Ultrastructure of Mucocysts in Peranema trichophorum (Euglenophyceae)1

The “mucigenic” or “muciferous” bodies of Peranema trichophorum are further characterized here as unique extrusive organelles, the mucocysts. Intracellular and ejected mucocysts have characteristic shapes that may represent different developmental stages. Mucocysts found near the Golgi apparatus are membrane-bounded, elongate, tubular structures with amorphous contents of low electron density. Subpellicular mucocysts are often aligned with pellicular striae and have dense contents, which are separated by an electron-lucent zone from granular material at the tips. Ejected mucocysts are uniform in structure and consist of an inner tube with helical striations, an outer tube with a diamond-shaped pattern, and a dense middle band. Fine fibrils, visible only after mucocyst discharge, emanate from the tips. Mucocysts may also protrude through the pellicle and discharge mucilaginous materials into the medium. Acid phosphatase activity is localized within the subpellicular mucocysts, suggesting that they may be involved in release of hydrolytic enzymes into the medium.     

Diazo-reaction positive substance observed in the cortex of Chattonella antiqua

In order to investigate the causative factors responsible for removal of mucous coat from the gill lamellae of young yellowtail, Seriola quinqueradiata by red tide, diazo-reactions were employed for planktons and their media. The concentration of NO2- in the medium containing the raphidophyceae, Chatonella antiqua (ca 2000 cells/ml), was 0.70 +/- 0.05 (mu g/ml +/- SEM). In addition, diazo-reaction positive substances (NOx) which may degenerate the mucous, was highly concentrated in the cortex (perikaryon) of Chattonella antiqua. Morphologically, mucocysts, and chloroplats were likewise present in the cortex. Mucocysts were packed with fine fibrous content. Histochemically, the mucocysts were stained with PAS and had an abundance of nitrogen oxides (NOx). We observed discharge of the fibrous material from the mucocysts. These results suggest that when Chattonella antiqua is passing between the gill lamellae, NOx discharged from the mucocysts may act on the mucous, leading to the degeneration and concomitant removal of the mucous coat from gill lamellae.     

On the reliability of the lead salt precipitation method of acid phosphatase localization in plant cells

Summary Fixation of cells with glutaraldehyde (5.0%, pH 6.7) was found to facilitate both the penetration of substrate (p-nitrophenyl phosphate) into cells and the leaking out of intracellular phosphate ions. 64% of the original activity survived the fixation for at least 24 hours. Lead ions added to the incubation medium at 6 mM neither accelerated nonenzymatic hydrolysis of the substrate, nor completely inactivated the enzyme activity. Lead ions at concentrations above 6 mM formed an insoluble compound with p-nitrophenyl phosphate, resulting in a decrease in the concentration of free substrate and lead ions. Phosphate ions liberated from substrate could not be completely trapped by lead ions even at above 6 mM, suggesting the possibility of intracellular migration of phosphate ions.In the presence of 4 mM p-nitrophenyl phosphate, 6 mM lead nitrate, and 0.2 M sucrose at pH 6.5, lead salt precipitates were deposited on the outer surface of cell walls, within cell walls, at tonoplast membranes, in nuclei, and occasionally in proplastids. No deposition of lead salt was formed in the control test from which the substrate was omitted. When cells were treated at first with lead nitrate and then with potassium phosphate, lead salt deposits were formed in the same sites as those of cells incubated in a complete reaction medium.It is concluded that although the result of the lead salt precipitation procedure reflects the presence of enzyme activity, it cannot directly show the site of the enzyme.     

ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE MEMBRANES OF THE SQUID NERVE FIBER

This investigation deals with the localization of sites of ATPase activity, especially of transport ATPase, in nerve fibers of the squid Doryteuthis plei, at the subcellular level. Splitting of ATP liberates inorganic phosphate which reacts with lead to form a precipitate in the tissue. The reaction was made on nerve fibers fixed with glutaraldehyde. Frozen slices were incubated in Wachstein-Meisel medium containing ATP and Pb(NO₃)₂. Deposits of reaction product were found in the axolemma (towards its axoplasmic side), Schwann cell membranes (mainly at the channels crossing the layer), and mitochondria. Control experiments revealed that no deposits were observed in nerve fibers fixed in osmium tetroxide prior to incubation in the medium containing ATP, or in nerve fibers incubated without substrate or with adenosine monophosphate, adenosine diphosphate, glycerophosphate, or guanosine triphosphate as substrate. For evaluation of transport ATPase activity, these findings were compared with results obtained with nerve fibers treated with G-strophanthin or K-strophanthoside before or after glutaraldehyde fixation. The cardiac glycosides produced a disappearance or diminution of the deposits. The largest inhibitory effect was observed in the axolemma. The findings indicate that the highest ATPase activity is localized in the axolemma and may be due primarily to transport ATPase.     

