Incubation In Acid Research Articles

Synergistic effects of oxidative environments and mechanical stress on in vitro stability of polyetherurethanes and polycarbonateurethanes.

The in vitro structural stability of polyetherurethanes (PEUs) and polycarbonateurethanes (PCUs and PCUUs) was examined under strong oxidative conditions (0.5N HNO3, pH 0.3; and NaClO, 4% Cl2 available, pH approximately 13) and in the presence of a constant strain state. Solvent-cast dog-bone shaped specimens were strained at 100% uniaxial elongation over extension devices and completely immersed in the oxidative solutions at 50 degrees C for 15 days. Unstrained polyurethane (PU) samples were treated in the same way for comparison. The modification of the PU molecular structure was determined by DSC, GPC, ATR-FTIR, static contact angle, and surface roughness analyses. The incubation in nitric acid and sodium hypochlorite brought about a greater degradation of samples tested under the applied strain with the exception of PEU treated with nitric acid. PEU was the most affected material, showing bulk deterioration in NaClO and significant modifications in nitric acid, with the appearance of new IR bands, which were assigned to oxidation products. A higher phase separation between soft and hard domains occurred in PCUs upon incubation in nitric acid, the treatment with NaClO gave rise to new bands in the IR spectra, denoting the presence of oxidation products at the surface. The surface roughness greatly increased in strained PCUs with SEM evidence of deep cracks and holes or ragged and stretched fractures perpendicular to the direction of stress. PCUU underwent complex chemical modifications with a marked decrease of N-H and urea IR absorptions and showed a lower degradation than PEU and PCUs under mechanical constraint. From these results, sodium hypochlorite appears to be able to create an ESC-like degradation for PUs that are resistant to other aggressive chemical environments.

Matrix-assisted laser desorption/ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrylamide gels.

A new strategy for the characterization of Coomassie Brilliant Blue stained SDS-PAGE separated proteins by UV-MALDI-MS is reported. The proteins are extracted directly from the polyacrylamide gel by treatment with an organic solvent mixture consisting of formic acid, acetonitrile, isopropanol and water in an ultrasonic bath. A fraction of the supernatant is then mixed directly with the matrix solution and measured by MALDI-MS. High quality spectra could be obtained from gels which were loaded with 6 pmol of myoglobin. Compared to other methods based on electroblotting or electroelution this method is much simpler and less time consuming. The sensitivity is higher than or comparable to the Coomassie Blue staining procedure for proteins up to about 25 kDa. Another advantage is that mass shifts due to charging effects of the membranes, which are common if membranes are mounted directly on the sample target, can be avoided. However, all proteins studied showed slightly higher masses than expected which reduces mass accuracy to 0.2-0.3%. This is presumably partly due to formylation of serine or threonine residues during incubation in formic acid. Gel electrophoresis induced modifications can contribute as well. The possibility of further characterizing the remaining part of the supernatant after extraction by means of proteolytic digestion is also demonstrated. The knowledge of both molecular weight of the whole protein and of the proteolytic fragments increases specificity for protein identification by searching in sequence databases.

Influence of anionic compounds on adsorption behavior of bovine serum albumin at oxide-water interfaces

The influence of anionic compounds on the adsorption behavior of bovine serum albumin (BSA) at oxide-water interfaces was studied in buffer solutions composed of carboxylic acids, phosphate, or KNO 3 at the isoelectric point (IEP) of BSA, using silicon dioxide (SiO 2), titanium dioxide (TiO 2), and aluminum oxide (Al 2O 3) particles. The amount of adsorbed BSA (Γ) decreased in the presence of buffer salt ions, depending on the kind of buffer anion. The degree of decrease in Γ was more pronounced on Al 2O 3 which has a higher positive surface charge density, and less on negatively charged SiO 2. The intrinsic viscosity of BSA increased with increasing ionic strength in all the buffer solutions. The densities of the positive surface charges of the oxides decreased markedly, accompanied by shifts in their IEP's to acidic values, as a result of preincubation in carboxylic acid- and phosphate-buffered solutions, but not in KNO 3 solution. The charge profiles of the BSA-covered oxides obtained in the citrate-buffered solution were more acidic in character, giving more acidic IEP's, compared with the profiles obtained in KNO 3 solution. It was also found that partial desorption of BSA preadsorbed on Al 2O 3 took place during incubation in carboxylic acid- and phosphate-buffered solutions but not in KNO 3 solution. From these results, the repression of BSA adsorption onto oxides was ascribed to the masking of adsorbing sites on the oxide surfaces and partial desorption of adsorbed BSA induced by adsorption of anions of carboxylic acids and phosphate, as well as to the expansion of the BSA molecule due to the charge-shielding action of buffer salt ions.

