Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

Abstract. The level of peroxidase activity utilizing ascorbic acid changes during the development of the green alga, Acetabularia mediterranea. During development almost parallel levels of peroxidase activity and ascorbic acid content are detectable: both steadily decrease as algae progress from very young, slowly growing cells to the rapid growth stage and then to cells exhibiting differentiation into primordium and cap. Changes in the levels of the enzyme and its substrate in the cytoplasm and periplasm were demonstrated using biochemical and cytochemical procedures. Concomitant with these developmental changes, we also observed changes in the stage-specific patterns of ascorbic acid concentration: growing algae exhibit a pronounced negative apicobasal gradient of ascorbic acid. Acetabularia cultivated at 1,200 lux (the normal intensity in a 12-h-light/12-h-dark cycle) and at 700 lux (intensity at which growth is reduced, and cap formation is delayed) were also compared. The higher light intensity induced a moderate decrease in the ascorbic acid content without noticeable changes in the compartmental distribution in the cytoplasm and periplasm, and an increase in the level of periplasmic peroxidase activity with little change in the total peroxidase activity. Catalase was found to be present at very low levels and is unlikely to play a role in H₂O₂ catabolism. Possible roles for ascorbic acid and peroxidase in the development of Acetabularia are discussed.     

c-AMP and Morphogenesis of Acetabularia

The c-AMP content has been found to double when Acetabularia develop from 5–10 mm long to grown or almost full-grown algae.
The biological significance of this fact has been approached by studying the effects of drugs known to influence the intracellular c-AMP content on the development of Acetabularia. When grown in the presence of theophyllin or papaverin, inhibitors of phosphodiesterase, the Acetabularia display a striking response during the exponential growth period; the final length, however, is not affected. Both substances increase the c-AMP content of the algea. Isoproterenol, which activates adenylate cyclase in many systems, also influences Acetabularia during the exponential growth period and, in addition, slightly affects cap formation.
The change in c-AMP content in the course of development and the effects of drugs influencing (theophyllin and papaverin) or likely to influence (isoproterenol) the c-AMP content of the algae suggest that this nucleotide plays a role at the time of intense growth.
The same phosphodiesterase activity has been found in the 5–10 mm and the 19–25 mm long algae, whereas two enzymes were found in cap-bearing Acetabularia.
The results are discussed as well as the involvement of c-AMP in the development of this alga.     

Changes in the Cyclic AMP Content during Growth and Development of Acetabularia

The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10 mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5–1.0 pmoles per 100 mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm.
Treatment with theophylline (2.10⁻³ M), a phosphodiesterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm.
The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.     

Cell age-related differences in the interaction of a potential-sensitive fluorescent dye with nuclear envelopes of Acetabularia mediterranea

Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC₆(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC₆(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.     

Temporal morphology and cap formation in Acetabularia--I. Light-dark cycle perturbations

Cap formation, a major developmental process in the alga Acetabularia, is influenced by a single perturbation of the entraining light-dark schedule and thus, presumably, of the circadian rhythms. This perturbation is brought about several weeks before cap formation, the most conspicuous expression of morphogenesis in Acetabularia. The effect is more pronounced on cap formation than on growth. It varies in importance with the circadian time at which the perturbation was brought about. The effect is dependent on the developmental state of the alga: transfer carried out during the logarithmic phase of growth produces a delay whose importance decreases with time. When carried out during the phase of slow terminal growth, the transfer induces a transitory acceleration of cap formation. When the algae approach their final length, no effect is elicited. Photoperiodism seems to be involved.     

Catalatic activity of yeast cytochrome b2: l-lactate dehydrogenase

Abstract Crude extracts of yeast exhibited two catalase activity bands on starch gel zymograms. Antibody prepared against catalase T specifically precipitated the fast-moving catalatic band of catalase T, but did not affect the slow-moving catalatic band of cytochrome b ₂. 3-Amino-1,2,4,-triazole, a specific inhibitor of catalase, inhibited the catalytic activity of cytochrome b ₂ but had little effect on its l -lactate dehydrogenase activity.     

