Accumulation Of Reaction Products Research Articles

Depolymerization of alginate by an extracellular alginate lyase from a marine bacterium: substrate specificity and accumulation of reaction products

Depolymerization of alginate by an extracellular alginate lyase from a marine bacterium: substrate specificity and accumulation of reaction products

Central projections from cat suboccipital muscles: a study using transganglionic transport of horseradish peroxidase.

Central projections of suboccipital muscle nerves were examined following exposure of cut peripheral nerves to the tracer horseradish peroxidase. Labelled fibers entered the C1 and C2 dorsal roots and accumulated in the dorsolateral part of the dorsal funiculus. Many labelled fibers entered the grey matter of C1 to C3 in ventrally directed bundles which passed medially to the base of the dorsal horn. No terminal labelling was apparent in superficial layers of the dorsal horn. However, labelled fibers ramified extensively throughout medial parts of the intermediate laminae, in and around the central cervical nucleus. Labelled fibers also projected toward the ventral horn. In cats subjected to ventral root section at the time of peripheral nerve exposure, a modest distribution of reaction product was observed deep in the ventral horn. In cats which did not undergo ventral root section, anterograde projections in the ventral horn were obscured by the simultaneous retrograde filling of motoneurons both in the ventromedial nucleus and on the medial and lateral borders of the gray matter. Labelled axons also coursed rostrally into the medulla where they formed a circumscribed bundle between the main cuneate nucleus and the spinal nucleus of V. Three consistent regions of HRP deposition could be identified at medullary levels. Dense accumulations of reaction product were present in circumscribed regions of the external cuneate nucleus (ECN) throughout its rostrocaudal extent. A second zone of dense labelling occurred in the intermediate nucleus of Cajal, where it appeared to form a continuing column rostral to the central cervical nucleus in C1-C3. Sparse labelling was restricted to a third zone in the ventrolateral part of the main cuneate nucleus.

Ultrastructural localization of several phosphatases with cerium.

Cerium ions have been used as the capture agent for inorganic phosphate released during the enzymatic hydrolysis of phosphate-containing substrates by a variety of phosphatases. Cerium phosphate reaction product accumulation is proportional to the amount of enzyme present in a cell-free model system. Ultrastructurally, cerium phosphate reaction product appears as a very fine electron-dense precipitate. Cerium appears to be a better capture agent for inorganic phosphate than lead in that reaction product is usually more uniform and more consistently reproducible when cerium is used. Furthermore, nonspecific deposits of reaction product that are commonly encountered in lead-based phosphatase reactions are virtually nonexistent when cerium is the capture agent.

Effects of secondary chemical reactions upon the performance of dye lasers

The effect of absorption at the lasing wavelength due to the accumulation of reaction products from the photodegradation of a laser dye is analyzed theoretically. This analysis is used as a basis for the representation of data from dye degradation experiments in terms of absorption at the lasing wavelength. Studies of the effects of flashlamp pulse rate and time on the inferred absorption for a number of different dyes reveal the influence of secondary chemical reactions. The most significant effects are shown to be associated with spontaneous reactions where the reactants and products have markedly different molar absorptivities at the lasing wavelength.

Immunocytochemical localization of enkephalin in the neostriatum of rat brain: A light and electron microscopic study

The peroxidase-antiperoxidase (PAP) immunocytochemical technique was used to determine the light and electron microscopic localization of antisera directed against either methionine [Met5]-or leucine [Leu5]-enkephalin in the neostriatum of brains from untreated rats. By light microscopy, neuronal perikarya and processes showing enkephalin-like immunoreactivity (ELI) were unevenly distributed throughout the neostriatum. The greatest accumulation of neuronal structures showing ELI was in the ventro- and caudo- lateral portions of the nucleus. The labeled perikarya measured 10--15 micrometer in diameter and constituted about 15--20% of the total neurons in the neostriatum. By electron microscopy, examination of three areas from horizontal and coronal sections revealed no regional differences in types of neurons showing ELI or in their synaptic organization. All labeled neurons showed a relatively low intensity reaction product which was diffusely distributed throughout the perikarya and dendrites. The cytoplasm contained relatively few organelles, which included mitochondria, endoplasmic reticulum and numerous "alveolate" vesicles. The dendrites had many spiny processes which formed asymmetric synapses with unlabeled axon terminals containing all small clear vesicles. In contrast to the perikarya and dendrites a dense accumulation of reaction product was present in a few myelinated and numerous unmyelinated axons and axonal varicosities. Approximately 75% of the labeled varicosities did not form a specialized synaptic junction in a single plane of section. The remaining 25% of the labeled terminals formed asymmetric junctions primarily with unlabeled dendrites and rarely with unlabeled perikarya or axons. The morphology and synaptic relations of the neurons showing ELI suggest that they may belong to the general group of medium-sized spiny cells characterized in Golgi studies by Kemp and Powell ('71a). At least some of the peptide-containing neurons may also have a myelinated efferent axons.

Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes.

The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs.

Cytochemical localization of acetylcholine receptors at the neuromuscular junction by means of horseradish peroxidase-labelled α-bungarotoxin

The cytochemical localization of acetylcholine (ACh) receptors at the neuromuscular junction was investigated with a procedure utilizing α-bungarotoxin (α-Btx) labeled directly with horseradish peroxidase (HRP). Following incubation of tissues in the conjugate and reaction for peroxidase, activity was observed on the junctional folds of the motor endplate. A uniform layer of reaction product < 15nm thick occurred over the apical portions of the junctional folds. Membranes at the bases of the cynaptic cleft showed only small amounts of reaction product. Non-junctional regions of the muscle fiber were unreactive. Activity was also observed in the membrane of the axon facing the muscle surface, often including the axolemma overlaying the “active zones” of the nerve terminal. Such presynaptic activity was still evident on nerve terminals disjuncted from the synapse by enzymatic treatment prior to incubation in the conjugate. Thes localization indicates the possible presence of presynaptic ACh receptors within the axolemma. In muscle denervated for 7–12 days, motor endplates were reactive and parajunctional sarcolemma showed slight activity, but most extrajunctional regions contained no obvious accumulations of reaction product. Activity at all sites was prevented by preincubation of tissues in native α-Btx prior to incubation in the conjugate and reaction for HRP. This procedure represents a simple and convenient method for the high resolution localization of ACh receptors.

Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons. Immunocytochemical localization by light and electron microscopy.

The monoamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and tryptophan hydroxylase (TrH) were immunocytochemical localized in dopaminergic, noradrenergic and serotonergic neurons of rat brain by light and electron microscopy. In dopaminergic and serotonergic neurons, the respective synthesizing enzymes. TH and TrH, were distributed throughout the cytoplasm of the neuronal perikarya, dendrites, axons and terminals. The most selective accumulation of reaction product for the specific enzyme was associated: (a) in perikarya with endoplasmic reticulum, Golgi apparatus and microtubules, (b) in processes with microtubules, and (c) in terminals with dense granules or clear vesicles. The labeled terminals were characterized by their content of labeled organelles and the absence of synaptic junctions. In noradrenergic neurons, both TH and DBH were localized in the perikarya, similar to TH in dopamine neurons. TH and DBH differed in their localization within proximal axons and dendrites in that TH was associated with microtubules but DBH was not. These results provide ultrastructural evidence to suggest that monoamines may be: (a) synthesized by enzymes which are associated with different organelles depending on the portion of the neuron and the type of enzyme; (b) synthesized in both axons and dendrites and (c) released from terminals without postsynaptic membrane specializations.

Role of molecular packing and structural defects in reactions of gases with organic solids: ozonolysis of trans-stilbene and αβ-diethyl-4,4′-dihydroxystilbene

Optical microscopic examination of partially ozonolysed and wet-etched single-crystal faces of trans-stilbene (1a) and αβ-diethyl-4,4′-dihydroxystilbene-(1b) reveal the importance of line defects (dislocations) in governing the rate of the (anisotropic) oxidative attack of the ethylenic double bond in these two solids. In the regions of enhanced reactivity at dislocations emergent on the (100) face of (1b) the rate of ozonolysis is considerably slower than at corresponding regions of the (001) face of (1a), a direct consequence of the protection afforded to the double bond in molecules of the former. As reaction product [benzaldehyde and p-hydroxypropiophenone for (1a and b) respectively] accumulates within each solid reactant, stresses are set up which generate more dislocations on well-defined slip planes (which are identified); these, in turn, function as new centres of preferred attack so that the reaction accelerates as ozonolysis proceeds. Very many new slip systems in crystals of (1a), and relatively few new ones in crystals of (1b), are activated by the accumulation of reaction product. This is explicable in terms of the crystallographic differences between the two solids. It is directly demonstrated that numerous new families of dislocations may be introduced during the course of a gas–solid reaction; and many of the slip systems identified here for (1a) have not hitherto been detected in other aromatic solids (such as naphthalene and anthracene) which also belong to the space group P21/a.

