bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbonic anhydrase distribution in rodent embryos and its relationship to acetazolamide teratogenesis.
Authors: Schreiner C M , Hirsch K S , Scott W J
Published: April 2011

The carbonic anhydrase inhibitor, acetazolamide, leads to a unique distal postaxial right forelimb deformity in rats and CBA/J mice, but SWV mice are completely resistant. Using Hansson's histochemical method, the distribution of carbonic anhydrase and its inhibition by acetazolamide in rat, CBA/J mouse, and SWV mouse embryos were compared. Carbonic anhydrase activity was demonstrable in many tissues of sensitive rat and CBA/J mouse embryos and in resistant SWV mouse embryos. The forelimb buds of resistant and sensitive embryos possess carbonic anhydrase activity in the area between the ectoderm and adjacent mesenchyma with no localization of enzyme activity corresponding to the malformation seen in acetazolamide teratogenesis. This suggests that carbonic anhydrase in the forelimbs is not the primary site of action for acetazolamide. A distinctive staining pattern of nucleated erythrocytes in resistant embryos indicated the presence of a low activity form of carbonic anhydrase in nearly half of the erythrocytes. A five-to tenfold greater amount of acetazolamide was needed to completely inhibit carbonic anhydrase activity in nucleated erythrocytes from resistant embryos than in those from sensitive embryos. The existence of a low activity form of carbonic anhydrase in SWV embryo erythrocytes may be the basis of resistance to acetazolamide teratogenesis.
Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems.
Authors: Wood G S , Warnke R
Published: April 2011

As biotin-avidin systems continue to be developed for applications involving single cells, cell suspensions, and especially tissue sections, the need arises for a method of blocking endogenous avidin-binding activity. One such method is described and its proposed mechanism is discussed. Utilizing this method, endogenous avidin-binding activity was detected and suppressed in selected human and murine tissues, thus facilitating the interpretation of specific immunohistochemical staining utilizing hybridoma monoclonal antibodies in a biotin-avidin-horseradish peroxidase detection system.
Epidermal growth factor and renin in mouse submandibular glands.
Authors: Tanaka T , Gresik E W , Barka T
Published: April 2011

Epon sections of the submandibular gland of SWF/J male mouse were stained immunocytochemically for epidermal growth factor (EGF) and renin. Most cells of the granular convoluted tubules (GCT) contained both EGF and renin. However, examinations of adjacent semithin or thin sections stained for EGF and renin, respectively, revealed a small population of GCT cells that contained EGF but no renin. Within a cell all secretory granules contained both EGF and renin. The renin-negative/EGF-positive cells may represent a subpopulation of tubular cells that do not express, or carry, the renin gene.
Protein A, avidin, and biotin in immunohistochemistry.
Authors: Hsu S M , Raine L
Published: April 2011

Conjugated and unlabeled peroxidase antibody methods have proven to be quite satisfactory in localizing sites of antigen-antibody reaction. The use of avidin-biotin-peroxidase complex (ABC), as well as protein A, can contribute significantly to the field of immunohistochemistry. The sensitivity and specificity of several immunohistochemical methods is compared. In general, the ABC method produced the most intense staining and the least background staining of any method tested. The unlabeled antibody (peroxidase-antiperoxidase: PAP) method also yielded satisfactory results, but it was less intense than the ABC method. In comparison to the PAP method, the indirect conjugated method presented slightly inferior staining intensities and significantly higher background staining. Protein A techniques produced a range of staining sensitivities similar to or inferior to the PAP technique. The main disadvantage in using protein A is that it reacts with intrinsic immunoglobulin (Ig) G, thus producing an intense background. Therefore, its use is not recommended on tissues that have either abundant immunoglobulins in their interstitium or numerous IgG-containing plasma cells.
A comparative study on the preparation of immunoglobulin-galactosidase conjugates.
Authors: Deelder A M , de Water R
Published: April 2011