The cytochemical demonstration of NaK dependent adenosine triphosphatase at electrocyte level in Eigenmannia virescens (gymnotidae)

Summary ATPase activity (E.C. 3.6.1.3) has been studied by electron microscopy with the help of several cytochemical techniques on Eigenmannia virescens electrocytes. Incubation was carried out with in two different media containing paranitrophenyl phosphate (p-NPP) or adenosine triphosphate (ATP) as substrate. With p-NPP the phosphate freed is captured at alkaline pH, either by strontium chloride or by lead citrate. With ATP the phosphate freed is captured at a pH close to neutrality by the lead nitrate. NaK ATPase activity was only demonstrated with the medium containing ATP; the positive results obtained with this technique were sensitive to ouabain. — The enzyme is situated both on the membrane of the posterior face which is innervated and on that of the anterior face of the electrocytes. The cytoplasm of the anterior face is occupied by a strong concentration of tubules on whose membranes the enzyme is also present. The localisation of the enzyme on the tubules can explain biochemical results which indicate that 70% of the total NaK ATPase of the electrocytes is situated at the level of the anterior face.     

LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE RAT SPERM TAIL AS REVEALED BY ELECTRON MICROSCOPY

The epididymides of rat testis were fixed in glutaraldehyde and cut as frozen sections. The sections were incubated in lead nitrate solution containing as a substrate either ATP, AMP, creatinine phosphate, beta glycerophosphate, or phenyl phosphate. Then they were postfixed in osmium tetroxide, embedded, sectioned, and examined with the electron microscope. In the sperm tail, when ATP is used as a substrate the reaction product (lead phosphate) is observed both in the tail filament complex and on the surface membrane of the mitochondrial helix of the middle piece. In the tail filament complex, this product is seen near the nine paired peripheral and two central filaments, and in the matrix between the outer coarse fibers. But the product is not observed within these filaments and fibers. In longitudinal sections, no periodicity of the deposits in the complex is observed. When the other phosphate compounds are used as substrates the reaction products appear on the surface membrane of the mitochondrial helix, and are not found in the tail filament complex. No distinctly different localization of the reaction products is observed when substrates other than ATP are used. Possible relationships between the structure and the function of the sperm tail are discussed in the light of these findings.     

A Gelatin Film Method for Improved Histochemical Localization of Dehydrogenases in Plant Cells

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases diffused from frozen sections of Vicia faba embryos during histochemical incubation. In the liquid incubation medium, the dehydrogenases catalysed the oxidation of substrate and reduction of NADP. NADPH₂ thus formed could lead to artifactual deposition of formazan in frozen sections. The addition of 20% polyvinyl alcohol to the incubation medium was found unsatisfactory in preventing this loss which appeared to be overcome by incorporating the reaction mixture into a gelatin film. Equal volumes of 10% gelatin solution in 0.05 M phosphate buffer at pH 7.8, and the enzyme reaction medium containing twice the normal concentration of substrate (0.014 M), of 0.007 M pyridine nucleotide, of 0.02 M KCN and of 0.0024 M NBT in the buffer, were mixed and layered onto polyethylene, and allowed to set in the dark at room temperature for 30-60 min. The solidified medium and its support were cut into strips and layed onto unfixed, frozen sections of plant tissues which were incubated at 20 C. Evidence is presented to support the supposition that the enzymes are retained in the sections during the reaction.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.     

Acid hydrolases in the suckling rat small intestine. II. On the importance of alkaline phosphatase inhibition in the histochemical localization of acid phosphatase activity.

Phosphatase activities against beta-glycerophosphate, I-naphthyl phosphate and naphthol AS-TR phosphate were investigated, at acid and aldaline pH levels, using unfixed and fixed cryostat sections of suckling rat jejunum. The use of 10 mm EDTA and 10 mm NaF as inhibitors indicated that alkaline phosphate is predominantly located in the microvillous region of the adsorptive cells, while acid phosphatase is located in small particles distributed between the brush borders and the nuclei of these cells. Alkaline phosphatase activity was found to interfere with the localization of acid phosphatase unless EDTA was included in the incubation medium. A modified Gomori medium, containing 10 mm EDTA and additional lead nitrate, is described. Latency experiemtns using this medium, with unfixed sections, indicated the lysosomal nature of particulate acid phosphatase. The discussion stresses the importance of including an aldaline phosphatase inhibitor in incubation media designed to localize extralysosomal acid phosphatase activity.     