Fold surface of polyethylene single crystals as assessed by selective degradation studies. II. Refinements of the nitric acid degradation method

AbstractDetailed critical examinations of three aspects of nitric acid treatment of polyethylene are reported. (1) The endgroups introduced by the nitric acid are examined. It is concluded that in samples degraded to a single‐crystal traverse stage, carboxyl features only as endgroup, and conversely each chain‐end is terminated by such a group. Consequently, the carboxyl content can be used for quantitative characterization of the cut chain length. The function and position of the nitro group remains unexplained. (2) The morphological selectivity of the nitric acid‐induced degradation is scrutinized. It is concluded that the crystal core is attacked and thinned by the acid; the slowing down of the reaction is principally due to accumulation of reaction products. Accordingly, the usual distinction between a vulnerable amorphous layer and a resistant crystal core is not justified on the basis of such degradation experiments. It follows that meaningful structural information is obtained only when the relevant crystal properties are studied as a function of chain cutting, assessed from the molecular weight distribution (GPC), as opposed to that of degradation time alone. (3) The role of sample consistency and reagent strength has been examined. It is demonstrated that for the reaction to be sufficiently uniform throughout the sample for the results to be meaningful, the reaction rate has to be slow compared to the rate at which the reagent diffuses into the specimen. To realize these conditions, finely dispersed samples and weak reagents were found to be favorable

Some aspects of transport and digestion in the lens of the normal young adult rat

The normal young-adult rat lens was studied morphologically and histochemically. Morphological findings included the presence of dense bodies, smooth and coated vesicles, and multivesicular bodies. These were present in greater number in the epithelium and bow region than elsewhere. Acid phosphatase was demonstrated histochemically. The bow and epithelium were the most reactive parts of the lens. Accumulations of reaction product were observed within dense bodies, Golgi vesicles and cisternae, a few multivesicular bodies, and some elements of smooth endoplasmic reticulum. Injections of cadmium-free ferritin or horseradish peroxidase into the posterior aqueous chamber of the eye were used to demonstrate the extent and mode of transport of protein molecules into the lens. At intervals ranging from 90 sec to 4 hr following injection, eyes were removed and their lenses fixed in glutaraldehyde. Ferritin did not appear to penetrate the lens capsule. The peroxidase penetrated the capsule and entered portions of the intercellular space. It was incorporated in vesicles at the cell surface and, subsequently, in multivesicular bodies and dense bodies.

Use of horseradish peroxidase-labeled antibody for light and electron microscope localization of reovirus antigen.

Horseradish peroxidase-labeled antibody was used for light and electron microscopic localization of reovirus antigen in tissue culture. Reaction product in infected cells was easily detected in the cytoplasm, and the procedure was as sensitive as the fluorescent-antibody technique. At the electron microscopic level, infected and enzyme-labeled antibody-treated cells showed accumulations of reaction product at the sites of viral replication and around the viral particles. Reaction product was not detected in unstained infected cells, in stained uninfected cells, or in cells infected with an unrelated virus.

Use of Horseradish Peroxidase-Labeled Antibody for Light and Electron Microscope Localization of Reovirus Antigen

Horseradish peroxidase-labeled antibody was used for light and electron microscopic localization of reovirus antigen in tissue culture. Reaction product in infected cells was easily detected in the cytoplasm, and the procedure was as sensitive as the fluorescent-antibody technique. At the electron microscopic level, infected and enzyme-labeled antibody-treated cells showed accumulations of reaction product at the sites of viral replication and around the viral particles. Reaction product was not detected in unstained infected cells, in stained uninfected cells, or in cells infected with an unrelated virus.

INDOPHENOL OXIDASE AND PEROXIDASE ACTIVITIES IN MAMMALIAN ERYTHROCYTES.

Cytochrome oxidase and peroxidase activities in mammalian red cells are suggested by G- and M-Nadi reactions in rabbit, mouse, and human erythrocytes. Peroxidase activity was present in both the hemoglobin and the nonhemoglobin constituents of the red cells. Removal of oxygen stopped the G-Nadi reaction but not the M-Nadi reaction. Potassium cyanide and sodium azide at a concentration of 10–3-10–4 M partially inhibited G- and M-Nadi reactions. Microscopic examination of reacting cells revealed accumulation of reaction products at the cell periphery. This was not evident when the cells were incubated in a suspension of indophenol blue. Mammalian erythrocytes reduced the tetrazolium salt nitro-BT. Reduction was augmented by removal of oxygen and by cytochrome oxidase inhibitors. Accumulation of formazan at the surface of reacting cells was evident upon microscopic examination. The Nadi reactions were similar whether Ehrlich ascites tumor cells or stroma of hemolyzed erythrocytes were used. Reactions of stroma or whole erythrocytes with nitro-BT were essentially similar. Fixation or heating slowed or abolished all the reactions described.

