A simplified aluminum stain in paraffin sections of bone from hemodialysis patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

Niagara Blue 4B As A Fast Simple Stain for the Adenohypophysis

For differentiation of cells of the adenohypophysis, the Niagara blue 4B method requires no special preliminary fixative nor very fresh tissue, and requires no more time than routine hematoxylin-eosin (H-E) staining. The method requires fixation in 10% formalin. After processing to paraffin wax, deparaffinise and hydrate the sections and stain in 1% aqueous Niagara blue 4B solution for 2 min. Stain afterwards with hematoxylin for 1 min then differentiate, wash, dehydrate, clear and mount. This method can be used also for staining old HE slides by removing the covers, applying the Niagara blue 4B and restaining with eosin. The Niagara blue 4B combined with H-E gives the best and most colorful result. This method allows special staining of the adenohypophysis from human post-mortem material to become routine.     

CDRI-85/287: studies on competition to estrogen binding sites in the immature rat uterus.

Ability of compound CDRI-85/287, a new nonsteroidal antiestrogen with negligible inherent estrogenicity, to inhibit uptake of 3H-estradiol (3H-E2) by the immature rat uterus in vivo was investigated. Different doses of 85/287 were administered either intraperitoneally 30 min before 3H-E2 or orally 1 and 6 hr before 3H-E2. A dose dependent inhibition in 3H-E2 uptake was observed after administration of the compound by either route and was 69% at 50 micrograms/rat ip dose and 80% at 2.5 mg/kg po dose. In in vitro competitive binding assay, however, the compound showed poor affinity (RBA 0.42% of estradiol-17 beta) for cytosolic estrogen receptors. Considering the potent anti-estrogenic as well as anti-implantation efficacy of the compound, its action in vivo appears to be mediated via its active metabolite(s).     

The calcification of cartilage matrix in chondrocyte culture: studies of the C-propeptide of type II collagen (chondrocalcin)

We have shown that when chondrocytes are isolated by collagenase digestion of hyaline cartilage from growth plate, nasal, and epiphyseal cartilages of bovine fetuses they rapidly elaborate an extracellular matrix in culture. Only growth plate chondrocytes can calcify this matrix as ascertained by incorporation of 45Ca2+, detection of mineral with von Kossa's stain and electron microscopy. There is an extremely close direct correlation between 45Ca2+ incorporation in the first 24 h of culture and the content of the C-propeptide of type II collagen, measured by radioimmunoassay, at the time of isolation and during culture. Moreover, growth plate cells have an increased intracellular content of the C-propeptide per deoxyribonucleic acid and, during culture, per hydroxyproline (as a measure of helical collagen) compared with nasal and epiphyseal chondrocytes. In growth plate chondrocytes 24,25-dihydroxycholecalciferol (24,25-[OH]2D3), but not 1,25-dihydroxycholecalciferol alone, stimulates the net synthesis of the C-propeptide and calcification; proteoglycan net synthesis is unaffected. Together, these metabolites of vitamin D further stimulate C-propeptide net synthesis but do not further increase calcification stimulated by 24,25-(OH)2D3. These observations further demonstrate the close correlation between the C-propeptide of type II collagen and the calcification of cartilage matrix.     

Involvement of peripheral TRPV1 in TMJ hyperalgesia induced by ethanol withdrawal

Ethanol withdrawal increases nociception after the injection of formalin into the rat's temporomandibular joint (TMJ). Little is known about the neurological basis for hyperalgesia induced by ethanol withdrawal, but it has been reported that ethanol can potentiate the response of transient receptor potential vanilloid receptor-1 (TRPV1) in superficial tissues. The present study was designed to test the hypothesis that peripheral TRPV1 could be involved on nociceptive behavioral responses induced by the injection of formalin into the TMJ region of rats exposed to chronic ethanol administration and ethanol withdrawal. Behavioral hyperalgesia was verified 12 h after ethanol withdrawal in rats that drank an ethanol solution (6.5%) for 10 days. In another group submitted to the same ethanol regimen, the selective vanilloid receptor antagonist capsazepine (300, 600 or 1200 microg/25 microl) or an equal volume of vehicle were injected into the TMJ regions 30 min before the TMJ formalin test. The local injections of capsazepine reduced the increased nociceptive responses induced by ethanol withdrawal. The effect of capsazepine on rats that did not drink ethanol was not significant. These results indicate that the peripheral TRPV1 can contribute to the hyperalgesia induced by ethanol withdrawal on deep pain conditions.     

Enhancement of the periodic acid--Schiff (PAS) and periodic acid--thiocarbohydrazide--silver proteinate (PA-TCH-SP) reaction in LR white sections

LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination: 1) The PAS reaction in semithin sections turns out stronger after partial (70% ethanol) than complete (100% ethanol) dehydration of the tissue before its transfer to 100% LR White. 2) Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end. 3) The use of hot silver proteinate (50 degrees C) plus strong silver enhancement (15-20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.--Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.     