For the application of beta-D-galactosidase-immunoglobulin conjugates in the enzyme-linked immunosorbent assay (ELISA), four techniques for the preparation of such conjugates were compared. Sheep immunoglobulin (Ig) (against soluble egg antigens of the trematode Schistosoma mansoni) was coupled to beta-D-galactosidase by means of 1) glutaraldehyde treatment, 2) the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and 3,4) two different procedures using the coupling agent m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS). The prepared conjugates were then fractionated by gel filtration on Sepharose 6B and the resultant molecular weight fractions were tested in an ELISA for the detection of S. mansoni antigen. Optimal results were obtained with a conjugate that was synthesized according to one of the two techniques using MBS. With this conjugate, 10(-9) g antigen/ml could still be detected in an ELISA with a chromogenic substrate, which was at least ten times as sensitive as with the other conjugates. Application of a fluorogenic substrate resulted in a lower detection level of 10(-10) g antigen/ml.
Studies on the localization of the glycoprotein GP-2 within the renal glomerulus in vivo and in cultured kidney cell strains in vitro.
Authors: Oberley T D , Chung A E , Murphy-Ullrich J E , Mosher D F
Published: April 2011

Positive staining for the glycoprotein GP-2 was demonstrated in the kidney glomerulus by use of the indirect peroxidase-labeled antibody technique. At the ultrastructural level, heaviest staining for GP-2 was demonstrated along the lamina rara externa and lamina rara interna of the glomerular and tubular basement membranes, demonstrating definite molecular organization for structures which appear amorphous even at the electron microscopic level. However, GP-2 was also present in the lamina densa of the glomerular basement membrane, but not of the tubular basement membrane. The staining for GP-2 is in contrast to the predominantly mesangial staining for fibronectin. Using the indirect immunoperoxidase techniques for kidney cells cultured in vitro, it was demonstrated that cell surfaces of specific subpopulations of glomerular cells stained heavily for both fibronectin and GP-2, while renal medullary cells did not stain at all using specific antiserum to these molecules. GP-2 was present extracellularly and showed moderate staining in glomerular cell culture, while fibronectin showed heavy staining in this location.
Hemocyanin linked to protein A as an immunochemical labeling reagent for electron microscopy.
Authors: Miller M M , Strader C D , Raftery M A , Revel J P
Published: April 2011

Hemocyanin-protein A (Hcy/A) is an immunochemical marker suitable for both transmission and scanning electron microscopy. A method is described for the preparation of highly monomeric Hcy/A where protein A and hemocyanin are present in the conjugate in a nearly equal molar ratio. Examples of Hcy/A labeling of several cellular antigens are provided.
Uptake of 5-hydroxytryptamine by gonadotrophs of the bat's pituitary: A combined immunocytochemical radioautographic analysis.
Authors: Nunez E A , Gershon M D , Silverman A J
Published: April 2011

Serotonergic mechanisms were examined in the bat pituitary. A seasonal fluctuation in the concentration of serotonin in the pituitary was found; the concentration rose just prior to hibernation, fell during hibernation, and transiently rose at the time of arousal from hibernation. This pattern is consistent with serotonin being stored within a cell that becomes inactive while bats hibernate. The uptake of 3H-serotonin was examined utilizing electron microscopic radioautography and the simultaneous light microscopic radioautographic localization of 3H-serotonin and the immunocytochemical localization of adenohypophyseal peptides. Uptake of 3H-serotonin was found to be a saturable process, inhibited by fluoxetine, but neither mimicked nor inhibited by norepinephrine. Cells responsible for uptake of 3H-serotonin contained luteinizing hormone immunoreactivity and were identified as gonadotrophs. The seasonal fluctuation and the presence of a specific serotonin uptake mechanism suggests that serotonin plays a role in the function of the adenohypophysis.
Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison.
Authors: Davies P F , Kuczera L
Published: April 2011

