Inactivation of prions by acidic sodium dodecyl sulfate

Prompted by the discovery that prions become protease-sensitive after exposure to branched polyamine dendrimers in acetic acid (AcOH) (S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay, F. C. Szoka, F. E. Cohen, S. B. Prusiner, and M. R. Scott, J. Virol. 75:3453-3461, 2001), we investigated the inactivation of prions by sodium dodecyl sulfate (SDS) in weak acid. As judged by sensitivity to proteolytic digestion, the disease-causing prion protein (PrPSc) was denatured at room temperature by SDS at pH values of < or =4.5 or > or =10. Exposure of Sc237 prions in Syrian hamster brain homogenates to 1% SDS and 0.5% AcOH at room temperature resulted in a reduction of prion titer by a factor of ca. 10(7); however, all of the bioassay hamsters eventually developed prion disease. When various concentrations of SDS and AcOH were tested, the duration and temperature of exposure acted synergistically to inactivate both hamster Sc237 prions and human sporadic Creutzfeldt-Jakob disease (sCJD) prions. The inactivation of prions in brain homogenates and those bound to stainless steel wires was evaluated by using bioassays in transgenic mice. sCJD prions were more than 100,000 times more resistant to inactivation than Sc237 prions, demonstrating that inactivation procedures validated on rodent prions cannot be extrapolated to inactivation of human prions. Some procedures that significantly reduced prion titers in brain homogenates had a limited effect on prions bound to the surface of stainless steel wires. Using acidic SDS combined with autoclaving for 15 min, human sCJD prions bound to stainless steel wires were eliminated. Our findings form the basis for a noncorrosive system that is suitable for inactivating prions on surgical instruments, as well as on other medical and dental equipment.     

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: myosin-V assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 oC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.     

Bioconversion of municipal solid waste to glucose for bio-ethanol production

Selected biodegradable municipal solid waste fractions were subjected to fifteen different pre-hydrolysis treatments to obtain the highest glucose yield for bio-ethanol production. Pre-hydrolysis treatments consisted of dilute acid (H₂SO₄, HNO₃ or HCl, 1 and 4%, 180 min, 60°C), steam treatment (121 and 134°C, 15 min), microwave treatment (700 W, 2 min) or a combination of two of them. Enzymatic hydrolysis was carried out with Trichoderma reesei and Trichoderma viride (10 and 60 FPU g⁻¹ substrate). Glucose yields were compared using a factorial experimental design. The highest glucose yield (72.80%) was obtained with a pre-hydrolysis treatment consisting of H₂SO₄ at 1% concentration, followed by steam treatment at 121°C, and enzymatic hydrolysis with Trichoderma viride at 60 FPU g⁻¹ substrate. The contribution of enzyme loading and acid concentration was significantly higher (49.39 and 47.70%, respectively), than the contribution of temperature during steam treatment (0.13%) to the glucose yield.     

Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections.

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl(2) for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120 C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl(2), 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor alpha (ERalpha) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERalpha immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl(2) at 40 C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.     

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.     

Irreversible thermoinactivation of ribonuclease‐A by soft‐hydrothermal processing

Ribonuclease (RNase), which often represents molecular biological contamination, is a thermostable enzyme. When RNase is heated at 121°C by autoclave sterilization for 20 min, it does not lose its activity. However, the nature of the molecular events by which the irreversible denaturation occurs remains unknown. The purpose of this study was to elucidate the molecular mechanisms of irreversible thermal denaturation of RNase A and to develop an advanced sterilization method using soft‐hydrothermal processing, which has the advantages of improved safety and cost‐efficiency. The enzymatic activity of RNase was measured using polyacrylamide gel electrophoresis with torula yeast RNA. We evaluated the temperature and time course of irreversible thermoinactivation of RNase by normal autoclaving, hot‐air sterilization, and soft‐hydrothermal processing that had been controlled to the desired steam saturation ratio. The results indicated that RNase A was deactivated by autoclave sterilization (121°C, 20 min) immediately after treatment, but was reactivated over time. Hot‐air sterilization (180°C, atmospheric pressure, 60 min) produced results similar to that of autoclave sterilization. In contrast, RNase A was irreversibly thermoinactivated by soft‐hydrothermal processing (110°C, 20 min) at 100% steam saturation ratio. We also determined that the mechanism of irreversible thermoinactivation of RNase A involved hydrolysis and deamidation under this condition at a steam saturation ratio of more than 100%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Comparative Study of Antigen Retrieval Heating Methods: Microwave, Microwave and Pressure Cooker, Autoclave, and Steamer