CGMP Is Required for Gibberellic Acid-Induced Gene Expression in Barley Aleurone.

The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell.

Reconstitution of lactate proton symport activity in plasma membrane vesicles from the yeast Candida utilis.

Lactic acid transport was studied in plasma membrane vesicles from the yeast Candida utilis IGC 3092 which were fused with liposomes containing cytochrome c oxidase. After the addition of an electron donor system, these hybrid membrane vesicles were able to generate a proton-motive force of about--150 mV, inside alkaline and negative. In vesicles prepared from lactic acid-grown cells, the uptake of labelled lactic acid, at pH 6.2, under energized conditions, was expressed by a kinetics consistent with the involvement of a mediated transport system. This carrier exhibited a substrate specificity pattern identical to the one found for the lactate-proton symport in intact cells. The transport of labelled lactic acid was accumulative and strongly sensitive to the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, consistent with the involvement of the proton-motive force in acid uptake, hence with the presence of a proton symport for lactate. Dissipation of the transmembrane electric potential by valinomycin did not have a significant effect on lactate accumulation, whereas abolishing the transmembrane pH gradient (delta pH) by nigericin prevented the accumulation and led to a rapid efflux of the accumulated acid. The data support that the delta pH is the main component of the proton-motive force involved in the transport of the acid and its accumulation. The lactate-proton symport stoichiometry was 1:1, being independent of the pH. Vesicles prepared from glucose-grown cells did not display the capacity to transport and accumulate lactate. However, activity for the carrier was also reconstituted in vesicles obtained from glucose-grown cells after incubation in buffer containing lactic acid. These results were consistent with those obtained in intact cells, which demonstrated that the lactate-proton symport of the yeast C. utilis is inducible.

Effect of age on the fatty acid composition of phospholipids in human lymphocytes.

We have examined the fatty acid composition of phospholipids of unstimulated and PHA-stimulated T cells from young and old donors. Our results demonstrate that aging is accompanied by decreases in the saturated fatty acids, myristic acid, and palmitic acid, and a concomitant increase in the unsaturated arachidonic acid. Following activation with PHA for 24 h, age-associated differences in fatty acids could no longer be detected. In contrast to the lymphocyte, aging did not affect the fatty acid composition of either serum or neutrophil phospholipids. Exposure of lymphocytes from old donors to myristic acid complexed medium increased the levels of myristate in the phospholipids to levels similar to that seen in lymphocytes from young donors. We conclude from these studies that aging is accompanied by an alteration in the fatty acid profiles of phospholipids, and that incubation in myristic acid complexed medium modulates these profiles. These alterations are unique to lymphocytes and may contribute to the age-related declines in lymphocyte function.

A simple colorimetric assay for muramic acid and lactic acid

The Barker and Summerson method of assaying lactic acid colorimetrically is modified to provide a simple and fast method of measuring lactic acid and other compounds such as muramic acid and glyceraldehyde that will release acetaldehyde on incubation in hot sulfuric acid. The assay can be done with open tubes and no more complicated equipment than a spectrophotometer. A further modification allows a relatively specific determination of formaldehyde.

Methyl jasmonate and bean leaf abscission

The effect of rac-methyl jasmonate, both in solution and as a vapour, on the separation of pulvinar and petiolar tissues in explants containing the distal abscission zone of primary leaves of Phaseolus vulgaris var. Contender was investigated. The effects of rac-methyl jasmonate were compared to those of (±)-abscisic acid, α-naphthalene acetic acid, ethylene and 2-chloroethylphosphonic acid. Abscission times were determined in explants prepared from 14-day-old control plants and in explants prepared from plants that had been pretreated for 24h with the ethylene-action inhibitor, silver thiosulphate. While silver-pretreatment, or treatment with α-naphthalene acetic acid delayed abscission, treatment with ethylene or 2-chloroethylphosphonic acid accelerated tissue separation. However, (±)-abscisic acid delayed abscission under these conditions. In all instances, treatment with rac-methyl jasmonate had no apparent effect on abscission. The loss of chlorophyll from bean leaf discs incubated in the dark was enhanced by treatment with 2-chloroethylphosphonic acid or (±)-abscisic acid and was retarded in discs incubated in benzyl adenine. While incubation in α-naphthalene acetic acid was without effect, incubation in solution of rac-methyl jasmonate also retarded chlorophyll loss when compared to water controls.