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.
Four unlabeled antibody bridge techniques: a comparison.
Authors: Ordronneau P , Lindström P B , Petrusz P
Published: April 2011

Four unlabeled antibody immunocytochemical techniques, the "single bridge" (Avrameas S: Immunocytochemistry 6:825, 1969; Mason TE, Phifer RF, Spicer SS, Swallow RS, Dreskin RD: J Histochem Cytochem 17:190, 1969a; Sternberger LA, Cuculis JJ: 1969), the "single peroxidase-antiperoxidase (PAP)" (Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG: J Histochem Cytochem 18:315, 1970), the "double PAP" (Vacca LL, Rosario SL, Zimmerman EA, Tomashefsky P, Ng P-Y, Hsu KC: J Histochem Cytochem 23:208, 1975) and the "double bridge" (Ordronneau P, Petrusz P: Am J Anat 158:491, 1980) were compared at both the light and electron microscopic levels. The "double" procedures involved repeating incubations with the bridge antibody, in this case, sheep anti-rabbit gamma globulin, followed either by a second PAP step for the "double PAP" or a second anti-horseradish peroxidase step and a single incubation in horseradish peroxidase for the "double bridge." At both the light and electron microscopic levels the staining intensity was greater with the "double" techniques than with the "single" ones. This is probably due to amplification achieved with the second sheep anti-rabbit gamma globulin step, permitting an increase in the number of horseradish peroxidase molecules bound for each molecule of tissue-bound primary antibody. Also, the quality of the various commercial PAP preparations tested was variable. With the weaker ones the staining intensity could be increased by performing an incubation in fresh horseradish peroxidase after the PAP step. Finally, in electron microscopic studies, the reaction products formed in both the bridge and PAP procedures were identical in shape and size.
Immunocytochemical investigations on the submandibular glands of developing and adult mice using a specific antiserum on protease A.
Authors: Gresik E W , Schenkein I , Barka T
Published: April 2011

The submandibular glands of developing and adult mice were studied immunocytochemically by the unlabeled antibody peroxidase-antiperoxidase and the colloidal gold-protein A methods, using an antiserum to a highly purified esteroprotease (protease A, EC 3.4.4) of mouse submandibular gland origin. A thin subluminal rim of immunoreactivity, seen in striated duct cells throughout development, persisted in adulthood. From 15 days of age onwards, striated duct cells with diffuse cytoplasmic staining also occurred; such cells increased in number with age. A clear sexual dimorphism of the submandibular gland was first discernable by 25 days of age, when the developing granular convoluted tubule (GCT) cells of males were slightly larger than those of females; this size difference became more pronounced at later ages, resulting in a distinct dimorphism by 50 days of age. In adults, the principal sites of immunoreactivity were the GCTs, whose component cells stained with different intensities. Electron microscopic immunocytochemical techniques revealed that deposits of oxidized diaminobenzidine or particles of celloidal gold were restricted to the secretion granules of GCT cells; all other organelles were unstained. Acinar and intercalcated duct cells were negative.
Immunocytochemical detection of serotonin with monoclonal antibodies.
Authors: Consolazione A , Milstein C , Wright B , Cuello A C
Published: April 2011

The development of the monoclonal antibody YC5/45 HLK (YC5/HLK) against a 5HT-bovine seroalbumin immunogen and its application for immunocytochemistry is described. The YC5/HLK antibody is the product of a rat x rat hybrid myeloma, producing a heavy chain and two light chains. In hemagglutination tests, the antibody cross-reacts to entirety with dopamine, serotonin, and tryptamine at high concentrations. The serotonin-albumin conjugate is 20,000 times more effective in displacing the binding antibody, while albumin itself goes unrecognized by the antibody. In fixed preparations of brain tissue, immunofluorescence is observed only in neurons known to contain serotonin, while no reaction is observed in dopamine-rich neurons. All immunofluorescence is extinguished by the use of agents that inhibit the biosynthesis of 5HT, but not of the catecholamines.
Microfluorometric analysis of protein thiol groups with a coumarinylphenylmaleimide.
Authors: Sippel T O
Published: April 2011

