Collidine Buffer Research Articles

Series of Mn Complexes Based on N‐Centered Ligands and Superoxide – Reactivity in an Anhydrous Medium and SOD‐Like Activity in an Aqueous Medium Correlated to MnII/MnIII Redox Potentials

AbstractTwo crystal structures are described in this article: (a) the structure of a monomeric MnII complex with the tridentate N‐centered N3 ligand tris[(1‐methyl‐2‐imidazolyl)methyl]amine (TMIMA) ([MnII(TMIMA)2]2+); and (b) the structure of a monomeric MnIII complex with the tridentate N‐centered N2O ligand 2‐{[(1‐methyl‐2‐imidazolyl)methyl]amino}phenolate (PI–)2 ([MnIII(PI)2]+) (5). The latter was isolated both in the MnII and in the MnIII state, although only MnIII crystals were successfully grown. They are part of a series of Mn complexes prepared as SOD mimics, namely [Mn(BMPG)(H2O)]+ (2) {BMPG = N,N‐bis[(6‐methyl‐2‐pyridyl)methyl]glycinate}, [Mn(IPG)(MeOH)]+ (3) {IPG = N‐[(1‐methyl‐2‐imidazolyl)methyl]‐N‐(2‐pyridylmethyl)glycinate}, [Mn(BIG)(H2O)2]+ (4) {BIG = N,N‐bis[(1‐methyl‐2‐imidazolyl)methyl]glycinate}. The reactivity of MnII complexes 1 and 2 in an anhydrous medium is described and compared to that of complexes 3 and 4, the data for which was previously published. The cyclic voltammograms of the whole complex series were recorded in an aqueous medium (collidine buffer). Their SOD‐like activities were estimated by the McCord–Fridovich test (IC50 with 22 μM cytc FeIII: 1.6 ± 0.1 μMol L–1 for 1, 1.2 ± 0.5 μmol L–1 for 2, 3.0 ± 0.2 μmol L–1 for 3, 3.7 ± 0.6 μmol L–1 for 4, 0.8 ± 0.1 μmol L–1 for 5). IC50 values were converted into the corresponding kinetic constant kMcCF values. A linear correlation between Ea and log(kMcCF) was obtained, indicating that in this series the conversion to MnIII is probably the rate‐limiting step. This is of substantial importance for further Mn–SOD mimic design in this series. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Variable Conformation of GAP Junctions Linking Bone Cells: A Transmission Electron Microscopic Study of Linear, Stacked Linear, Curvilinear, Oval, and Annular Junctions

There is a marked variability in the conformation of bone cell gap junctions in newborn murine cortical bone as defined by transmission electron microscopy (TEM). Studies were done in newborn BALB/c mouse and Sprague-Dawley rat femurs and tibias. Femoral and tibial cortices were dissected into 1 mm3 fragments and prepared in standardized fashion using modified Karnovsky fixation, 7.5% EDTA decalcification, 1% osmium tetroxide-sym collidine buffer with 1% lanthanum nitrate postfixation, Epon resin, 60 nm sections, lead citrate/uranyl acetate staining, and examination at 60 kV. Previous TEM descriptions of bone junctions have, with rare exceptions, noted only isolated linear or mildly curvilinear structures. In this study we noted gap junctional shapes on thin-section TEM preparations of osteoblasts and osteocytes to be extremely variable and complex encompassing linear, curvilinear, stacked linear, oval, and annular conformations. Multiple observations revealed linear gap junctions linking surface osteoblast cell bodies; linear, curvilinear, stacked linear, and oval junctions linking osteoblast processes in osteoid; linear and curvilinear junctions where cell processes joined with osteocyte cell bodies and each of the five conformations linking osteocyte processes within canaliculi. The annular junctions were found within osteoblast and osteocyte cytoplasm and in osteocyte cell processes within canaliculi. The annular junctions are intracellular, degenerating structures which appear as ultrastructural markers of gap junction involution. The more complex shapes reported here must be considered in (1) interpreting quantitative studies using freeze-fracture replicas, thin sections, and confocal microscopy immunolabeled junction connexin-43 components and (2) assessing gap junction biogenesis and turnover. 3-D reconstruction of bone junctions will enhance our understanding of these complex conformations.