In Vitro cDNA Amplification from Individual Intestinal Crypts: A Novel Approach to the Study of Differential Gene Expression along the Crypt-Villus Axis

The mouse small intestine is lined with a monolayer of continuously renewing epithelial cells. Cells of four distinct epithelial lineages are derived clonally from the stem cell zone, located near the crypt base, from which cells differentiate and migrate to the villus tip. The kinetics of the multilineage process are well understood. However, the molecular mechanisms underlying gene expression during lineage commitment and cell proliferation and differentiation remain obscure. A novel approach to the problem is presented here. Single intact epithelial crypts were isolated by incubation in ethylenediaminetetraacetic acid and mechanical vibration of everted mouse intestinal or colonic segments. Crypts isolated in this manner were suitable for mRNA-directed polymerase chain reaction, thus generating crypt epithelium-specific cDNA. The fidelity of transcript amplification was confirmed by Southern blot hybridization with cloned intestinal transcripts. To demonstrate the potential utility of crypt-specific cDNA, the amplified transcripts from a single jejunal crypt were used to construct a cDNA library, characterization of which revealed a high representation of cryptdin-1-related transcripts. This study presents a technique which will facilitate comprehensive analyses of gene expression in the differentiating mammalian intestine.

Fibre type distribution in the muscle spindles of cat jaw-elevator muscles

Small samples of the masseter and temporalis muscles of six adult male cats were dissected free, snap-frozen and cryosectioned. Serial sections from areas with muscle spindles were histochemically stained for myofibrillar ATPase after incubation in acid and alkali buffer, and for reactivity to two different myosin isoform-specific antibodies. Differential reactivity to these four stains identified bag 1, bag 2 and chain intrafusal fibre types. The most typical distribution of fibres within the spindle was 1 bag 1, 1 bag 2 and 3–4 chains. Although a few spindles were found in the anterior area of the temporalis that attaches to the coronoid process of the mandible and in the anterior area of the superficial masseter, the vast majority were found in the deepest portion of the masseter. Here spindles were characteristically with a number of simple spindles fused together into complex units.

Purified human neuromelanin, synthetic dopamine melanin as a potential model pigment, and the normal human substantia nigra: characterization by electron paramagnetic resonance spectroscopy.

Neuromelanin is a poorly understood pigment that accumulates in catecholaminergic neurons during normal aging. Electron paramagnetic resonance spectroscopy, an especially effective technique for investigating melanins, is used in the present study to show unambiguously that neuromelanin is a melanin; however, it is not well modeled by synthetic dopamine melanin and thus is an atypical melanin. Some of the unusual features of neuromelanin can be explained by postulating two distinct sources for its free radicals, the dominant one possibly derived from a precursor containing sulfur. Examination of human substantia nigra by electron paramagnetic resonance spectroscopy during the purification of neuromelanin also demonstrates, contrary to some other studies, that a portion of the paramagnetic metal ions in this tissue are bound to the pigment in situ. Combined with previous histochemical data, these observations have implications for the mechanism through which neuromelanin accumulates in vivo and are consistent with its having a cytoprotective function under normal conditions, but a cytotoxic role at advanced ages and in patients with Parkinson's disease. Other results of this study show that homogenizing tissues during the purification of any natural pigment may cause contamination of the pigment by extraneous metal ions and that subsequent incubation in hot acid, though most effective in removing metal ions and hydrolyzing proteins, leads to degradation of melanin. A purification procedure using incubation in acid at room temperature, however, is well suited for identifying and characterizing unknown natural pigments by electron paramagnetic resonance spectroscopy.