Characteristics of thiol adducts formed by the fluorogenic maleimide, N-(4-(7-diethylamino-4-methylcoumarin-3-yl) phenyl) maleimide (CPM), (Sippel: J Histochem Cytochem 29:314, 1981) were determined in solution, on the epithelia in beef cornea paraffin sections, and with an egg white model. Absorption was found maximal at 385-390 nm with a molar absorbancy of not less than 30,000 cm2/mmol, while emission peaked at 465 nm; the spectra and output were hardly affected by hydrolytic opening of the succinimide ring. Fluorescence measured in an epi-illuminating microfluorometer faded rapidly at high magnifications, but the initial output from sections of increasing thickness under a 10 x objective was proportional to the thiol density (concentration x thickness) up to a limit equivalent to an absorbance of nearly 0.5 at 387 nm. The staining included less than 5% nonspecific fluorescence, as determined on duplicate sections blocked by 2,2'-dithiopyridine. Factors affecting the use of CPM for quantitation of both thiols and disulfides are discussed.
Enzyme-incorporated erythrocyte ghosts: a new model system for quantitative enzyme cytochemistry.
Authors: Raap A K , Van Duijn P
Published: April 2011

The preparation and properties of a new microscopic model system for quantitative enzyme cytochemistry are described. The enzyme to be studied is entrapped in human erythrocyte ghosts by a simple hypotonic procedure. After fixation in suspension the ghosts can be analyzed both biochemically and cytochemically. The system has been tested with alkaline phosphatase. It is demonstrated that an azo method that uses naphthol AS-MX phosphate as substrate and 4-aminodiphenylamine diazonium salt as coupling agent can detect very low levels of enzymic activity. The biochemical activity determinations of alkaline phosphatase loaded erythrocyte ghosts were found to correlate linearly with cytophotometric activity determinations. The possible use of the erythrocyte ghost model system for other cytochemical applications is briefly discussed.
Whole-Body Autoradiography
Authors: Larsson Bengt , Ullberg Sven
Published: April 2011

Ever since its introduction in the early 1950s whole-body autoradiography has been used quite extensively for mapping the fate of various substances in the body, especially in the fields of pharmacology and toxicology. A detailed description of the whole-body autoradiographic technique is given. Some factors of general interest are dealt with—such as radionuclides and labeled substances, followed by a number of illustrations providing details and specific references.
Autoradiography of Diffusible Substances: Verification of the Specificity of the Localization by Correlated Physiological, Biochemical, and Pharmacological Studies,
Authors: Mills John W.
Published: April 2011

Recent advances in autoradiographic technique make it possible to localize quite a diverse class of soluble and/or diffusible substances. However since the molecules in question could move or be displaced at any time during preparation it is necessary to employ chemical or physiological tests to prove the validity of the localization. For two classes of compounds this can be done quite readily. 3H-ouabain, a cardiac glycoside that binds specifically to the Na pump, can be monitored for its effect on Na transport and the binding itself can be controlled by manipulating specific ligands known to modulate the ouabain-Na pump interaction. Acetylcholine can be readily analyzed by chemical means and its solubility characteristics allow one to extract it even from osmium-fixed embedded sections. Thus it is possible to monitor for the presence of acetylcholine in tissue throughout the processing for autoradiography and even chemically characterize small areas of tissue by microdissection of sectioned material.
Double Labeling Autoradiography.
Authors: Schultze Brigitte
Published: April 2011