A new dynamic model system for the study of capture reactions for diffusable compounds in cytochemistry. I. Description of the model with special attention to phosphate capture in acid phosphatase cytochemistry

A model system is described for the investigation of the dynamics of precipitation processes in a matrix. In this system a solution containing the molecular species to be precipitated and the precipitating medium are pumped along opposite sides of a polyacrylamide film. The solutions flowing continuously along the film, interact and can form a precipitate inside the film. The applicability of the model was tested on the capture reaction for phosphate ions by the Gomori type medium for acid phosphatase. Precipitation of lead phosphate in the film occurred only at a phosphate concentration above a certain value. The dependence of this minimal phosphate concentration on various parameters was studied and the results were compared with values found in earlier model studies and calculations concerning phosphate concentrations that can be built up in lysosomes during the Gomori reaction. The system seems promising for obtaining fundamental data about other cytochemical enzyme trapping reactions as well as for the matrix facotrs involved in bone calcification and shell formation.     

Selection for and Enrichment of Non-Discharge Mucocyst Variants of Tetrahymena thermophila 1,2

The present protocol for selection for and enrichment of potential non-discharge mucocyst variants of Tetrahymena thermophila is based on the ability of wild-type cells to discharge their mucocyst contents simultaneously when stimulated with alcian blue 8 GS. Under appropriate ionic conditions, the discharged mucocyst contents form a capsule around each cell and prevent its locomotion. Non-discharge variants unable to shed a capsule are assumed to retain their ability to swim and are simulated in this study by cells not induced to shed their capsule. For mass phenotype screening, the conditions for maximum capsule shedding were established for wild-type cells. One hour starvation in Wagner's solution rendered 100% of the cells competent to shed a capsule when triggered with a 0.4% solution of alcian blue 8 GS. Decontamination of the shedding mixture by addition of egg albumin in a final concentration of 0.1% guaranteed survival of >95% of these cells that were now encapsulated, but allowed up to 5% of the cells to escape their capsule and swim freely. Cells with intact mucocysts and cells with emptied mucocysts were separated in reconstruction experiments by density-gradient centrifugation in which 95% of the cells with intact mucocysts appeared in a discrete band. Using the same protocol, the efficiency of separation was tested with mixtures of morphologically marked (food vacuoles stained with India ink) and genetically marked (resistance to cycloheximide) cells. Using 1:1 mixtures of marked cells with intact mucocysts and cells with emptied mucocysts (or vice versa), the cells with intact mucocysts were efficiently separated from other cells; one cell with emptied mucocysts per 100 cells with intact mucocysts was found in the upper discrete gradient band.     

Measurement of the proton-motive stoichiometry of the respiratory chain of rat liver mitochondria: the effect of N-ethylmaleimide

The apparent proton-motive stoichiometry as measured by the oxygen-pulse technique in KCl medium is depressed by the rapid uptake of inorganic phosphate, unless endogenous phosphate is depleted or uptake is inhibited. In sucrose or choline chloride media, where the internal pH is more acid than in KCl media, uptake may be greatly diminished. In the absence of significant phosphate uptake, the observed stoichiometry of around 8, obtained with no added substrate or respiratory inhibitors, appears to be characteristic of NADH oxidation without significant participation of the proton-translocating NAD(P) transhydrogenase. A mechanistic stoichiometry of at least 8 is indicated.     

Influence of CO2 and pH on the attachment response of the cercaria of Diplostomum spathaceum (trematoda) (author's transl)]

1. The attachment of the cercaria to artificial substrates (offered via dialyzing membranes) in definite media was investigated under conditions of variable pH and [CO2]. 2. A decrease of the pH of the substrate releases only attachments in CO2 containing media and consequently acts via CO2 systems of the medium. 3. As effective components of CO2 systems, dissolved CO2 + H2CO3 are confirmed. 4. The sensitivity of the reaction on gradients of the CO2 partial pressure (in solution) could be established by offering substrates with lowered pH in CO2 containing media. Thus, by raising the CO2 partial pressure from ca. 0,04% to 0,15% maximal fixation rates were obtained (Fig. 3). 5. The carboanhydrase inhibitor acetazolamide, when added to the medium, had no direct influence on the CO2 receptors.     

The causes of the biological action of electrochemically activated solutions by changes in the growth of Escherichia coli cells

To study the causes of the biological effect of electrochemically activated solutions, nutrient growth media M 9 were prepared using catholyte and anolyte solutions containing separate components of the nutrient medium, such as distilled water, phosphate buffer, phosphate buffer with chlorides (NaCl, NH4Cl), and chlorides. The biological activity of different nutrient media was assessed by a comparison with the stimulation or inhibition of the growth of Escherichia coli cells in the catholyte and anolyte of the complete nutrient medium M 9. It was shown that medium M 9 prepared on the catholytes of different initial solutions acquired the stimulating properties only if the initial solution contained salts containing chlorine. The stimulating effect of the initial solution was 18-24%. Electrochemical treatment of solutions containing no chlorides (distilled water, phosphate buffer) and subsequent addition of the components of nutrient medium to exposed solutions had neither a stimulating nor the inhibiting effect on cell growth. The cultivation of cells in a nutrient medium based on the catholyte of preliminarily treated hydrochloric acid showed that it is the presence of chlorine ions in solution during electrolysis that causes the stimulating effect of the nutrient medium based on the catholyte. The formation of oxidizers and the inhibitory effect of the anolyte described previously was also observed if the solution contained chlorine ions during electrolysis. Possible mechanisms of the biological effect of catholytes containing chlorides during electrolysis were discussed.     