Changes in the cyclic AMP content during growth and development of Acetabularia.

The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5--1.0 pmoles per 100mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm. Treatment with theophylline (2.10(-3) M), a phosphodieterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm. The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.     

Characterization of the catalase of the genus Porphyromonas isolated from cats

Crude cell extracts from members of the genus Porphyromonas isolated from catswere examined in SDS-PAGE and nondenaturing PAGE. In eachof the species catalase activitywas detected as a single band with characteristics of typical bacterial catalases, i.e. each catalase functioned over a broad pH range (pH 5—10), was not inhibited by chloroform-ethanol, did not possess detectable peroxidase activity, and was irreversibly inhibited by3-amino-1,2,4 triazole. The catalase enzyme of P. gingivalis VPB 3492, P. circumdentaria NCTC 12469^T, P. salivosa VPB 3313 and VPB 3444 was inactivated at 71, 66·5, 63·5 and 57°C respectively. The molecular weights of the enzymes from P. gingivalis VPB 3492, P. circumdentaria NCTC 12469^T, P. salivosa NCTC 11632^T and P. salivosa VPB 3444 were 200000, 216000, 209000 and 200000 Da respectively.     

海藻中清除氧自由基的物质

新鲜海藻的提取液含有超氧物歧化酶（SOD）活性物质,能清除超氧自由基（O2-）。海藻的SOD活性通常为60—280Ug-1FW,而在孔石莼（Ulvapertusa）、江蓠（Gracilariaverrucosa）和凤尾菜（G．eucheumoices）中活性较高,约为300Ug-1FW。一般来说,海藻的SOD活性和稳定性为：绿藻＞红藻＞褐藻。绿藻的SOD以CuZn-型为主,而蓝藻的SOD以Fe-型为主。以江蓠琼枝（Eucheumagelatinae）提取液作PAGE并SOD活性染色时,除了观察到SOD同工酶带之外,还发现在前沿指示剂附近有一区域,此区域与高效自由基清除剂SPD（Superphycodismutas）的电泳行为和对氮蓝四唑（NBT）负染色的抑制相同,可能两者为同一种物质。     

TIMING AND LIGHT REGULATION OF APICAL MORPHOGENESIS DURING REPRODUCTIVE DEVELOPMENT IN WILD-TYPE POPULATIONS OF ACETABULARIA ACETABULUM (CHLOROPHYCEAE)

In the giant unicellular green alga, Acetabularia acetabulum (L.) Silva, development is altered by light. For example, blue light induces the vegetative apex to produce whorls of hairs that encircle the stalk and, later, blue light may trigger reproductive onset. The two goals of this study were to determine when changes in apical shape occur during formation of the reproductive structure, or "cap," and to determine which of these differentiation events require light. The first visible indication of cap initiation was a rounded swelling of the apex, which we call a knob-shaped apex (time = 0 hours). Subsequent changes in shape were a hyaline, knob-shaped apex, reached by 50% of the population 3 h later, and the formation of a whorl of unilobed chambers at 16 h. These chambers became bilobed at 33 h and trilobed at 34 h. Successive sets of cap hairs grew from protuberances found on the surface of the uppermost lobes of the chambers (superior corona). After knob, the remainder of cap formation was largely independent of light. However, the initiation of each set of cap hairs required light. If a recently initiated cap was amputated, the individual recapitulated development, repeating a portion of vegetative morphogenesis (i.e. it made whorls of sterile hairs) before initiating a new cap. The developmental sequence between amputation and initiation of a new cap required light. A model for light-regulated changes in shape at the apex of Acetabularia acetabulum, which integrates whorl and cap formation and encompasses both vegetative and reproductive development of this organism, is presented.     

Accumulation of peroxidase in the cap rays of Acetabularia during the development of gametangia.