Evidence that intermediate‐like filaments are found in the micronucleus of Paramecium bursaria during prophase

AbstractThe micronuclear spindle apparatus in Paramecium bursaria was studied by electron microscopy during prophase, metaphase, and anaphase of the first meiotic division. During prophase, the spindle apparatus consists mostly of intermediate‐like filaments, relatively few spindle microtubules, and unique cone‐shaped structures termed microlamellae. Microlamellae join the ends of chromosomes to the fibrous elements of the spindle. The capacity to preserve the intermediate‐like filaments is largely dependent upon the use of collidine buffer during fixation. In contrast, during metaphase and anaphase, microtubules are the dominant fibrous element of the spindle. The microtubules interact with chromosomes during these phases by joining to true kinetochores. Neither treatment with cytochalasin B or fixation with a low concentration of osmium tetroxide affects the development of intermediate filaments during prophase. Because intermediate‐like filaments are abundant during prophase and microtubules are more common during metaphase and anaphase, the structural differences may reflect differences in the mechanisms for chromosome movement.

Effect of collidine (2,4,6-trimethylpyridine) on the osmiophilia and chromaffin reaction in the synaptic vesicles of rat pineal nerves.

Rat pineal nerve endings contain a population of small and of large synaptic vesicles that are either electron lucent or have electron-dense cores. It has been reported that their osmiophilia is eliminated when collidine buffer is used in the fixation procedure. We investigated this effect and found that osmium tetroxide and potassium dichromate reactivity were abolished when excised pineal glands were briefly incubated with collidine buffer before glutaraldehyde-cacodylate fixation. Such an effect was not observed when collidine was applied after fixation. Glands that had been fixed in glutaraldehyde or osmium tetroxide buffered with collidine exhibited a peripheral zone containing reactive synaptic vesicles and a deeper, central zone where such reactivity was absent. These results indicate that the effect of collidine is due to depletion of monoamines rather than to chemical blockage of their reactivity, and further suggest that collidine has a higher rate of penetration into tissues than the tested fixatives.

Technical Evaluation of Four Common Embedding Resins Used in Electron Microscopy

Today, numerous types of embedding media are available for use in the electron microscopy laboratory. Laboratories generally choose a particular embedding media based on their own practical experiences with it. This study was performed to compare and evaluate four common resins found in electron microscopy laboratories.Skeletal muscle was removed from rats perfused with 3% glutaraldehyde in 0.1M collidine, cut into rectangles and allowed to remain in fixative for 6 hours. Tissue was then washed in 0.1M collidine buffer and post fixed for 1 hour with 1% osmium tetroxide in 0.1M collidine. After rinsing in collidine buffer the tissue was divided into groups for processing according to established procedures for the following resins: Araldite, Epon, Mollenhauer, Spurr (see formulas).

Effect of K+ and Na+ on calcium-dependent electron-dense particles in the monoaminergic synaptic vesicles of rat pineal nerves fixed in Ca2+-containing solutions.

The effect of K+ and Na+ on the Ca2+ binding site in the dense core of monoaminergic vesicles of pineal nerves was investigated in the rat. Rat pineal glands, bisected immediately after decapitation, were incubated at room temperature in solutions containing high K+ or high Na+ in the presence or absence of Ca2+. Fixation was performed in glutaraldehyde-osmium tetroxide in collidine buffer, with and without CaCl2. It was confirmed that, after fixation in Ca2+-containing solutions, an electron-dense particle, located in the vesicle core, which can be considered a calcium deposit, appears within the synaptic vesicles. It was observed that this Ca2+ deposit may be modified by incubation in a high K+ or high Na+ milieu before fixation in Ca2+ containing solutions. When the incubation was carried out with high K+ and high Ca2+ simultaneously, Ca2+ deposits were considerably increased. With K+ alone, no Ca2+ deposits were apparent, as when electrical stimulation is applied before fixation. This effect was not observed when the incubation was done in high Na+. Consecutive incubations in high K+ and high Na+, respectively, restored the capability of the vesicle cores to bind Ca2+. Prolonged incubation in high Na+ before fixation increased Ca2+ deposits within the vesicles. These findings are in line with data on the effect of these ions upon the storage and release of biogenic amines and suggest that these ions modify the capability of synaptic vesicles to bind Ca2+.

