethasone treatment on the lymphoid and stromal composition of the mouse thymus: a flowcytometric and immunohistological analysis

The effect of injection of a range of doses of dexamethasone on the distribution of T-cell subpopulations and stromal cells in the thymus of BALB/c mice was investigated with flowcytometry and immunohistology. To this purpose we used monoclonal antibodies directed to the T-cell differentiation antigens Thy-1, T200, Lyt-1, Lyt-2, T4, MEL-14, and monoclonal antibodies directed to various classes of stromal cells. Injection of dexamethasone in increasing doses of 5-130 mg/kg body weight gradually leads to a depletion of the cortical thymocyte population, i.e., bright Thy-1 + ve, dull T-200 + ve, bright Lyt-2 + ve, and bright T4 + ve cells. These cortical cells are very dull MEL-14 + and express variable numbers of Lyt-1 molecules. Also the medulla is affected by dexamethasone although to a lesser extent. Dexamethasone injection at 130 mg/kg selects for a dull Thy-1 + ve, bright T-200 + ve, and bright Lyt-1 + ve medullary population. These cells are either T4 + ve Lyt-2-ve or T4-ve Lyt-2 + ve. Under these conditions, MEL-14 + ve cells were no longer present in the cortex but accumulated in medullary perivascular spaces. Staining of sequential sections showed that this particular subpopulation has a typical "helper" phenotype. This observation provides strong evidence that perivascular compartments are an exit pathway for emigrating T cells. The medullary population contains a phenotypically distinct, dexamethasone-sensitive subpopulation. This conclusion is based on two findings: 130 mg/kg dexamethasone depletes the thymus of all but 4% of the thymocytes, which form a much smaller subpopulation than the population of dull Thy-1 + ve cells (amounting to 15% of the total thymocytes). The medulla contains a subpopulation of dull Lyt-2 + ve cells, which are resistant to 20 mg/kg dexamethasone, but depleted by 130 mg/kg. Dexamethasone also has a severe effect on thymic nonlymphoid cells. Even at low doses, dexamethasone induces TR4 + ve cortical epithelial-reticular cells to become spherical ("nurse cell-like") structures, depleted of lymphoid cells. These stromal cells no longer express MHC antigens in a membrane-bound fashion. In contrast, the medullary epithelial cells appear morphologically unaffected even at a dexamethasone dose of 130 mg/kg.     

A histone 1-like antigen is a component of the nuclear envelope

Rabbit antibodies have been raised against rat liver nuclear envelopes. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titer antiserum specific for the nuclear envelope preparation. Immunocytochemical studies showed that the antiserum stained the nuclear envelopes, but not intra-nuclear components of HEp-2 (human malignant epithelial) cells. When electrophoretically separated peptides were tested by immunoblotting techniques, the rabbit antiserum specifically stained proteins with molecular masses of 26 and 28 kD. These peptides had similar mobilities to purified histone 1 (H1). Indeed purified calf thymus H1 recognized the antiserum. The antigens are not loosely bound to the nuclear envelope, as they could not be extracted with low salt. Therefore, we have established that the 26 and 28 kD nuclear envelope peptides are not contaminants of the nuclear envelope preparation and that they express determinants that are immunologically cross-reactive with purified H1, but not with intra-nuclear H1.     

Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni

The outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections.     

Immediate and transient inhibition of the respiration of Escherichia coli under hyperosmotic shock

Abstract A range of isolates of Serpula lacrymans show identical molecular profiles by Western Blotting or lectin staining. One isolate, BF-050, showed differences in silver stained protein profiles from old mycelium but profiles from young mycelium of BF-050 were identical to the type strain of S. lacrymans FPRL 12C. Both S. lacrymans and the related organism, Serpula himantoides , shared different profiles for old and young mycelium confirming that growth phase antigens/proteins are not limited to S. lacrymans . Lectin staining profiles could distinguish between S. lacrymans FPRL 12C, BF-015B, S. himantoides and all other basidiomycete organisms tested. Differences between strains of S. lacrymans including BF-050, were not apparent.     