Museum fish specimens and molecular taxonomy: A comparative study on DNA extraction protocols and preservation techniques

Museum fish specimens are invaluable resources for genetic studies, but extraction of high quality DNA is often problematic. In this study, hairtail fishes of the genera Trichiurus and Lepturacanthus (family: Trichiuridae) representing a wide range of preservation histories and three different methods of preservation were analyzed for mitochondrial DNA (mtDNA) extraction, amplification and sequencing of marker genes. A total of six protocols, including a commercially available kit, were compared in this study. Amplification of conserved genes such as16S rRNA and 12S rRNA were done using polymerase chain reaction with sequence analyses using automated capillary sequencing techniques. The results show that mtDNA extraction, amplification and sequencing of conserved genes could be obtained successfully from frozen (?20°C) preserved specimens (1–5 years) and also from ethanol (95%) fixed specimens (2–5 years) but not from any of the formalin (10%) fixed specimens (3–4 years). However, specimens that have been fixed for only 7 days in buffered formalin (10% formalin with phosphate buffer containing 173 mm salt) and ethanol (95%) could yield successful mtDNA extraction, amplification and sequence information of both 16S rRNA and 12S rRNA.     

Experimental calcification in rat submandibular gland.

The present study describes the phenomenon of calciphylaxis, rapid calcification due to treatment with sensitizer dihydrotachysterol (DHT) and challenging agent 5-hydroxytryptamine (5-HT) in the rat submandibular gland (SMG) in terms of light and electron microscopy, and histochemistry. For biophysical analysis of the calcified bodies, X-ray microanalysis (XMA) and X-ray powdered diffraction methods were used. The calcified lesions in the salivary glands were histologically divided into 3 types: type 1, calcification of basal membranes in duct-like structures; type 2, granular calcified materials with remarkable necrotic changes in cell, containing 3 kinds of small vesicular structures observed in electron microscopy; and type 3, von Kossa's positive structures containing needle-like crystalline and electron-dense amorphous materials. Con A and UEA-1 lectin staining reactions were strong in the type 1 and 2 lesions. These findings suggest that the calcification matrix may contain mannose, fucose and glucose. The X-ray microanalysis of calcified materials revealed the magnesium whitelockite pattern, the type 3 displayed high quantities of Ca, P, and Mg ions comparing with the type 1 and 2, and the X-ray diffraction showed the hydroxyapatite pattern. We suggest that the above changes may be categorized as dystrophic calcification due to necrotic alterations brought about by the hypercalcaemic condition.     

Concomitant Localization of Dopa Oxidase and Adenosine Triphosphatase in Cryostat Sections

Cryostat sections fixed 10 min in calcium formalin were incubated sequentially in 0.1% DOPA, 2 hr at 37 C, and ATPase substrate 1% hr at 37 C. The enzymatically produced calcium phosphate was visualized by 0.2% glyoxal-bis(2-hydroxyanil) (GBHA) in alkaline ethanol as a red precipitate. Nonspecific protein-bound calcium ions which obscured active sites in such formalin-fixed material were successfully removed by treatment with 0.5% ethylenediaminetetraacetic acid at pH 7.0 for 1 min before treatment with GBHA. Phosphatase sites were red; DOPA active sites, black. The method was also successfully applied to the demonstration of alkaline phosphatases. Acetone fixation inhibited both enzymes; fixation in 70% alcohol suppressed the DOPA reaction and partially inhibited ATPase.     

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about one-third those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried material fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

Summary A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about onethird those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried metarial fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Protective effect of long term high fiber diet consumption on rat exocrine pancreatic function after chronic ethanol intake

The effects of ethanol administration on exocrine pancreas have been widely studied, but little is known about the effect of dietary fiber in combination with chronic ethanol on exocrine pancreatic function. The aim of this work was to examine the chronic effects of a high fiber diet, ethanol ingestion, and a combination of both on the function of the rat exocrine pancreas. Four groups of rats were fed for six months the following diets: 1.- NW: standard laboratory diet; 2.- FW: high fiber diet (15% cellulose); 3.- NE: standard laboratory diet and 20% ethanol in the drinking water; and 4.- FE: high fiber diet and 20% ethanol. Cholecystokinin (CCK) and acetylcholine (Ach) effects on amylase release and intracellular calcium mobilization in pancreatic acini were studied. In rats fed a 20% ethanol (NE), both the basal amylase release and the basal [Ca(2+)](i) were significantly increased; nonetheless, CCK and Ach-induced amylase release were significantly reduced compared with control rats. Ach- but not CCK-stimulated [Ca(2+)](i) increase in NE rats was significantly decreased compared with NW. In rats fed a combination of ethanol and a high fiber diet (FE) all the parameters under study were not significantly affected compared to control rats (NW). In conclusion, high fiber consumption does not alter the function of the exocrine pancreas. However, it ameliorates the deleterious effect of chronic ethanol consumption on pancreatic amylase secretion and, at least partially, reverses the ethanol-induced alterations on [Ca(2+)](i) in the rat exocrine pancreas.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