Vasectomy in rhesus monkeys. IV. Electron microscopic studies of the seminiferous epithelium.

AbstractStudies were undertaken to assess the fine structural effects of vasectomy upon the rhesus monkey testis, using 27 monkeys subjected to one of three surgical techniques: control sham operation (COS), unilateral silk vasoligation (USV), or unilateral clasp vaso‐occlusion (UCV). The monkeys were sacrificed from 1 to 66 weeks after surgery; tissues were fixed in glutaral‐dehyde in collidine buffer and processed for electron microscopy (EM) using routine techniques. No alterations were noted in the seminiferous epithelium of any COS animal; only focal lesions were found in vasectomized animals. These changes correlated with length of time post‐vasectomy, a finding consistent with earlier light microscopic studies (Heidger et al., '78). Even at the longest post‐operative periods studied, spermatogenesis appeared in most animals to be normal. One animal exhibited areas in which the seminiferous epithelium consisted of Sertoli cells and spermatogonia, only. The seminiferous epithelium of approximately one‐third of other animals, both on the vasectomized and contra‐lateral sides, exhibited such alterations as development of extensive infoldings and duplication within the basal lamina, and presence within the basal Sertoli cell cytoplasm of late spermatids and sperm tails. These alterations were observed in both UCV and USV animals. The presence of such late germ cells in the basal Sertoli cell cytoplasm was noted by light microscopy; however, lu‐minal spermiophages encountered in one animal at the light microscopic level were not detected in our EM study, which underscores the limitations of EM as a survey technique. All UCV animals developed spermatic granulomas of the vas, whereas only 3 of 13 USV animals developed such granulomas. It does not appear that the alterations exhibited by the animals in this study would necessarily dispose toward impaired spermatogenesis following vasovasostomy.

Morphology and cytochemistry of acinar secretory granules in normal and isoproterenol-treated rat submandibular glands.

Several fixation procedures have been utilized in a fine-structural study of rat submandibular glands with the aim of correlating the morphology of granule substructure with its composition. Procedures included fixation with aldehyde in a variety of buffers, with or without fixation additives such as tannic acid or calcium chloride. Both immersion and perfusion fixation studies were performed. Osmium tetroxide postfixed tissue was compared with non-postfixed tissue. Thin sections were stained for carbohydrate-containing constituents by either periodic acid-thiocarbohydrazide-silver proteinate or tannic acid-ferric chloride sequences. The morphology and cytochemistry of acinar secretory granules were highly dependent upon the fixation procedure utilized. In postfixed tissue, fine filaments and vesicles were the major granule constituents. Filaments often aggregated into fibrils in fixatives containing phosphate buffer or calcium, whereas vesicles were prominent with fixatives containing collidine buffer. Tannic acid, as a fixation additive, imparted enhanced density to the peripheral rim of aggregated filaments. If post fixation was eliminated, an amorphous meshlike material was the major granule component. This stained readily with methods for carbohydrate whereas the granules of post-osmicated tissue did not. Following chronic isoproterenol treatment the degree of filament aggregation in postfixed tissue was augmented, and this corresponded to an increase in amount of material stainable for carbohydrate in non-post-osmicated tissue.

Beef kidney 3-hydroxyanthranilic acid oxygenase. Purification, characterization, and analysis of the assay.