Accumulation of peroxidase was demonstrated by light and electron microscopy to occur in Acetabularia in certain regions of the cap rays in relation to the development of the gametangia (cysts). Peroxidase was found to be incorporated into special, cell wall-like obstructions that separate the cap rays from the stalk when the secondary nuclei have settled in the cap rays. It is assumed that peroxidase acts as an anti-microbial protectant of the gametangia.     

Vasopressin in tissue basophils of the dura mater of white rats

By an indirect immunohistochemical method with fluorescein-isothiocyanate (FITC) and horse radish peroxidase as markers (HRP) the presence of vasopressin was shown in cells of dura mater in white rats. Mast cells were identified after staining with methylene blue by the metachromatic granularity of the cytoplasm. It was shown that the number of cells found by means of FITC luminescence corresponds with their number found by means of methylene blue. The use of conjugate with HRP unveils a lesser number of vasopressin-containing cells.     

Lipid composition ofAcetabularia mediterranea (Lamour.) Variations during cap morphogenesis

Summary YoungAcetabularia mediterranea cells without cap have a fatty acid composition different from other green algae currently used for biological research. They contain important quantities of palmitic and oleic acid, but are very poor in polyunsaturated fatty acids such as linoleic and linolenic acid. (These polyunsaturated fatty acids are predominant in higher plants and many green algae.)     

Characterization of Nervana, a Drosophila melanogaster Neuron-Specific Glycoprotein Antigen Recognized by Anti-Horseradish Peroxidase Antibodies

Abstract: Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identify neurons in Drosophila and other insects. We are interested in characterizing the gene product(s) recognized by anti-HRP antibodies because it may be important for nervous system function and/or development. Here we identify and purify from adult Drosophila heads an anti-HRP-reactive M_r 42K glycoprotein that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between M_r 38K and 42K that vary as a function of developmental age. We have collectively named these components Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system. It should now be possible to undertake a full biochemical and functional characterization of Nervana in Drosophila .     

Chromogenic substrates for horseradish peroxidase

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.     

Identification and immunolocalization of superoxide dismutase isoenzymes of olive pollen

Gametophytic tissues of plants are an area largely neglected in the broad literature on free radical processes in plants. In order to study the mechanisms of protection against oxidative stress in pollen, the presence of the key antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) was investigated. Crude extracts of olive tree ( Olea europaea L.) pollen were subjected to native PAGE in 10% polyacrylamide gels. The SOD activity staining of gels showed the presence of four isoenzymes. All the SODS were completely inhibited by 2 m M KCN and 5 m M H₂O₂, and therefore belong to the family of CuZn‐SODS. Isoelectric focusing (pH 3.5‐7) of crude extracts and further detection of SOD activity allowed determination of isoelectric points for the four isoforms, namely 4.60, 4.78, 5.08 and 5.22. The cross‐reactivity of pollen extracts with a polyclonal antibody to cytosolic CuZn‐SOD from spinach leaves was assayed by western blotting. After SDS‐PAGE and immunoblotting, a major polypeptide band of about 16.5 kDa was detected, which is characteristic of the subunit of most CuZn‐SODS. Immunocytochemical studies at TEM level using the same antiserum showed that CuZn‐SOD was localized in the cytoplasm of both vegetative and generative cells, and also in material adhered to the pollen wall. The olive pollen CuZn‐SODS could function in the protection against oxidative stress during pollen development.     

Some aspects of glycoprotein metabolism in Acetabularia: Spatio‐temporal activity of β‐galactosidase and physiological effects of tunicamycin

Abstract

β‐galactosidase has been chosen as an indicator of glycoprotein metabolism in Acetabularia, an unicellular and uninucleate green alga. This catabolic enzyme was quantified by fluorecence spectrometry. It was found at all developmental stages, but the activity levels differed, peaking at the end of the growth phase, at the time of cap morphogenesis initiation, β‐galactosidase activity is also subjected to periodic modulation, displaying a bimodal rhythm with a prominent peak at 16 h. The distribution of the enzyme was examined by cytochemistry, using a substrate analogue (X‐gal). It is present both in the cytoplasm and in the cell wall. No apico‐basal gradient was detectable. The physiological role of glycoproteins was assessed with tunicamycin, an inhibitor of N‐linked glycoprotein synthesis. Two pulses of 3 or 4 h of inhibitor (10 μg ml‐1) always inhibited growth, but more severely during the light period. One pulse may inhibit growth during the light period and stimulate it during the dark one; it may also have little effect, in both periods. Cap formation is inhibited between time 0 and 7. During the dark or subjective dark period, it is often stimulated or not affected. The same results were obtained in constant light. Cap formation is also inhibited in anucleate algae treated during the light period.     

