A histological and histochemical study of the cotyledons ofPhaseolus vulgaris L. during germination

Summary The cotyledon ofPhaseolus vulgaris L. comprises four tissues: epidermis, abaxial hypodermis, storage parenchyma, and procambium. A complex intercellular space system is present throughout the storage tissue and comprises about 16% of the cotyledon volume. All the cells contain protein bodies, and the hypodermis and storage parenchyma also contain starch grains. The epidermal cells are at the 2 C level of DNA, those of the hypodermis at the 4 C level, and the storage cells vary from 8 C to 32 C. During germination stomata differentiate in the epidermis. Reserve mobilization begins in the cells furthest from the epidermis and from the vascular tissue. Protein is removed from these cells with little or no coalescence of protein bodies. The DNA content of the nuclei decreases. The cell walls swell and then decrease in thickness as material is removed. Finally the nuclei and cytoplasm disappear and the cells collapse. In the cells near vascular bundles the protein bodies coalesce before losing their protein. The DNA content of the nuclei declines but nuclei and cytoplasm are still present at abscission. These cells do not collapse. Cytoplasmic RNA content is highest near the abaxial surface. Most of the RNA is removed during the first three days of germination.     

Histology and histochemistry of the cotyledons of pisum arvense L. during germination

Summary The mature cotyledon of Pisum arvense L. comprises several distinct tissue regions; these are the epidermis, hypodermis, storage parenchyma and procambium. The storage parenchyma includes two zones: an outer abaxial zone and an inner adaxial zone. The cells of both zones contain abundant starch grains and protein bodies. Scattered through the storage tissue but increasing in frequency towards the periphery are certain cells which differ to a slight extent from the majority of the parenchyma cells. They have a more opaque, granular cytoplasm and a higher level of cytoplasmic RNA. the cotyledon has a complex, reticulate vascular system. Differentiation of the conducting elements from the procambium appears to begin about 12 hours and to be completed 48 hours after the commencement of imbibition. Differentiation of phloem preceeds that of xylem. The relationship between the timing of vascular differentiation and various physiological events in the cotyledon is discussed.Mobilization of the reserves in the storage parenchyma is initiated at the periphery of the cotyledon and proceeds inwards. There appears to be a correlation between the breakdown of the reserves and changes in DNA and RNA content of the cells.     

Changes in the activity of trout lysosomal enzymes during fasting

Activity of lysosome enzymes (acidic phosphatase, beta-glucosidase, DNAase, RNAase and cathepsin D) is determined for its variation in different organs of rainbow trout during complete fasting. It is shown that the activity of most enzymes of concern almost in all organs except skeletal muscles is on the higher level in trouts fasted for 30 days than in the control ones. With an increase of the fasting term to 60 days the acid phosphatase, DNAase, RNAase activity decreases while the glucosidase and cathepsin D activity in some organs increases. Variations detected in the enzyme profile of the trout lysosomes under fasting are of adaptive character.     

Nuclear Changes in the Cotyledons of Pisum arvense L. during Germination

In the cells of the cotyledons of Pisum arvense L. there isa close correlation between cell volume, nuclear volume, andnuclear DNA level. The cells of the epidermis are at the 2Clevel of DNA, those of the hypodermus vary from 2C to 4C, whilethose of the storage tissues range from 4C to 16C. During germinationthe nucler increase in size In the storage tissues there isa three to fourfold increase, which is accompanied by an increasein lobing of the nucler Simultaneously the DNA level of thenucler decreases by about 50 per cent. There are parallel changesin nuclear histone levels. Initially nuclear RNA level is lowbut in any given cell it increases to a maximum at the timethe storage reserves of the cell begin to disappear, after whichit declines. The level of cytoplasmic RNA is high initiallyin the tissues at the abaxial side of the cotyledon. Duringthe first few days of germination it declines until the over-alllevel is uniformly low, after which there is a further smalldecline.     