We present a study comparing the most popular beating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies; the microwave pressure cooker, extended microwave heating (5 min × 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min × 2 J. Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for “maximal retrieval” as a common end point for all laboratories.     

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4°C, and more than a year at ?20°C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

Retention of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4 degrees C, and more than a year at -20 degrees C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis

Abstract: Paired helical filaments (PHFs), a characteristic neuropathologic finding in Alzheimer's disease brain, are abnormal fibrillary forms of hyperphosphorylated tau (PHF-tau), which have been shown to be highly resistant to calpain digestion. Either excessive phosphorylation or fibrillary arrangement of tau proteins in PHFs may play a role in proteolytic resistance by limiting access to calpain recognition/digestion sites. To determine the contribution of the fibrillary conformation, isolated PHFs were subjected to treatment with either formic acid or guanidine. Both procedures effectively abolished the fibrillary structure of PHF but preserved PHF-tau immunoreactivity using a panel of antibodies that recognize nonphosphorylated and phosphorylated epitopes. These treatments also significantly increased the sensitivity of PHF-tau polypeptides to calpain proteolysis as shown by significant decreases in the half-life ( t _1/2) from the infinite with native PHF to 44 min and 4.4 min in formic acid- or guanidine-treated samples, respectively. In contrast, the sensitivity of normal fetal tau (3.4 min) was either decreased (5.9 min) or unaffected (3.6 min) by similar treatment. Our results indicate that after guanidine treatment, the sensitivity of PHF to calpain resembles that of fetal tau. These results strongly suggest that the fibrillary structure of PHF-tau, rather than hyperphosphorylation, is the major factor responsible for the resistance of abnormal filaments to calpain-mediated proteolysis.     

Optimum conditions of autoclaving for hydrolysis of proteins and urinary peptides of prolyl and hydroxyprolyl residues and HPLC analysis

A method for urinary peptide(s) and protein hydrolysis, involving autoclaving at 15psi (121 degrees C) for 60min, is described. Using three candidate proteins (bovine serum albumin, casein and gelatin) and urine specimens, the effect of autoclaving with respect to the optimum time required for hydrolysis under both acidic (6N HCl) and alkaline (6N KOH) conditions was studied. Recoveries of total amino acids from proteins and urine hydrolysate(s) suggest that complete hydrolysis of proteins and urinary peptides could be achieved by autoclaving for 30-60min instead of 16h of incubation at 110 degrees C. Further, stability of some of the individual amino acids was also studied. The observed differential stability of amino acids under acidic and alkaline conditions, as demonstrated in this study by HPLC analysis, makes it imperative to choose the appropriate hydrolytic condition while studying the composition of any given amino acids in urinary peptide(s)/protein hydrolysates. Further, the finding that both Pro and Hyp were stable under alkaline conditions of hydrolysis by autoclaving renders this method suitable for assaying these two amino acids from urine hydrolysates, hence its utility in the study of urinary peptide derived Hyp and Pro in bone/cartilage disorders.     

Molecular hybridization with RNA probes in concentrated solutions of guanidine thiocyanate

Hybridizations with RNA probes were performed in 3-6 M guanidine thiocyanate concentrations where cells can be solubilized. At these concentrations the melting temperature of hybrids and the optimum temperature of hybrid formation are greatly reduced. The rate of hybridization in GuSCN at room temperature was greater than that in 50% formamide at 42 degrees C. Hybridizations were performed on DNA and RNA in cells dissolved in GuSCN without prior nucleic acid purification.     