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

Esterase from the oil-degrading Acinetobacter lwoffii RAG-1: Sequence analysis and over-expression in Escherichia coli

Abstract The antigenic properties of the surface layer (S-layer) proteins of various Campylobacter rectus strains including 24 clinical isolates and the type strain ATCC 33238 were examined. S-layer proteins were extracted from whole cells by acid treatment according to the method of McCoy et al. (Infect. Immun. 11, 517–525, 1975). The acid extracts from 23 of the isolates and ATCC 33238 contained two major proteins with molecular masses of 130 kDa and 150 kDa, both of which were identified as subunits of the S-layer after comparison with the protein profiles of acid-treated (S-layer-deficient) cells. An S-layer protein from one isolate (CI-808) demonstrated a different molecular mass (160 kDa). Both the 150-kDa proteins of ATCC 33238 and isolate CI-306 and the 160-kDa protein of CI-808 were purified by ion-exchange chromatography in the presence of urea. In Ouchterlony immunodiffusion experiments with these purified proteins and rabbit antiserum raised to each purified protein, both common and strain-specific antigenic determinants were identified in the C. rectus S-layer proteins.     

Human thymus-leukemia related antigen(s): detection by a nonhuman primate antiserum

A nonhuman primate antiserum to human thymus cells can detect an antigen which is specific for human thymus cells. In addition, this antiserum can also detect antigens which are cross-reactive between thymocytes and cells from patients with lymphocytic leukemia and perhaps some antigens which are present on actively dividing cell populations like cultured lymphoid cell lines derived from normal individuals. The relevance of these data to other human leukemia-associated antigens and murine TL antigens is discussed.     

Conservation of diagnostic antigen epitopes among biologically diverse isolates of Trichinella spiralis

Crude and immunoaffinity-purified excretory-secretory antigens derived from a domestic pig isolate of Trichinella spiralis were used in an enzyme-linked immunosorbent assay to test serum from mice infected with 25 different pig and wild animal isolates of T. spiralis sspp. All of the sera were found positive by ELISA using either of the antigen preparations, indicating all isolates shared certain antigen epitopes. Excretory-secretory antigens were prepared from 3 distinct isolates of T. spiralis sspp.--Trichinella spiralis spiralis (pig isolate), Trichinella spiralis nativa (polar bear isolate), and Trichinella spiralis pseudospiralis--and compared by electrophoresis and monoclonal antibody binding. While protein profiles varied among the isolates, a monoclonal antibody recognizing a major immunodiagnostic antigen epitope bound all 3 antigen preparations. However, this antigen epitope occurred on different molecular weight excretory-secretory proteins from the different isolates.     

A2B5+/GFAP+ Cells of Rat Spinal Cord Share a Similar Lipid Profile with Progenitor Cells: A Comparative Lipidomic Study

The central nervous system (CNS) harbors multiple glial fibrillary acidic protein (GFAP) expressing cell types. In addition to the most abundant cell type of the CNS, the astrocytes, various stem cells and progenitor cells also contain GFAP+ populations. Here, in order to distinguish between two types of GFAP expressing cells with or without the expression of the A2B5 antigens, we performed lipidomic analyses on A2B5+/GFAP+ and A2B5?/GFAP+ cells from rat spinal cord. First, A2B5+/GFAP? progenitors were exposed to the leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP) to induce their differentiation to A2B5+/GFAP+ cells or A2B5?/GFAP+ astrocytes, respectively. The cells were then analyzed for changes in their phospholipid, sphingolipid or acyl chain profiles by mass spectrometry and gas chromatography. Compared to A2B5+/GFAP? progenitors, A2B5?/GFAP+ astrocytes contained higher amounts of ether phospholipids (especially the species containing arachidonic acid) and sphingomyelin, which may indicate characteristics of cellular differentiation and inability for multipotency. In comparison, principal component analyses revealed that the lipid composition of A2B5+/GFAP+ cells retained many of the characteristics of A2B5+/GFAP? progenitors, but their lipid profile was different from that of A2B5?/GFAP+ astrocytes. Thus, our study demonstrated that two GFAP+ cell populations have distinct lipid profiles with the A2B5+/GFAP+ cells sharing a phospholipid profile with progenitors rather than astrocytes. The progenitor cells may require regulated low levels of lipids known to mediate signaling functions in differentiated cells, and the precursor lipid profiles may serve as one measure of the differentiation capacity of a cell population.     