Chick embryo anchored alkaline phosphatase and mineralization process in vitro.

Bone alkaline phosphatase with glycolipid anchor (GPI-bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate, but containing calcium and GPI-bALP substrates, served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate, arising from the hydrolysis of a substrate by GPI-bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from 1.5 h to 144 h. The measurements of Ca/Pi ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures, while the analysis of X-ray diffraction data allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine, glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate by GPI-bALP produced hydroxyapatite in a manner similar to that of beta-glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation, then poorly formed hydroxyapatite crystalline structures were observed, followed by the presence of hydroxyapatite crystals after 6-12 h incubation time. However, the hydrolysis of either ATP or ADP, catalysed by GPI-bALP in calcium-containing medium, did not lead to the formation of any hydroxyapatite crystals, even after 144 h incubation time, when hydrolysis of both nucleotides was completed. In contrast, the hydrolysis of AMP by GPI-bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on the ability of GPI-bALP to hydrolyze the organic phosphate but also on the nature of substrates affecting the nucleation process or producing inhibitors of the mineralization.     

A Petroleum-Degrading Freshwater Alga Phormidium foveolarum Gom

The freshwater blue-green alga Phormidium foveolarum Gem. was grown on medium containing hydrocarbons. Measurements of O2 consumption during degradation of the N-tetradecane by Phormidium foveolarum Gom. and residues of the hydrocarbon substrate were made by Warburg manometry and gas-chromatography. The results show that: (1) Phormidium foveolarum Gom. has ability to absorb and degrade the N-tetradecane and then the concentration of N-tetradecane decreased. (2) Under the dark condition the O2 consumption increased with the reaction time for hydrocarbon degradation. (3) Notable changes in cell contents of Phormidium foveolarum Gom. have been observed after it has continuously grown on the substrate containing hydrocarbon. The oil droplets appeared and more large granules have been found in the cells.     

Reaction rate studies of glucose-6-phosphate dehydrogenase activity in sections of rat liver using four tetrazolium salts

Summary The reaction rate of glucose-6-phosphate dehydrogenase activity in liver sections from fed and starved rats has been monitored by the continuous measurement at 37 C of the reaction product as it is formed using scanning and integrating microdensitometry. Control media lacked either substrate or both substrate and coenzyme. All reactions were nonlinear; however, subtraction of either of the controls from the test response produced linearity. Differing responses in sections of livers from fed and fasted rats indicate that the appropriate control medium for use in the assay of this dehydrogenase is one lacking both substrate and coenzyme rather than a medium containing coenzyme. The reaction rate was the same with each of the final acceptors. Problems with the diffusion of the formazan of BPST and with the failure to precipitate the formazan of Neotetrazolium make Tetranitro BT and Nitro BT the tetrazolium salts of choice in quantitative dehydrogenase assays.     

Model for prebiotic pyrophosphate formation: Condensation of precipitated orthophosphate at low temperature in the absence of condensing or phosphorylating agents

Summary The formation of pyrophosphate (PPi) by condensation of orthophosphate (Pi) at low temperature (37°C) in the absence of condensing or phosphorylating agents could have been an ancient process in chemical evolution. In the present investigation the synthesis of³²PPi from³²Pi was carried out at pH 8.0 and PPi was found in larger amounts in the presence of insoluble Pi (with calcium or manganese ions) than in its absence (with magnesium ions, or with no divalent cations present). After 10 days of incubation in the presence of precipitated calcium phosphate, about 1.6 nmol/ml of PPi was formed (0.057% yield relative to insoluble Pi). The hypothesis that the reaction is dependent on precipitated Pi was reinforced by the effect of adding dimethyl sulfoxide (2.1–9.9 M) in the presence of magnesium ions: the amount of magnesium phosphate precipitated in the presence of the organic solvent was proportional to the amount of PPi formed. The large and negative activation entropies found in aqueous media with calcium ions and in a medium containing 11.3 M dimethyl sulfoxide with magnesium ions suggest that the reaction was favored by a hydrophobic phenomenon at the surface of solid Pi. This reaction could serve as a model for prebiotic formation of PPi.