Effects of ethanol on deep pain evoked by formalin injected in TMJ of rat

It has been reported that ethanol can alter nociceptive sensitivity from superficial tissues, such as skin and subcutaneous region. However, the influence of ethanol on deep pain conditions is not understood. The aim of this study was to demonstrate the acute, chronic and ethanol withdrawal effects on nociceptive behavioral responses induced by the injection of formalin into the temporomandibular joint (TMJ) region of rats. In experiment 1, rats were injected with ethanol (2,5 g/Kg, i.p.) or an equal volume of saline 15 min before the administration of formalin (1.5%) into the TMJ. Rats pretreated with ethanol showed a decrease in nociceptive behavioral responses. In experiment 2, rats were given an ethanol solution (6.5%) or tap water to drink for 4 and 10 days. On day 4, the animals (ethanol group) showed amounts of analgesia when submitted to the TMJ formalin test. Tolerance to the antinociceptive effects was observed on day 10. Behavioral hyperalgesia was verified 12 hr after withdrawal in another group that drank ethanol for 10 days. These results show that ethanol can affect the nociceptive responses related to deep pain evoked by the TMJ formalin test.     

Ethanol-induced increases in [Ca2+]i and inositol (1,4,5) triphosphate in rat hepatocytes

Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.     

Enhancement of phase II enzyme activity by purpurin resulting in the suppression of MeIQx-DNA-adduct formation in mice

We previously demonstrated using a bacterial system that the antigenotoxic activity of the anthraquinone compounds purpurin and alizarin was due to the suppression of microsomal enzyme activity involved in the activation of mutagens. In the present study we determined the effect of purpurin and alizarin on (i) MeIQx-DNA-adduct formation in mouse tissues and (ii) the activity of phases I and II enzymes in liver fractions, the liver being the target tissue of MeIQx. The amount of MeIQx-DNA adduct formed was determined using 32P-postlabeling methods. Methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) enzyme activities, which reflect CYP 1A activity, were measured as markers for phase I enzymes, and UDP-glucuronyltransferase (UGT) and glutathione S-transferase (GST) activities were determined as markers for phase II enzymes. Mice fed with a diet containing 0.5% purpurin for 3 days prior to MeIQx administration had 70% fewer MeIQx-DNA adducts in the lung and kidney, and fewer DNA adducts (insignificant, statistically) in the liver compared with mice fed a diet lacking purpurin. MROD and EROD activities in the liver of these mice increased six- and eight-fold, respectively, and were higher than those determined for the control mice within 1 day following commencement of purpurin treatment. These elevated activities were maintained during treatment and declined immediately following removal of purpurin from the diet. GST and UGT activities gradually increased 2.5- and 3-fold, respectively, following purpurin treatment, and were maintained at significantly high levels even after purpurin administration ceased. Alizarin did not significantly affect DNA-adduct formation and enzyme activity, except in the case of UGT. Taken together, our results show that purpurin reduced MeIQx-DNA-adduct formation by maintaining elevated phase II enzyme activities, thereby facilitating accelerated excretion of MeIQx.     

Multi-component system of estrogen binding proteins from the liver: characterization of binding properties of high molecular weight protein from rat liver similar to uterine estradiol receptors]

A comparative study of the hormonal specificity of the affinity, the equilibrium association constant (Ka) and the kinetics of [3H]-estradiol (3H-E2) interaction with high molecular weight specifically binding E2 proteins from liver cytosol of male and female rats and with uterine estrogen receptors was carried out. The hormonal specificity of the affinity for the E2-binding proteins from the three sources was found to be similar, i.e. only the compounds possessing the estrogen activity competed with 3H-E2 for the binding sites. The values of the apparent equilibrium constants (Ka) for the proteins from male rat liver and female rat liver and uterus were equal to (6,6 +/- 1,2) . 10(9) M-1, (7,4 +/- 0,9) . 10(9) M-1 and (11,2 +/- 2,3) . 10(9) M-1, respectively. The dissociation kinetics of the 3H-E2--protein complexes from the three tissues at 0--4 degrees were two-phase: during the first 8--12 hours the dissociation processes were characterized by the dissociation rate constants (k-1) equal to (4--5) . 10(-5) S-1; then the k-1 values were decreased approximately by one order of magnitude. The kinetics of 3H-E2 association with the three types of proteins are presumably two-phase as well. During the first 10--15 min the association process can be characterized by association rate constants equal to (8--27). .10(5 M-1 S-1; then these values decreased about 4-fold. The data obtained suggest that the high molecular weight estrogen--binding proteins from different tissues are similar in their E2-binding properties on the one hand, and may be interpreted as evidence for the heterogeneity of the populations of E2-binding proteins in various tissues, on the other.