Killing and Preserving Nematodes in Soil Samples with Chemicals and Microwave Energy

Three basic procedures for treating nematode-bearing soil samples for international shipment or from areas under quarantine were tested for their killing effect and recovery of nematodes by sugar flotation for diagnostic and advisory purposes. These were: fumigation with methyl bromide followed by storage at -15 C; microwave treatment (2450 MHz, 630 w, 2-5 min) followed by addition of FAA + picric acid or 5% Formalin; and adding chemical preservatives (FAA + picric acid, 5% Formalin, NAN₃, and 2-phenoxyethanol) directly to the soil. Larvae of Heterodera glycines in eggs within cysts were stimulated to hatch by 2-rain exposure to microwaves, and an exposure of 5 rain was required to kill them. Soil type and moisture significantly affected microwave effectiveness. Direct saturation of soil samples with preservative chemical solutions (FAA + picric acid or 5% Formalin) was most effective, and often increased the number of nematodes recovered. The high concentration (2%) of NaN₃ a required for soil sterilization is too hazardous for routine work. NaN₃, therefore, is not recommended for this purpose.     

Changes in the fine structure of microbial cells induced by chaotropic salts

The electron microscopic examination of the thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37 degrees C for 3-5 h or at 100 degrees C for 5-6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

Isolation of Low-Molecular-Weight Proteins from Amyloid Plaque Fibers in Alzheimer''s Disease

During aging of the human brain, and particularly in Alzheimer's disease, progressive neuronal loss is accompanied by the formation of highly stable intra- and extraneuronal protein fibers. Using fluorescence-activated particle sorting, a method has been developed for purifying essentially to homogeneity the extracellular amyloid fibers that form the cores of senile plaques. The purified plaque cores each contain 60-130 pg of protein. Their amino acid composition shows abundant glycine, trace proline, and approximately 50% hydrophobic residues; it resembles that of enriched fractions of the paired helical filaments (PHF) that accumulate intraneuronally in Alzheimer's disease. Senile plaque amyloid fibers share with PHF insolubility in numerous protein denaturants and resistance to proteinases. However, treatment of either fiber preparation with concentrated (88%) formic acid or saturated (6.8 M) guanidine thiocyanate followed by sodium dodecyl sulfate causes disappearance of the fibers and releases proteins migrating at 5-7,000 and 11-15,000 Mr which appear to be dimerically related. Following their separation by size-exclusion HPLC, the proteins solubilized from plaque amyloid and PHF-enriched fractions have highly similar compositions and, on dialysis, readily aggregate into higher Mr polymers. Antibodies raised to the major low-Mr protein selectively label both plaque cores and vascular amyloid deposits in Alzheimer brain but do not stain neurofibrillary tangles, senile plaque neurites, or any other neuronal structure. Thus, extraneuronal amyloid plaque filaments in Alzheimer's disease are composed of hydrophobic low-Mr protein(s) which are also present in vascular amyloid deposits. Current evidence suggests that such protein(s) found in PHF-enriched fractions may derive from copurifying amyloid filaments rather than from PHF.     

Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies

Formaldehyde fixation of biopsy specimens for routine purposes has often been held responsible for the poor reproducibility of immunohistochemical studies. Recently, antigen retrieval (AGR) using microwave irradiation was described as a potential tool to enhance immunostaining. A comparison of conventional staining and staining after microwave heating was performed for 52 markers, using tissues fixed in formaldehyde for 24 h, 1 to 6 weeks and 3 years respectively, as well as consultant case material. After adequate duration of fixation (24 h), only a few markers (17%) showed better results after AGR, but this percentage was increased to 50% when tissues were fixed for longer periods. Maximal enhancement was obtained in the group of consultant cases (58% of tested markers demonstrated better staining results), in which the period of fixation and tissue processing was unknown. To achieve reliable enhancement with AGR, continuous heating (100° C) should not be shorter than 20 min. In conclusion, AGR may become the most important tool to simplify and equalize immunohistochemical techniques, if critically evaluated.     