Abscisic Acid Enhances the Ability of the Desiccation-Tolerant FernPolypodium virginianumto Withstand Drying

Detached fronds of Polypodium virginianum L. survived loss of 65-70% of their fresh weight over 10 d of slow-drying. Drying over silica gel resulted in a faster rate of water loss, to a lower fresh weight, which the fronds did not survive. When fronds were incubated in abscisic acid for 24 h prior to silica-drying, the amount of water lost was reduced, resulting in survival of the fronds upon subsequent rehydration. Incubation in abscisic acid for at least 18 h was necessary for survival. Fronds in which the final fresh weight after drying was below a critical amount (i.e. to less than 25% original fresh weight) did not survive. A reasonable correlation could be drawn between electrolyte leakage upon rehydration and survival of some desiccation treatments, although this was not always clear-cut, especially in fronds incubated in abscisic acid for an insufficient time to ensure survival. Several polypeptides were synthesized during slow- and silica-drying, and in response to abscisic acid. No novel polypeptides were identified that were unique to the desiccation regimes which resulted in survival. Nor did ABA induce specific proteins in fronds desiccated after preincubation in this regulator for 24 h.

Conversion of hydrolysates of corn cobs and hulls into ethanol by recombinantEscherichia coli B containing integrated genes for ethanol production

Hemicellulose and residual starch in corn hulls from wet milling and hemicellulose in corn cobs were hydrolyzed by incubation in dilute sulfuric acid at 140°C to 160°C. These hydrolysates were efficiently fermented to ethanol by a genetically engineered derivative ofE. coli B, strain KO11. Fermentation of com hull hydrolysate was complete after 48 h with a final ethanol concentration of 38 grams per liter. Fermentation of corn cob hydrolysate was essentially complete after 24 h due to a lower concentration of sugars and higher levels of inocula. In both cases, ethanol produced was equivalent to 100% of the maximum theoretical yield (0.51 grams ethanol/gram sugar) based on momoner sugar content. ThusE. coli B strain KO11 appears to be an excellent candidate for the efficient production of ethanol from hydrolysates of corn residues.

Facile separation of the subunits of ovine and bovine gonadotrophins by high-performance liquid chromatrography: microheterogeneity and biological activity

The alpha and beta subunits of ovine and bovine gonadotrophins which undergo instant dissociation in 0.1% (v/v) trifluoroacetic acid in water could be separated into their multiple components by reverse-phase high-performance liquid chromatography on analytical and preparative scales. Several batches of highly purified ovine LH (oLH) separated into four alpha components (alpha 1 to alpha 4) and five beta components (beta 1 to beta 5). While the major alpha component (alpha 3) remained the same in all, the beta subunit was variable in all these preparations of oLH of equal biological and immunoreactivity. Various manipulations, such as prolonged incubation in dilute acid or alkali or conditions prone to air oxidation, did not change elution patterns or produce interconversions among the alpha or beta components, suggesting that microheterogeneity arose in the pituitary itself or during its collection. The subunits obtained on a preparative scale were of high yield and quality and recombined (100%) to produce fully active LH or FSH. Chemical deglycosylations in which 75-80% of the peripheral sugars were removed shifted the elution of all components (alpha as well as beta) slightly but modifications to the protein backbone by other reagents changed the profile of the alpha subunit. Obtaining the various alpha and beta components in high yield would permit a complete analysis of gonadotrophin microheterogeneity.

Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.

Differentiation of human variant small cell lung cancer cell lines to a classic morphology by retinoic acid : Doyle LA, Giangiulo D, Hussain A, Park H-J, Yen R-WC, Borges M. University of Maryland Cancer Center, 655 W. Baltimore St., Baltimore, MD 21201. Cancer Res 1989;49:6745-51

Variant small cell lung cancer (SCLC) is distinguished from the classic histology by changes in growth characteristics and morphology, c-myc amplification, a loss of some biochemical markers, and relative chemo- and radioresistance. Three variant SCLC lines were incubated in 1 microM all-trans retinoic acid. After 8-10 days, a marked change in morphology was noted in all three lines, with tight cell aggregation and central necrosis of large floating spheroids similar to classic SCLC. The retinoid-treated cells demonstrated decreases in growth rate and cloning efficiency to levels comparable with classic SCLC cell lines. Retinoic acid incubation caused a reproducible decrease in c-myc expression in variant SCLC cells, but was not noted to increase L-dopa decarboxylase, an enzyme which biochemically distinguishes classic from variant SCLC cell lines. Retinoid exposure led to an increase in HLA and Leu-7 antigens, but a reduction of antibody binding to 3-fucosyllactosamine, a dominant SCLC glycolipid antigen. Clonogenic assays revealed that the variant cells, after incubation in retinoic acid, became more sensitive to etoposide, but more resistant to Adriamycin. We conclude that exposure of variant SCLC cells to retinoic acid can lead to a morphologic phenotype similar to classic SCLC, but with substantial differences in cell biology. ?