Detection of cross-reactive antigens shared byFusarium oxysporum andGlycine max by indirect ELISA and their cellular location in root tissues

Pathogenicity test ofFusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium ofF. oxysporum. Polyclonal antisera were raised against the mycelial suspension ofF. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.     

Isolation and purification of Brucella antigens synthesized by Escherichia coli K12 cells]

The homogeneous preparations of the brucella protein antigens were isolated from the hybrid producer strains Escherichia coli 6SE579 and 6SE800 by the cold osmotic shock technique and further purification on immunosorbents. The 18 + 38 and 38 kDa antigens were obtained. The antiserum specific to brucella 38 kDa antigen was obtained and used for isolation of the 18 kDa antigen from the producer strain 6SE579 synthesizing two brucella antigens. The immunosorbent developed on the basis of BrCn-agarose conjugated with antibodies from the serum has permitted isolation of 18 kDa protein antigen preparation. Thus, the combined technique of cold osmotic shock and affinity chromatography on immunosorbents permits one to isolate highly purified individual antigens of brucella from Escherichia coli K12 producer cells.     

Characterisation of Bacteroides from sheep periodontal disease by SDS-PAGE of outer membrane proteins

Isolates of Bacteroides species obtained from a longitudinal study of developing periodontal disease in sheep were analysed by SDS-PAGE. Protein profiles of Sarkosyl-insoluble outer membrane extracts were compared within groups of isolates which had already been defined by conventional biochemical techniques. Heterogeneity was exhibited within most groups. Isolates of B. gingivalis and B. asaccharolyticus shown to be similar to human isolates by conventional biochemical tests, gave different protein profiles from the respective type cultures. The sheep B. gingivalis-like isolates were however homogeneous, while the B. asaccharolyticus-like organisms could be divided into 3 subgroups. SDS-PAGE appears to be a useful tool for the examination of bacterial flora and recognition of subgroups of subspecies.     

Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus

The preparation of antisera to the three purified sodium dodecyl sulfate (SDS)-treated polypeptide components (VP1, VP2, VP3) of adenovirus-associated virus (AAV) type 3H is described. In immunofluorescence tests (FA), these antisera stained heat-stable antigens with distinct morphologies in cells co-infected with either adenovirus or herpes simplex virus. Kinetic studies of antigen formation showed that VP1 antiserum first stained the cytoplasm (14 hr) and later (by 18 hr) stained both cytoplasmic and intranuclear areas. VP2 antiserum stained only discrete intranuclear areas, and VP3 antiserum stained nearly the entire nucleus. All three VP antigens appeared at about the 14th hr postinfection, about 2 hr prior to the appearance of whole virion antigen. The VP antisera cross-reacted in FA with AAV types 1 and 2 (all at one-eighth of the homologous titer), but did not react with other parvoviruses, i.e., rat virus, hemadsorbing enteric virus of calves, minute virus of mice, or H-1 virus. These non-neutralizing antisera reacted specifically with SDS-treated AAV virion antigens in complement fixation and immunodiffusion tests, and antiserum prepared against SDS-treated helper adenovirus structural polypeptides reacted with adenovirus polypeptide antigens. All antisera to SDS-treated polypeptides were specific for new antigens revealed on the dissociated peptides and did not react with whole virions, whereas whole-virion antisera did not cross-react with the polypeptide antigens. These findings suggest that antigens unique to the polypeptides of AAV are revealed by SDS treatment and that these antigens can be detected in cells prior to the folding of the polypeptides into the molecular configuration they possess as virion subunits. These results also indicate that at least one AAV polypeptide component is synthesized in the cell cytoplasm.