Microwave irradiation for rapid epidermis-dermis separation and improved epidermal cell immunodetection

We explored the effects of microwave irradiation on epidermal-dermal separation and subsequent immunostaining of epidermal cells. Epidermal sheets were obtained after incubation in 0.02 M EDTA in PBS and microwave irradiation with 4 pulses of 420 watts for 5 sec, with a total incubation period of 4 min. The control epidermal sheets were immunostained for Langerhans cells and dendritic epidermal T cells using a conventional immunoperoxidase method. The experimental immunodetection of these cells was assisted by incubating the primary antibodies for 10 min at 70 watts. We showed a simple and rapid method for separation of the epidermal-dermal junction and immunostaining of epidermal cells with optimal morphological preservation.     

Chemical, thermal and pH-induced equilibrium unfolding studies of Fusarium solani lectin

The effect of urea, guanidine thiocyanate, temperature and pH was studied on the conformational stability of Fusarium solani lectin. Equilibrium unfolding with chemical denaturants showed that the lectin was least stable at pH 12 and maximally stable at pH 8.0 near its pI (8.7). Guanidine thiocyanate (the concentration of denaturant at which the protein is half folded, D1/2 = 0.49 M at pH 12) was found to be an eight times stronger denaturant than urea (D1/2 = 3.88 M at pH 12). The unfolding curves obtained with fluorescence and CD measurements showed good agreement indicating a monophasic nature of unfolding and excluded the possibility of formation of any stable intermediate. The effect of pH on the lectin was found to be unusual as at acidic pH, the lectin showed a flexible tertiary structure with pronounced secondary structure, and retained its hemagglutinating activity. On the other hand, the lectin did not show any loss of conformation or activity upto 70 degrees C for 15 min. Moreover, thermal denaturation did not result in the aggregation or precipitation of the protein even at high temperatures. Thermal denaturation was also carried out in the presence of a low concentration of guanidine thiocyanate. Change in the enthalpy of transition (DeltaHm) varied linearly with transition temperature (Tm), which indicated that the heat capacity (DeltaCp = 3.95 kJ . mol-1 . K-1) of the lectin remained constant during the unfolding.     

Unmasking antigen determinants in amyloid

Rehydrated paraffin sections of formalin-fixed, amyloid-containing tissues were treated with denaturing agents (guanidine and urea) and reducing agents (DDT and mercaptoethanol) before immunostaining, in an attempt to expose antigenic determinants hidden in the rigid structure of amyloid fibrils. Pre-treatment overnight with 6 M guanidine or urea was found beneficial, especially in specimens from familial amyloid polyneuropathy of the Portuguese type. Addition of reducing agents had no major effect. Modifications of this method may be useful in unmasking other antigens that are polymerized or considered destroyed by fixation and paraffin embedding.     

Methods for staining amyloid in tissues: a review

The traditional way of identifying amyloid in tissue sections has been staining with Congo red and demonstration of green birefringence under crossed polarizers. The original method of Congo red staining, described by Bennhold in 1922, has undergone several modifications to improve its sensitivity, specificity, and reliability. The most common modification is the alkaline Congo red method described by Puchtler and co-workers in 1962. Specificity is improved by using freshly prepared stain and a staining solution fully saturated with sodium chloride. Amyloid proteins can be further distinguished by autoclaving or by treating the tissue with potassium permanganate or alkaline guanidine. Autoclaving the tissues at 120 C for 30 min causes protein AA to lose its affinity for Congo red. Prolongation of autoclaving to 120 min abolishes the Congophilia of protein AL, but prealbumin-related amyloid shows little or no change. Treatment of the tissue with potassium permanganate causes protein AA and B2-microglobulin amyloid to lose their affinity to Congo red. Protein AA fails to stain with Congo red after treatment with alkaline guanidine for 1 min and protein AL and systemic senile amyloid protein (SSA) after 2 hr. Familial amyloid protein (FAP), prealbumin type, can stand 2 hr of alkaline guanidine treatment without losing its ability to stain with Congo red. Other methods of detection of amyloid include fluorescent stains, e.g., thioflavin T or S, and metachromatic stains such as crystal violet. Immunofluorescence and immunoperoxidase methods are used to identify and classify amyloid proteins in tissues. Antibodies against the P component, proteins AA and AL and FAP have been used with great precision. Due to cross-reactivity, these methods do not differentiate between some types of familial and senile systemic amyloidosis.