Effect of hyperthermia on selective expression of HL-60 heat shock proteins

Hyperthermia is used experimentally to treat human malignancy. The effect of heat delivery rate and thermotolerance on the expression of heat shock proteins (hsp) by the human HL-60 cell line before and after differentiation was studied. This leukemia cell synthesized multiple hsp in response to elevated temperatures. The most obvious and consistent proteins were within the highly conserved stress-inducible family of polypeptides hsp70 which resolved as a hsp69/72 doublet. Cells which were made thermotolerant by gradual heating selectively failed to express the hsp70 doublet even though other hsp were synthesized. Mature HL-60 cells induced to differentiate by incubation in retinoic acid expressed a full complement of hsp when exposed to immediate heat, but there was selective deletion of hsp70 with gradual hyperthermia. This model for selective induction of hsp confirms that synthesis of hsp and thermotolerance can be dissociated in the HL-60. It suggests that the hsp70 does not play an obligatory role in thermotolerance of this human leukemia cell.

Central GABAergic innervation of the pituitary in goldfish: A radioautographic and immunocytochemical study at the electron microscope level

The GABAergic innervation of the goldfish pituitary was studied at the light and electron microscope levels by means of radioautography after in vitro incubation in tritiated γ-aminobutyric acid (GABA) and immunocytochemistry using antibodies against GABA. Following incubation of pituitary fragments in a medium containing tritiated GABA, a selective uptake of the tracer was observed within the digitations of the neurohypophysis. Silver grain clusters were also observed in the adenohypophyseal tissue. At the electon microscope level, this uptake was found to correspond to nerve endings containing small clear and dense-core vesicles. These labeled profiles were located mainly in neurohypophyseal digitations in close apposition with the baement membrane separating the neurohypophysis from the adenohypophysis. However, they were also encountered in direct contact with most adenohypophyseal cell types in the different lobes. These results were confirmed by immunocytochemical data demonstrating the presence of numerous GABA immunoreactive fibers in both anterior and neurointermediate lobes. They were found either in the digitations of the neurohypophysis or in the adenohypophysis in direct contact with the glandular cells with a distribution and an ultrastructural aspect similar to those observed by radioautography. These data demonstrate that the pituitary of teleosts receives a massive GABAergic innervation. Although physiological data providing a functional significance for such an innervation are lacking, the present study suggests that, as already documented in mammals, GABA may be involved in the neuroendocrine regulation of pituitary functions in teleosts.

Rapid Simultaneous Estimation of Protoporphyrin and Mg-Porphyrins in Higher Plants

A rapid, inexpensive method has been derived that allows the simultaneous determination of Protoporphyrin and Mg-Porphyrins in the same extract.The procedure allows for accurate (> 90%), repeatable estimates in the nanomolar range.Analyses of experimental conditions known to alter porphyrin metabolism, such as illumination of etiolated material or incubation in 5-aminolevulinic acid, give results that are consistent with those reported by others. Using this procedure, it is shown that one characteristic of chilling induced chlorosis involves a non-lethal alteration in the ability of etiolated tissue to synthesize chlorophylls.

Isolation of intestinal mononuclear cells from colonoscopic biopsies for immunofluorescence analysis by flow cytometry.

A method for isolating and characterizing intestinal lymphoid cells from colonoscopic biopsies is presented. Intraepithelial lymphocytes were separated from the lamina propria by incubation in edetic acid (EDTA) and lamina propria lymphoid cells isolated by incubation in collagenase followed by Ficoll-Hypaque density flotation. Quantitation of T lymphocyte helper (OKT4) and suppressor (OKT8) cells was performed using monoclonal antibodies to cell surface markers and analyzed on a flow cytometer. The isolation procedure yielded approximately 400,000 lamina propria cells and 100,000 intraepithelial cells per sample, with better than 90% viability. Surface marker analysis demonstrated significant differences in the ratios of helper to suppressor cells between the intraepithelial lymphocytes and the lamina propria lymphocytes. These demonstrate the feasibility of lymphoid cell isolation from colonoscopic biopsy specimens for surface marker analysis by flow cytofluorimetry. These techniques could prove important in the study of immune mechanisms in inflammatory bowel diseases.