Beef kidney 3-hydroxyanthranilic acid oxygenase has been purified to homogeneity. It is a single subunit protein of Mr = 34,000 +/- 2,000 with a frictional coefficient (f/f0) of about 1.1. The enzyme readily aggregates to form, apparently inactive, higher molecular weight oligomers. The very rapid loss of enzyme activity during the assay was analyzed extensively. It was found to be due to inactivation of the enzyme by the substrate, 3-hydroxyanthranilate, and unrelated to enzyme turnover or oxidation of bound iron. The loss of activity was shown to be a first order decay process, and methods are given for obtaining accurate initial reaction rates under all conditions. Evidence was presented that the enzyme assumes a catalytically inactive conformation at pH 3.4, which only relatively slowly rearranges to an active form at pH 6.5; the rearrangement can be blocked by the presence of substrate. We have found that Fe2+, which is required for enzymatic activity, can equilibrate freely, albeit slowly, with the enzyme during the course of the enzyme reaction even in the presence of saturating 3-hydroxanthranilate. Under assay conditons, the Fe2+ has an apparent dissociation constant of 0.04 mM. The kinetic properties of the enzyme were found to be dramatically different in beta,beta-dimethylglutarate buffer and collidine buffer; both the rate of loss of activity during the assay and the substrate Km and Vmax were affected.

Technic for the Study of Small, Soft-Bodied Aurelia with Scanning EM

Rapid advances have been made in the preservation and preparation of cells and soft tissues of vertebrates for scanning electron microscopy (SEM). These technics, however, only rarely have been applied to small soft-bodied invertebrates. When an effort was made to preserve intact metamorphosing Aurelia polyps (strobilae) for SEM studies, it became apparent that special methodology was needed. The following technic was developed:Aurelia polyps are induced to metamorphose with 1 x 10-5M thyroxine. Strobilae in the desired stage of metamorphosis are fixed in 2 changes of 3% gluteraldehyde for 1 hr. each. They are rinsed 2X in collidine buffer, pH 7.4 (based on). The organisms are dehydrated in grades of acetone from 30% to 100%. To avoid disturbing the strobilae, acetone is added to the dilute acetone every 10 min. to achieve concentrations of 50, 70, and 95%. The strobilae are rinsed 2X in 100% acetone for 5 and 10 minutes each.

Comparative Examination Between the Chemical and Physical Methods to the Demonstration of the Ionic Localization in the Tissues. I. In the Case of Potassium Antimonate Method

The results involved in the histochemical technique for the detection of sodium(Na) ion have been analysed. Nonspecific precipitation occurs when potassium(K) antimonate reacts with the ethanol used for dehydration, and also may occur when the pH is below 6.8 during fixation. Since calcium(Ca) and magnesium(Mg) ions can form precipitates with antimonate(Sb) there is a possibility of interference by Ca and Mg ions in tissues. There nonspecific reactions must not be confused with the specific histochemical reaction. The K-phosphate and collidine buffer used to control the pH seems to inhibit the precipitation reaction of K-antimonate with Na ion. The reduced formation of precipitate with K in preserve of these buffers might be caused by an altered ionic balance between the dissociated antimonate and other correlating ions, such as K, H( pH below 6.8 ), or collidine. The non-buffered fixative, however, seems to good results at the electron microscopic observation. On the other hand, Legato et al reported on the Ca-ion localization in the cardiac muscle using the same method for the Na-detection.

Pores in the Plasma Membranes of Myocardial Cells

Heart muscle samples were collected from the right ventricles of twelve mature cattle and twelve pigs at slaughter and immediately prepared for electron microscopy by immersion in three different fixatives: 40% OsO4 dissolved in CCl4, 1.5% OsO4 in S collidine buffer, and in 2.5%. glutaraldehyde followed by 1.5%, OsO4. Some tissue from each animal was processed in each fixative and was embedded in either an epoxy resin or in a polyester resin. Sections were cut at 300 to 600 Å in thickness on a thermally advancing microtome, using glass and diamond knives, and were mounted on copper mesh grids. Uranyl acetate and lead citrate stains were used. Tissues were examined in an RCA, EMU-3F electron microscope.In both the cattle and pigs, some heart samples contained myocardial cells with plasma membranes which invaginated into the cells to form tubular appearing channels. These channels occurred in addition to the transverse sarcotubular system, and in addition to the pinocytotic vesicles. The basement membrane which surrounds myocardial cells did not extend into the invaginations, but bridged over their external apertures.