QUANTITATIVE LYSOSOMAL ENZYME ACTIVITY CHANGES IN THE NEURAL LOBE OF THE RAT FOLLOWING WATER DEPRIVATION AND LACTATION

—The activities of five lysosomal enzymes, acid phosphatase, β-glucosidase, β-glucuronidase, β-galactosidase and N-arylamidase (classified according to Marks (1970)) were measured by means of sensitive microchemical techniques in frozen-dried rat neural lobe tissue after experimental and physiological stimulation of hormone release from the hypothalamo–neurohypophysial system i.e. water deprivation (3 and 6 days), delivery and lactation (10 days). During all conditions of stimulation increases of 29 to 106 per cent were measured for lysosomal enzyme activity, expressed as mmol/ng DNA/h. With histochemical staining methods the acid phosphatase activity appears to be mainly localized in the pituicytes, but it was impossible to visualize the microchemically measured acid phosphatase activity increase within the two main compartments of the neurohypophysis, i.e. axonal endings and the neurohypophysial glial cells, the pituicytes.     

Polyamine synthesis during lymphocyte activation : Induction of ornithine decarboxylase and S-adenosyl methionine decarboxylase

Lymphocyte stimulation by phytohaemagglutinin (PHA) is accompanied by marked increases in the activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase, two key enzymes for the synthesis of polyamines. Both enzymes increase in a biphasic manner, with the rises in S-adenosyl methionine decarboxylase preceding the increases in ornithine decarboxylase. The initial rises precede the initiation of DNA synthesis, and seem to correlate with the increased rate of ribosomal RNA synthesis. Selective inhibition of ribosomal RNA synthesis inhibits the increases in the activity of both enzymes, especially ornithine decarboxylase, more than the increase in the overall rate of protein synthesis.Both enzymes are metabolically unstable and have half-lives of less than 1 h, although the half-life of ornithine decarboxylase depends on the amino acid concentration in the culture medium. While effects of PHA on the stability of the enzymes have not been ruled out, at least part of the PHA-dependent increases in activity are due to increased synthesis or activation of the enzymes. The synthesis of S-adenosyl-methionine decarboxylase declines rapidly after inhibition of RNA synthesis, but ornithine decarboxylase activity declines at about the same rate as protein synthesis as a whole.The activities of both enzymes also increase during lymphocyte stimulation by concanavalin A, lentil extract and staphylococcal filtrate.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.     

Gibberellin-like Substances in Developing Barley Grain and Their Relation to Dry Weight Increase

Gibberellin-like activity of two varieties of barley (Hordeum vulgare L.) at different stages of grain development was determined by barley endosperm bioassay (acid phosphatase bioassay). Activity of the acidic ethyl-acetate fraction (“free” GAs) in both varieties displayed two maxima, a first maximum at the 9th day and a second one 20 to 21 days after pollination. Activity of the n-butanol fraction (“bound” GAs) first dropped to a minimum level at the 9th day, then increased to reach a maximum 32 days after pollination, and finally decreased again towards maturity. From the 9th day after pollination, a conversion of “free” GAs to “bound” GAs has probably occurred. From the 12th day after pollination, the curves of rate of dry weight increase and of “free” GAs run nearly parallel, but the latter reached its maximum about 2–4 days earlier than the former. The results indicate that gibberellins may participate in the regulation of the accumulation processes into the barley grain.     

The effect of heat hardening on the heat resistance of some enzymes from plant leaves

Summary Heat hardening of leaves which leads to an increase in the heat resistance of their cells, also increases the heat resistance of their enzymes (urease, acid phosphatase, ATPase). As judged by the temperature reducing enzyme activity by 50%, the heat resistance increased by about 6° and 4°, respectively, for urease and acid phosphatase of cucumber, about 7° for acid phosphatase of wheat, and 1,5° for ATPase of Caragana. Increased heat resistance of acid phosphatase and ATPase caused by heat hardening was accompanied by a decrease in the activity ofthese enzymes. The activity of urease was not affected by heat hardening. It is assumed that the cause of this increase in thermal resistance of enzymes is a stabilization of protein macromolecules during heat hardening of leaves.     