Sensitivity limitations encountered in enhanced horseradish peroxidase catalysed chemiluminescence

Conditions for the enhanced horseradish peroxidase (HRP) catalysed reaction between luminol and hydrogen peroxide were optimized to determine detection limits for HRP conjugated to antibody fragment (HRP-Fab) in solution phase. Light output was linear with respect to HRP-Fab concentration but became nonlinear at low HRP-Fab concentrations when an accelerator (enhancer) of the reaction was used. para-Phenylphenol was a more effective enhancer than p-iodophenol at HRP-Fab concentrations below 20 pmol/l. The detection limit for HRP-Fab was 1.2 femtomoles in the absence of p-phenylphenol and 0.08 femtomole in the presence of p-phenylphenol. The acceleration of peroxidase activity at the lowest HRP-Fab concentrations occurred after an incubation time period of up to five minutes. This lag time limited the sensitivity and the mechanism for it was sought. Preincubation experiment results indicated that the lag time phenomenon may involve a reversible alteration in HRP catalytic activity and that enhancer, peroxide, luminol and HRP-Fab had to be incubated together some time before maximum activation could occur.     

Determination of safety levels of horseradish peroxidase-iodide system to human gingival keratinocytes and fibroblasts in vitro

The peroxidase-iodide (I-) system is a potential antimicrobial agent, and its bacteriocidal activity against various periodontal bacteria has been shown in many studies. The aim of this study was to investigate the possible cytotoxic effects of a non-physiological horseradish peroxidase (HRP)-I- system on human gingival keratinocytes and fibroblasts. Immortalized human skin keratinocyte cell line was used as a reference. Three indicators were studied: membrane permeability (trypan blue staining), cell growth (crystal violet staining) and metabolic activity (alamarBlue stain). The cells were cultured in microtitration plates, and the most commonly used exposure time to the HRP system was 1 h. The effects of HRP system on cell growth and metabolic activity were observed at lower I- and H2O2 concentrations than its effects on membrane permeability. Gingival fibroblasts were more prone to detachment than keratinocyte cell lines, but no differences in changes of growth or metabolic activities were observed between gingival fibroblasts and gingival keratinocytes. The highest concentrations of the HRP-I- system components which did not have any significant detrimental effects on the metabolic activity and cell growth of gingival keratinocytes and fibroblasts were: 50 microg/ml HRP, 500 micromol/L I- and 500 micromol/L H2O2. Although this system has been shown to be antibacterial against oral bacteria, no recommendations about the usage of the HRP-I- system in oral cavity can be made yet due to the in vitro nature of this study. Our results form the basis for future safety studies investigating the chronic toxicity of this system to oral epithelium.     

Intracellular marking of skeletal muscle cells with horseradish peroxidase in combination with a stain for cholinesterase

A procedure is described by which muscle fibers can be electrophysiologically studied, intracellulary marked with horseradish peroxidase (HRP), and stained for acetylcholinesterase (AChE) activity to identify nerve endings. Following electrophysiological characterization, 4% HRP is injected through the recording microelectrode with small depolarizing pulses. Muscles are fixed, washed, and stained with a Koelle procedure for AChE modified by extending the incubation period to 15 hours to identify small nerve endings. The HRP is visualized with 3,3'-diaminobenzidine tetrahydrochloride or benzidine dihydrochloride. Stained muscles are embedded in Epon and sectioned. Lucifer yellow dye is also demonstrated to be an excellent intracellular marker of muscle fibers but it cannot be combined with AChE staining.