FORMATE-PYRUVATE EXCHANGE REACTION IN STREPTOCOCCUS FAECALIS. I. FACTOR REQUIREMENT FOR INTACT CELLS.

Wood, N. P. (A. & M. College of Texas, College Station), and D. J. O'Kane. Formate-pyruvate exchange reaction in Streptococcus faecalis. I. Factor requirement for whole cells. J. Bacteriol. 87:97-103. 1964.-A factor present in plant and animal sources was found necessary for the incorporation of formate-C(14) into pyruvate by Streptococcus faecalis 10Cl. Yeast extract produced a response linear in the range between 10 and 30 mg/ml of reaction mixture. Soy peptone, beef peptone, and Brain Heart Infusion replaced yeast extract, but various intermediates, cofactors, amino acids, purines, pyrimidines, and peptides did not stimulate the reaction. A lag occurred in the rate of formate incorporation that was not influenced by anaerobic conditions or growth of cells in a medium containing pyruvate and formate. Phosphate or maleate buffer permitted rapid exchange velocities but tris(hydroxymethyl)aminomethane or collidine buffer was inhibitory. Heating yeast extract at 121 C for 15 min in 3 n H(2)SO(4) produced 66% inactivation of the factor(s), whereas treatment with 3 n KOH produced 97% inactivation. The factor(s) was insoluble in butanol, benzene, ethyl acetate, or chloroform. The material adsorbed on Dowex-1 (OH(-)) and Amberlite IR-120 (H(+)) but not on Amberlite IR-4B (OH(-)). The active component(s) was highly polar, nonvolatile, dialyzable, and had amphoteric properties.

L'activité aldolasique normale du liquide céphalo-rachidien

The values of the normal aldolase activity of the cerebrospinal fluid are much lower when incubated at 38° with Tris buffer, pH 8.6 than at 25° with collidine buffer, pH 7.2. Under the later conditions the normal aldolase activity of the cerebrospinal fluid appeared to be of the same order as that of blood serum.

An improved silver method for staining nerve fibres in decalcified sections of teeth

A silver staining method is described in which dewaxed sections of decalcified teeth are impregnated in a saturated solution of silver cyanate in dilute collidine buffer, pH 7.3, and developed in a buffered hydroquinone-sulphite solution. The impregnation-development process is repeated three or four times until a satisfactory intensity of staining is obtained. Non-myelinated nerve fibres are specifically stained black in a pale yellow background.

Aldolase in the culture form of Trypanosoma cruzi

1. 1. Aldolase has been demonstrated in homogenates of the culture form of T. cruzi by three methods for estimating the triose phosphate produced. 2. 2. The limits within which linearity of color production with respect to time and to concentration of enzyme exist have been determined. 3. 3. The Michaelis constant was found to be 1.2 × 10 −3 M HDP. 4. 4. Optimum pH was 7.3 in collidine buffer. 5. 5. Optimum temperature was about 45 °C. 6. 6. Aldolase in homogenates of T. cruzi was unstable when stored for several days at 5 °C. Freezing or dialysis hastened inactivation. Such inactive samples could not be reactivated by cysteine and/or ferrous ions. 7. 7. Copper sulfate inactivated the enzyme completely; pyrophosphate, 8-hydroxyquinoline, iodoacetamide somewhat less. 8. 8. Dipyridyl had no effect on the activity, and no evidence that this is a metal-activated aldolase was obtained. 9. 9. Aldolase activity per gram wet weight of organisms was equivalent to 0.03 Meyerhof units. This indicates that T. cruzi (culture form) has a very low aldolàse activity, and in connection with other calculations, indicates that the organism probably makes considerable use of non-glucose substrates. 10. 10. Indirect evidence for the existence of isomerase is presented.