Cytochemical Observations on Proteins,Alkaline and Acid Phosphatases,Adenosine Triphosphatase,and 5′-Nucleotidase in Chick Liver Cell Cultures Infected with Trichomonas vaginalis*, †

SYNOPSIS. By means of the ninhydrin-Schiff method for proteins a diffuse reaction as well as one localized in granular inclusions can be shown in the cytoplasm of fibroblasts, epithelial cells, and macrophages in trypsin-dispersed chick liver cell cultures. Nuclei and nucleoli also take the specific stain. A progressive loss of cytoplasmic and nuclear staining occurs in the fibroblasts in cultures infected with a relatively pathogenic strain of T. vaginalis. A loss occurs in epithelial cells in advanced stages of degeneration, but in less damaged cells, while the diffuse reaction disappears, the number and staining intensity of the cytoplasmic inclusions remain unchanged or possibly may increase somewhat. The intensity of the diffuse reaction and the number and size of the characteristic inclusions increase in the active, parasite-free, experimental macrophages, but phagocytes with trichomonads closely applied to their external surfaces and those containing the flagellates within their cytoplasm typically retain only a few weak-staining inclusions. Similar distribution of alkaline and acid phosphatases occurs in preparations treated according to Gomori's and Burstone's methods, except that no nuclear staining is obtained with the latter. Activity of both enzymes is localized primarily in inclusions which are dispersed thruout the cytoplasm of fibroblasts and epithelial cells and tend to accumulate along the cell membranes and around the nuclei. In the course of infection with T. vaginalis there is a progressive loss of alkaline phosphatase from both cell types; however, the acid phosphatase activity increases. In the control macrophages both enzymes are localized in mostly rather large, rounded cytoplasmic inclusions. The number of such inclusions increases in the parasite-free experimental macrophages, but only a few weak-staining granules remainin phagocytes with engulfed trichomonads and in those whose external surfaces are in direct contact with the parasites. The loss of the inclusions is less apparent in macrophages containing degenerated flagellates than in the ones with healthy trichomonads, but regardless of the condition of the parasites, the highest enzymatic activity is found around them. ATPase and 5′-nucleotidase are localized in small granules dispersed thruout the cytoplasm of fibroblasts and epithelial cells. The granules tend to accumulate along the periphery of the cells and around the nuclei. A diffuse cytoplasmic reaction is present in preparations processed for 5′-nucleotidase. Nuclei and nucleoli give positive reactions for both enzymes. In the course of infection with trichomonads, activity of the 2 enzymes declines in both culture cell types. Control macrophages have diffuse cytoplasmic reaction for ATPase and 5′-nucleotidase and these enzymes are localized also in rounded cytoplasmic inclusions. Activity of both enzymes increases in the parasite-free experimental phagocytes, but little if any diffuse staining and only a few characteristic inclusions are left in macrophages with engulfed healthy trichomonads and in those whose external surfaces are invested with the flagellates. The ninhydrin-Schiff-positive inclusions found in the macrophages appear to be the same as some of those which have acid phosphatase activity and may well be identical with the glycolipoprotein bodies noted by us previously. On the grounds of their chemical constitution and behavior it seems likely that the inclusions are lysosomes.     

Anesthesia and the golgi apparatus: enzyme activity changes in a golgi-rich fraction of rat liver after a short ether anesthesia.

Activity changes of enzymes in isolated rat liver Golgi preparations at different times (1-48 h) after a short ether anesthesia are reported. Activity of galactosyl-transferase showed a slight gradual increase but thiamine pyrophosphatase decreased sharply, and after 24 h increased to above control level. Arylsulphatase-A remained largely unchanged, and B was significantly decreased. Acid phosphatase activity did remain at the control level, but alkali phosphatase showed a gradual and highly significant increase. Five other enzymes representing probable contaminations from other subcellular organelles, have also been assayed. Correlation is sought between the enzyme activity changes and some other metabolic effects of anesthesia.     

