Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

Structure, biosynthesis, and function of asparagine-linked glycans on plant glycoproteins

In plants, glycoproteins with asparagine-linked glycans (oligosaccharides) are found in vacuoles, in the extracellular space or matrix, and associated with the endo-membrane system (endoplasmic reticulum, Golgi apparatus, plasma membrane, tonoplast). These glycans are of the high-mannose type, with a structure identical to that found in other organisms (mammals, yeast), or of the complex type with a β1–2 linked xylosyl residue not found in mammalian complex glycans. Asparagine-linked glycans play multiple roles by modifying the physicochemical properties of the polypeptides to which they are attached.     

An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco Bright Yellow (BY-2) cells.

In plant systems, the green fluorescent protein (GFP) is increasingly used as a marker to study dynamics of the secretory apparatus using fluorescence microscopy. The purpose of this study was to immunogold localize the GFP, at the electron microscopic level, in a line of tobacco BY-2-cultured cells, expressing a GFP-tagged Golgi glycosyltransferase. To this end we have developed a simple, one-step chemical fixation method that allow good structural preservation and specific labeling with anti-GFP antibodies. Using this method, we have been able to show that an N-glycan GFP-tagged xylosyltransferase is specifically associated with Golgi stacks of BY-2 transformed cells and is preferentially located in medial cisternae. As an alternative to cryofixation methods, such as high-pressure freezing, which requires specialized and expensive equipment not available in most laboratories, this method offers researchers the opportunity to investigate GFP-tagged proteins of the endomembrane system in tobacco BY-2 cells.     

A role for intracellular calcium and calmodulin in the release of triiodothyronine from human thyroid-cell monolayer cultures

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T₃. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intraceliular calcium mobilization, TMB-8, was found to inhibit the release of T₃ in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T₃ secretion by TSH and itself stimulated the release of T₃ to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23197 produced a biphasic effect on T₃ secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T₃ secretion but above a concentration of 1 M, T₃ secretion was increased. The calmodulin antagonist W7 was found to inhibit T₃ release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T₃ secretion.     

Calmodulin activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.     

Isolation, purification and cell-free synthesis of calmodulin from the pig anterior pituitary gland.

Calmodulin was extracted and purified from pig anterior pituitary gland. The protein was characterized by its migration on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of Ca2+ or EGTA, its U.V. spectrum between 240 and 290 nm and the activation of calmodulin-deficient cyclic AMP phosphodiesterase. The yield was 370 mg/kg wet wt. mRNA was also extracted from the same tissue and translated in a wheat-germ cell-free translation system. Translated calmodulin was identified by its heat-stability, its co-migration with authentic anterior-pituitary calmodulin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, its acidic isoelectric point (4.15) on flat-bed isoelectric focusing, its Ca2+-dependent binding to fluphenazine-Sepharose 6B, and its co-elution from this gel with authentic unlabelled calmodulin with EGTA buffer. Calmodulin was not translated as a precursor form. In this tissue it was calculated that calmodulin accounted for 0.5-1% of the total translated protein.     

Silver impregnation methods for reticulum fibers and reticulin: A re-investigation of their origins and specifity

Summary Maresch (1905) introduced Bielschowsky's silver impregnation technic for neurofibrils as a stain for reticulum fibers, but emphasized the nonspecifity of such procedures. This lack of specifity has been confirmed repeatedly. Yet, since the 1920's the definition of reticulin and studies of its distribution were based solely on silver impregnation technics. The chemical mechanism and specifity of this group of stains is obscure. Application of Gomori's and Wilder's methods to human tissues showed variations of staining patterns with the fixatives and technics employed. Besides reticulum fibers, various other tissue structures, e.g. I bands of striated muscle, fibers in nervous tissues, and model substances, e.g. polysaccharides, egg white, gliadin, were also stained. Deposition of silver compounds on reticulum fibers was limited to an easily removable substance; the remaining collagen component did not bind silver. These histochemical studies indicate that silver impregnation technics for reticulum fibers have no chemical significance and cannot be considered as histochemical technics for reticulin or type III collagen.     

Variation in response to cytotoxicity of cigarette smoke.

The cytotoxic effect of cigarette smoke condensate on human polymorphs was investigated in vitro by the method of vital dye exclusion. Exposure to 1/800 of the smoke from one high-tar cigarette killed a detectable proportion of a population of 10(6) cells. The response among the cells from 40 healthy people varied widely, the percentage of dead cells ranging from 3% to 66% and from 17% to 87% at exposure levels of 125 micrograms and 250 micrograms cigarette smoke condensate respectively. Differences in individuals'' responses were reproducible and unrelated to age or sex or smoking habit. The cells from 10 patients with irreversible obstructive airways disease and probable emphysema were significantly more sensitive than those from 10 patients with no respiratory disability matched for age and smoking habits. Genetically influenced variation in cellular response to cytotoxicity may be an important determinant of the risk of developing emphysema among smokers.     

Stimulation of the adenylate cyclase of A B16 melanoma cell line by pro-opiocortin-related peptides--a structure-activity study

The ability of alpha-melanotrophin (alpha-MSH or ACTH 1-acetyl-13 amide) and other structurally related peptides derived from the common precursor, pro-opiocortin, to stimulate adenylate cyclase activity in a pigmented B16 mouse melanoma was investigated. The peptides ACTH 1-39, ACTH 1-24, alpha-MSH, ACTH 1-13 amide and beta-MSH all stimulated the enzyme to a similar maximal extent and with similar potency (ED50 = 1.3 . 10(-6) M) except that ACTH 1-39 was slightly less potent (ED50 = 5 . 10(-6) M). ACTH 4-10 (ED50 = 4 . 10(-5) M) and gamma-MSH (ED50 = 5 . 10(-6) M) were partial agonists. ACTH 1-10 was no more effective than ACTH 4-10 in stimulating the enzyme whereas ACTH 1-13 amide was a full agonist. The peptides beta-endorphin and its derivatives, Met-enkephalin and melanotrophin potentiating factor (MPF), failed to stimulate the enzyme. We suggest that the B16 melanoma requires not only the sequence ACTH 4-10 but also some part of the sequence ACTH 11-13, or a similar sequence in the terminal portion of beta-MSH, for full activation of the receptor-linked enzyme.     

An ultrastructural and autoradiographic study of the immune response in Hyalophora cecropia pupae

Summary Membrane-bounded spherical vesicles found in rat Sertoli cells have been examined quantitatively during the cycle of the seminiferous epithelium. Most of the vesicles were localized to the basal and columnar portions of the Sertoli cell cytoplasm. The thin lateral projections of the Sertoli cells contained very few vesicles. Morphometric analysis of the basal portion of the Sertoli cell cytoplasm revealed that the volume density (V _v ) of the vesicles changed markedly during the cycle. The V _v was at its minimum (0.036) at stage VII and maximum (0.117) at stages XI-I. The vesicles were also smaller at stage VII compared to the vesicles at stages IX-V. The stage-dependent difference in the size of the vesicles was found both in the basal and the columnar portions of the Sertoli cells. At stage VII some of the vesicles appeared to be elongated much like the tubular elements of the smooth endoplasmic reticulum (SER) from which they are probably derived. The stage-dependent differences in volume density and size of the Sertoli cell vesicles may be related to cyclic biochemical variations in the Sertoli cells, and are further indications of a variation in Sertoli cell function during the cycle of the seminiferous epithelium. Whether or not this is due to an internal cycle of the Sertoli cell or to influences from adjacent germ cells remains to be determined.