The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt,Notophthalmus viridescens,during limb regeneration

Summary Previous work has shown that the monoclonal antibody 22/18 identifies progenitor cells (blastemal cells) which depend on the nerve for their division in the early stages of limb regeneration in the newt,Notophthalmus viridescens. This antibody also reacts with cultured cells derived from the newt limb, and the intensity of immunoreactivity appears related to cell density and differentiation into myotubes. We report here that the monoclonal antibody 22/18 recognizes a polypeptide (22/18 antigen) which is intracellular and filamentous. Double staining of cells with 22/18 monoclonal antibody and antibodies against various cytoskeletal components indicates that the epitope is expressed on an intermediate filament component. Although this antibody is specific for blastemal cells in cryostat sections of the regenerating limb, its reactivity on immunoblots is not confined to this tissue. The 22/18 antigen is differentially affected by aldehyde fixatives distinguished by the spacing of their reactive groups. While formaldehyde fixation impairs detection of the antigen, ethylene glycol-bis[succinic acid n-hydroxysuccinimide ester] reveals the antigen in sections of normal and regenerating limbs in a distribution that is consistent with the one obtained from immunoblots. We suggest that the 22/18 monoclonal antibody detects a change in protein conformation, probably related to changes in the physiological state of the cell, that occurs transiently during regeneration and possibly during development.     

De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines

Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed.     

Metabolic reduction of chromium,as related to its carcinogenic properties

At variance with Cr(III), Cr(VI) compounds easily cross cell membranes and exert genotoxic effects. No metabolic oxidation of Cr(III) could be detected, whereas Cr(VI) reduction was observed in the presence of body fluids and subcellular fractions of various tissues from several animal species. The differential efficiency of this process may account for the selection of target tissues in Cr(VI) carcinogenesis. For instance, reduction by saliva and gastric juice may explain a lack of carcinogenicity by the oral route; reduction inside erythrocytes may explain a lack of carcinogenicity at a distance from administration sites; reduction by the epithelial-lining fluid of terminal airways and by alveolar macrophages may be consistent with the occurrence of thresholds in lung carcinogenesis. Liver preparations displayed the top efficiency in reducing Cr(VI), whereas skeletal muscle, i.e., a typical target in experimental Cr(VI) carcinogenesis, had no detectable activity. Bronchial tree and peripheral lung parenchyma preparations from almost 100 individuals reduced Cr(VI) to a variable extent. The efficiency of lung parenchyma and of isolated alveolar macrophages was enhanced in cigarette smokers. In rats, Cr(VI) reduction by lung preparations was significantly stimulated by the repeated i.t. instillation of Cr(VI) itself. Among the electron donors (chiefly GSH) and enzymatic mechanisms responsible for the intracellular Cr(VI) reduction, such as cytochrome P-450 reductase, glutathione redactase, and aldehyde oxidase, an important role can be ascribed to cytosolic DT diaphorase activity, usually catalyzing a 2-electron reduction.     

Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1

The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.     

Polyamine oxidase activity and polyamine content in maize during seed germination

Polyamine oxidase (PAO, EC 1.5.3.3) activity and polyamine content in the cell wall and soluble fractions obtained from embryos, endosperms and shoots and roots of etiolated or green seedlings of maize ( Zea mays L. cv. WF9) during the first 7 days of germination were investigated. Polyamine content was also determined in the trichloroacetic acid-soluble (free polyamines) and trichloroacetic acid insoluble (bound polyamines) fraction obtained from the same tissues. PAO activity, determined by the radiometric method based on the recovery of the labelled reaction product 1-pyrroline, was mostly localized in the cell wall fraction. The activity was very low in embryos and endosperms and present in traces in roots. In etiolated shoots PAO activity increased sharply, while in green shoots it was low and increased slowly. No polyamines were found in the cell wall fraction and only putrescine was detected in the soluble fraction, with the exception of the embryo, where spermidine and spermine were also present. In the TCA-soluble fraction of embryos, putrescine increased during imbibition, while spermidine and spermine decreased; in the endosperm no relevant changes in polyamines occurred. In the same fraction of green and etiolated seedlings, putrescine increased, giving a peak at days 3–5, while spermidine decreased to very low levels. The amount of bound polyamines was 1–4% of the free ones. The pattern of PAO activity seems to be unrelated to endogenous free polyamine content, which is the same in shoots and roots of etiolated and green seedlings. Enzyme activity, very low in ungerminated seeds, increased continuously during the progression of germination, especially in etiolated shoots, indicating a possible involvement in cell wall formation.     

Development of tool use in a macaque and a gorilla

The development of the capacity to use a stick as a tool was tested in a macaque (Macaca fuscata) and a gorilla (Gorilla gorilla gorilla) infants that had previously shown to be able to use strings and supports as dragging tools. Subjects were tested between 15 and 38 months of age. Different levels of competence between the subjects emerged over testing. The macaque developed a stereotyped strategy to cope with the problem, only getting random successes, whilst the gorilla developed a flexible strategy and revealed to be able to mentally represent the solution of the problem. In fact, when not successful using the stick, the gorilla thought out an alternative strategy, choosing and adapting a new object to use it as a tool.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.