Examples of the multiple applicability of the double labeling method with 3H- and 14C-TdR are demonstrated. Double labeling with 3H- and 14C-TdR makes it possible to determine the cycle and its phases with high precision by modifying the usual percent labeled mitoses method with a single injection of 3H-TdR. This results in a considerable improvement of the resolution of the percent labeled mitoses curve. In addition, data is provided on the variances of the transit times of the cells through the cycle phases. For example, in the case of the jejunal crypt cells of the mouse, the transit times through successive cycle phases are uncorrelated. In the case of glial cells the double labeling method provides cell kinetic parameters despite the paucity of proliferating glial cells. In the adult untreated animal, glial cell mitoses are so rare that the percent labeled mitoses method can not be utilized. However, the S-phase duration can be measured by double labeling and the cycle time can be determined by the so-called method of labeled S phases. With the latter method the passage through the next S phase of the 3H-TdR-labeled S phase cells can be registered by injecting 14C-TdR at different time intervals following 3H-TdR application. In this way an S-phase duration of about 10 hr and a cycle time of about 20 hr was found for glial cells in the adult untreated mouse. An exchange of glial cells between the growth fraction and the nongrowth fraction has also been shown by double labeling. A quite different application of the double labeling method with 3H- and 14C-TdR is the in vivo study of the cell cycle phase-specific effect of drugs used in chemotherapy of tumors. Up to now studies of the effect of cytotoxic drugs on cells in different cycle phases were confined to in vitro experiments, since such studies need synchronized cells. However, the double labeling method, which leads to well-defined subpopulations of differently labeled cells in different phases of the cycle, allows such studies to be carried out under in vivo conditions. The effect of vincristine on these cells has been studied. Vincristine affects cells in S and G2 in such a manner that they are arrested during the next metaphase and subsequently become necrotic. It has no effect on G1 cells.
Autoradiographic Studies of Steroid Receptor Sites in Embryonic Tissues,
Authors: Gasc Jean-Marie
Published: April 2011

An autoradiographic technique adapted to diffusible compounds was used to localize steroid receptor sites in various tissues of the chicken embryo. In this article are presented results obtained on the gonads, the reproductive tract, the bursa of Fabricius, and the anterior pituitary after injection of either 3H-estradiol or 3H-dihydrotes-tosterone into 5 1/2- to 15-day-old chicken embryos. Target cells for steroid hormones, either estrogen or androgen, or both, are present in these organs early in the development. The precocity of the receptors suggests that sex steroids may influence embryonic differentiation earlier than currently recognized. The presence of the receptors in a variety of organs or tissues, known or unsuspected target for steroids, emphasizes the very diversified roles of sex steroid hormones in the differentiation processes. Conditions for these receptors to be active are discussed with respect to particularities of the embryonic system.
Improving the Resolution of the 2-Deoxy-D-Glucose Method,
Authors: Pilgrim Ch. , Wagner H.-J.
Published: April 2011

A modified thaw-mount technique is described for the autoradiographic localization of 2-(3H) deoxy-D-glucose in the rat brain. The demonstration of highly labeled individual cell bodies in characteristic sites proves that a resolution on the cellular level has been achieved. Attempts to recover these cells in autoradiographs of semithin sections after resin embedding by vascular perfusion of fixatives, dehydrating, and embedding media were unsuccessful.
Autoradiographic and Biochemical Studies of Hormone Receptor Localization,
Authors: Sheridan Peter J. , Buchanan James M. , Anselmo Vincent
Published: April 2011

Autoradiography has been used in the past to locate presumptive receptor systems for a number of steroids. It has provided information in rather complex tissues that could not have been obtained by biochemical procedures. In these studies we made use of autoradiography to redirect our biochemical efforts to study androgen receptors in the primate heart. Castrated-adrenalectomized female rhesus monkeys and baboons were injected with 1 μg of 5 α-dihydro-[1,2,4,5,6,7-3H]-testosterone per kilogram of body weight. The animals were killed 1 hour later and the hearts were removed and processed for autoradiography. A nuclear localization of androgen was found in most of the myocardial cells, but in few if any of the interstitial cells. After reviewing the autoradiographic data, we began a new series of biochemical studies using a new buffer system and unlabeled androgen to stabilize the receptor. This in combination with postlabeled gradients allowed us to demonstrate for the first time 8S binding on sucrose density gradients. We feel that autoradiography can be a useful adjunct to biochemical studies even in “less complex” tissue such as the cardiac muscle.
Quantitative Assessment of Steroid Hormone Binding Sites by Thaw-Mount Autoradiography,
Authors: Stumpf W.E. , Sar M. , Zuber T.J. , Soini E. , Tuohimaa P.
Published: April 2011