EMBRYO STRUCTURE AND STORAGE RESERVE HISTOCHEMISTRY IN THE PALM WASHINGTONIA FILIFERA

The seed of Washingtonia filifera (Lindl.) Wendl. is hemispherical and has a smooth testa. The embryo is located on the rounded side of the seed near the raphe. The embryo consists of a prominent single cotyledon, an epicotyl, and a small root apex. The shoot apex is oriented at a right angle to the long axis of the embryo and possesses 2 to 3 leaf primordia. The cotyledon functions as a storage organ and is composed of three cell types with similar ultrastructure. These three types—the parenchyma, protoderm, and procambium—can be distinguished on the basis of position, size, and shape. The procambial strands in the cotyledon consist of a ring of bundles grouped into two distinct sympodia and extend from beneath the shoot apical meristem to the tip of the cotyledon where they are situated very close to the surface. The most prominent organelles within all cell types are protein bodies, lipid bodies, and crystalline protein fibers. The protein bodies contain small crystalline inclusions which are presumed to be phytin. Protein bodies in the protoderm were smaller, denser-staining, and contained fewer crystalline inclusions than those in the parenchyma or procambium. On a volume basis, the parenchyma was shown to be 43% protein bodies, 25% lipid bodies, 15% cytoplasm, 7% cell wall, 4% intercellular space, 2% nuclei, and 4% other organelles (mitochondria and plastids).     

Fine structure,pattern of division,and course of wound phloem in Coleus blumei

The wound phloem bridges which have developed six days after interrupting an internodal vascular bundle contain wound sieve-elements, companion cells, and phloem parenchyma cells. An analysis of the meristematic activity responding to the wounding clearly demonstrates that three consecutive divisions are prerequisite to the formation of phloem mother-cells. Companion cells are obligatory sister cells of wound sieve-elements, connected to the latter by specific plasmatic strands and provided with a dense protoplast. Six days after wounding most of the wound sieve-elements are still at a nucleate state of development, but already have characteristic P-protein bodies and plastids containing sieve-element starch. Their cytoplasmic differentiation corresponds to the changes recorded during maturation of ordinary sieve elements. Sieve-plate pores penetrate through preexisting parenchyma cell walls, only, and develop from primary pitfield-plasmodesmata. Wound sieve-elements do not connect to preexisting bundle sieve-elements, they open a new tier of young sieve elements produced by cambial activity.     

Histochemical and biochemical studies on the localization and activity of lysosomal enzymes in fracture callus

Summary Quantitative biochemical studies on the activities of four lysosomal hydrolases during different stages of fracture healing in the rat were performed, and the results obtained were integrated with those of histochemical observations relating to changes in the localization of acid phosphatase in the same tissue.The findings showed presence of all the four lysosomal enzymes assayed in the callus; during early callus formation the enzyme activities calculated on a DNA basis increased up to about 12 days after the fracture. The enzyme activities appeared to be roughly reflected histochemically by the acid phosphatase staining. The increasing activity during early callus formation seemed to depend on the presence of numerous macrophage-like cells in the tissue containing many large lysosomes. A decrease in enzyme activity was found after day 12. Comparison with the histochemical and ultrastructural findings suggested that this decrease was due to a reduction in the number of macrophage-like cells and a concomitant increase in osteogenic cells with a lower enzyme content.     

Activity of enzymes of arginine metabolism in the cotyledons of developing and germinating pea seeds

Ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and arginase activity were measured in extracts from cotyledons of developing and germinating seeds of Pisum sativum L. The course of activity of these four urea cycle enzymes showed a similar pattern during seed development. The activity per cotyledon increased sharply initially and reached a maximum about 5 weeks after anthesis, when the relative water content of the seeds was about 60%. About 8 weeks after anthesis, the seeds were mature (air-dry) and had enzyme activities which were much lower. The activities of the enzymes differed considerably. Ornithine carbamoyltransferase showed the highest activity, followed in order of decreasing activity by arginase, argininosuccinate lyase, and finally argininosuccinate synthetase.

The course of the activity of the four enzymes was different during germination. Arginase activity increased sharply 7 hours after the onset of germination and remained at a constant level during the following days. Argininosuccinate synthetase activity decreased; the other enzymes showed a small increase in activity and a subsequent decrease. Results are discussed in relation to the regulation of the arginine metabolism during pea seed development and germination.