THE PARTICULATE HYDROLASES OF MACROPHAGES. II. BIOCHEMICAL AND MORPHOLOGICAL RESPONSE TO PARTICLE INGESTION.

The influence of phagocytosis on the morphological and biochemical properties of macrophage hydrolase-containing granules has been studied in vitro. Following the uptake of large numbers of heat-killed bacteria, an intracellular rearrangement of hydrolytic enzymes occurred. This was associated with the solubilization of 50 to 60 per cent of the total cell content of acid phosphatase, cathepsin, lysozyme, beta glucuronidase, acid ribonuclease, and acid desoxyribonuclease and with a corresponding decrease in granule-bound enzyme. With more prolonged incubation the majority of the soluble intracellular pool of acid ribonuclease and lysozyme was lost to the extracellular medium. No change in the total content of any of the hydrolases was noted during 180 minutes of incubation in vitro. The morphological fate of the granules was studied by a histochemical method for acid phosphatase. After the phagocytosis of yeast cell walls there was a disappearance of acid phosphatase-positive granules and an accumulation of reaction product about the ingested particle. Experiments employing macrophages which were supravitally stained with neutral red also demonstrated the loss of neutral red-positive granules and the accumulation of the dye about the yeast cell walls. These results strongly suggest that lysis of macrophage granules occurs following phagocytosis and that a portion of the granule contents are then resegregated within the newly formed phagocytic vacuole.

Fineness and Water-cement Ratio in Relation to Volume and Permeability of Cement

Four factors that largely determine the end product obtained when cement andwater are mixed are the chemical composition of the cement, the fineness towhich the cement is ground, the amount of mixing water used, and the procedureused in mixing the slurry. In order to study the effect of two of thesefactors, the other two, the chemical composition and method of mixing, are keptconstant, leaving as variables fineness and water-cement ratio. Through years of experimentation and usage it has been well established thatthe reduction of cement clinker to a very fine state by grinding improve itscementing value, principally because the reaction between cement and watertakes place only at the surface of the cement particle, further action beinghindered by the accumulation of reaction products that tend to coat theunreacted material. Therefore, the more finely ground a cement, and the greaterthe surface exposed in proportion to its mass, the more rapid is the hydrationrate. Similarly, the greater the surface exposed, the greater the proportion ofthe cement that reacts, and the lower the amount of residue that is unreactedand so remains inert and unable to make any contribution to cementingproperties. Although it is more expensive to manufacture a finely ground cement than acoarser cement, the advantages of fine grinding of construction cements were somarked that the fineness of cement has increased considerably in past years.When the manufacture of special oil-well cements was begun, it was necessary toconsider properties of cement that previously were considered of minorimportance, foremost of these being pumpability. Since, with the water-cementratio constant, a finely ground cement will produce a neat slurry of higherconsistency and shorter time of pumpability than will a coarser cement, thereis a tendency to grind oil-well cements to a lower degree of fineness.Therefore, in order to produce oil-well cements that will make a slurry havinga long time of pumpability, cements are manufactured that because of theircoarseness generally are poorer cementing materials than some of the morefinely ground construction cements. On the other hand, often it is necessary topump oil-well cements for long periods of time to place them properly behind astring of casing in a deep well, and since high strength is not of foremostimportance for such cement, there is room for considerable controversyconcerning the proper fineness range. T.P. 1266

Some Properties of Two-Bath Developers for Motion Picture Film

It is well known that the properties of developers change during use as a result of depletion of the developing agents and accumulation of reaction products. This change is greatest for low-energy developers such as those of the borax type, and results in (1) a reduced development rate, and (2) a lowering of effective emulsion speed. These two effects may be offset to some extent by increasing the development time and by adding booster solutions to revive the developer. Another method of securing a more uniform degree of development throughout the life of a developer consists in using two developer baths in succession. — Three types of two-bath developers have been investigated, as follows: (1) bath A contained all the developing agents plus sodium sulfite; bath B, all the alkali plus the balance of the sulfite. (2) Both baths contained developing agents. (3) Both baths were of identical composition, the first bath being replaced by the second as it became exhausted. The results of the investigation showed that Type I is the most satisfactory developer combination and with this method it is possible to obtain an almost constant gamma with only a slight loss of emulsion speed over a fairly wide range of time of development. A formula is also suggested for the development of variable density sound negatives. The application of two-bath developers to machine, and rack and tank systems is described.