     

The induction of phosphatase activity in thin slices of Jerusalem artichoke tissue by treatment with indole acetic acid

Summary Prolonged washing of thin slices of Jerusalem artichoke (Helianthus tuberosus) did not result in any apparent increase in the activity of the phosphatase enzymes, although the washing process is known to stimulate the activity of many other enzymes. However, treatment of the tissue in either 3×10^–5M 2,4-dichlorophenoxyacetic acid or 10^–4M indole acetic acid resulted in a 3-fold increase in the phosphatase activity. Significant stimulations of activity were detectable one hour after placing the tissue in the auxin. Treatment of the tissue in either kinetin or gibberellic acid failed to stimulate the activity of the enzyme. The enhancement of phosphatase activity caused by auxins could not be prevented by adding cycloheximide to the treatment solution an it is concluded that the stimulation occurred as the result of the activation of enzyme already present in the tissue rather than by the de novo synthesis of new enzyme protein.     

The influence on enzyme activity of storage of tissue blocks at −70° C

Summary Blocks of tissue from various organs of the rat have been chilled by precipitate immersion in n-hexane cooled to –70° C, and then stored at –70° C. At various intervals (up to 14 days) after chilling, cryostat sections were prepared from these blocks and assayed for the activity of a variety of enzymes. Enzyme activity was measured by scanning and integrating microdensitometry. With the exception of acid phosphatase and cytochrome oxidase, all enzymes assayed were stable for at least 7 days after storage at –70° C and most were stable for 14 days. Storage of fresh-frozen sections at –30° C in the cabinet of the cryostat, for up to 24 h, had little effect on enzyme activity.     

The Effect of Potassium on Cotyledon Expansion Induced by Cytokinins

Potassium has been found to enhance greatly the expansion response of cucumber cotyledons to cytokinins. A reduction of the response to kinetin is obtained with increasing age of the cotyledons. The lesser response is associated with lower levels of potassium remaining in the cotyledon. A high level of KCI in the incubation medium offsets the lower potassium content of the tissue and enables a much larger response to the cytokinins. At 40 mM KCI the response to kinetin is 4.2 times greater than in the absence of KCI. Calcium increases the effect of potassium on the response to kinetin. When incubated in 40 mM KCI and 10 mM CaCI₂ with 10 mg/I 6-benzylamino-purine, the final weight of the cotyledons is 6.8 times the initial weight after just 4 days. This KCI-CaCI₂ combination is also found to promote chlorophyll synthesis in the usual cucumber cotyledon bioassay.     

Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in the developing chick brain: partial characterization and levels during development.

Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase is largely expressed in chick brain tissue during development. The enzyme was purified from brain extract prepared from 19-day-old chick embryos and from adult chickens using ammonium sulfate fractionation, gel filtration on Sephadex G-75 and two DEAE-Cellulose ion-exchange chromatography steps. The purified enzymes from embryo and adult chick brains show identical molecular weight values (about 18-20 kDa) and biochemical and structural properties such as substrate specificity, sensitivity to inhibitors, and number of free reactive sulphydryl groups. These data suggest that they are the same enzyme protein. Although the total acid phosphatase activity does not change appreciably during development, the activity associated with the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase markedly increases after birth and reaches the adult values within the first week of life. Taken together, our results suggest an involvement of the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in postnatal development and maturation of chick brain tissue. The variations in tyrosine phosphorylation profile of chick brain polypeptides analyzed by Western blotting at the same developmental stages are also reported.     

Changes in the activity of different classes of enzymes in the cerebral cortex of rats in ontogeny and after the transplantation of embryonic nerve tissue

The activity of lactate dehydrogenase (LDH), indophenol oxidase, aspartate aminotransferase (AsAT), alkaline phosphatase, acid phosphatase and aldolase at different stages of rat development was measured. We have also determined changes in the activity of these enzymes resulting from transplantation of embryonic nerve tissue (ENT) into the brain of adult animals. During development from the embryo to the adult animal, LDH and AsAT activities increased, while alkaline phosphatase activity diminished. After ENT transplantation, the most prominent changes were in the alkaline phosphatase activity whereas the activity of LDH, AsAT and acid phosphatase remained unchanged and similar to that in the brain cortex of intact adult animals. Changes in the enzyme activity resulting from ENT transplantation changed in a manner characteristic of the transplant. Local brain damage did not change the activity of the studied enzymes fifty days after surgery.