A procedure for the quantitative assessment of nuclear receptors for steroid hormones—and other substances—in individual cells is presented. Thaw-mount autoradiography, a procedure developed earlier in our laboratory, is utilized. The silver grain yield (specific activity) is 16.6 disintegrations per silver grain as determined for tritium in guinea pig uterine tissues. An integrated formula is presented and applied for 3H-estradioI, 3H-diethyl-stilbestrol, and 3H-aldosterone in sampled tissues. A comparison with data derived from the literature that are based on the homogenization of whole uteri and biochemical analysis shows comparable values with the autoradiographic data if the latter are pooled. The pooled data indicate 12–14,000 molecules of 3H-estradiol per uterine nucleus, while subpopulations of target cells vary between 5,000 and 28,000 per nucleus.
Evaluation of Systems for Quantitation of Autoradiograms,
Authors: Beckman W.C.
Published: April 2011

Automated systems for the evaluation of autoradiograms were compared with the traditional method of manual counting. The automated systems included televison image analysis systems, reflectance/densitometric systems, and semiautomatic digitizing pad systems. Highly automated analysis systems were considered applicable for quantitation of autoradiograms when the subcellular location of the radioactivity is not an important consideration, when background is low, and when operator interaction with the system can be kept at a minimum. In many cases the relationship between silver grains and cell compartments is important for interpretation of the results. Under these conditions, the most rapid, reproducible and reliable method would combine measurement of cellular dimensions with a digitizing pad and manual counting of silver grains by the observer. Such a system should be connected to a computer for formatting and statistical evaluation of the data. Initial testing of a quantitative autoradiographic technique indicated that the autoradiographic slide is a reliable estimator of the radioactivity present in the tissue of experimental animals injected with tritium labeled compounds. Accuracy in counting was lost when silver grains became coincident. Thus, a system for the quantitation of silver grains in relation to cellular morphology requires careful selection of exposure time, computer assisted measurement of cellular dimensions, operator evaluation of silver grains and computerized statistical analysis of the data.
Cytochemical hybridization with fluorochrome-labeled RNA. I. Development of a method using nucleic acids bound to agarose beads as a model.
Authors: Bauman J G , Wiegant J , van Duijn P
Published: April 2011

A new procedure to label RNA at the 3'-terminus with a fluorochrome molecule is described. The thiosemicarbazides derived from tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC) were prepared by reacting these compounds with hydrazine in dimethylsulfoxide (DMSO):pyridine, 99:1 (v/v). They coupled efficiently to the aldehydes generated by periodate oxidation of RNAs. We determined, using Sepharose to which different nucleic acids and proteins had been bound, that the label added no specific binding properties to the RNA, and did not interfere with duplex formation of labeled poly(U) and poly(A). The stability of the fluorochrome-RNA bond under conditions generally used for hybridization was investigated. The bond was found to be unstable at 66 degrees C in 3 x SSC, 0.1% SDS (50% loss within 45 min) but stable for at least 40 hr at 23 degrees C in 70% formamide/3 x SSC. The hybridization characteristics of complementary RNA, both fluorochrome- and 3H-labeled, were investigated using DNA-Sepharose beads as a cytochemical model. Hybridization was measured by scintillation counting of microliter quantities of beads and quantitative fluorescence microscopy of individual Sepharose beads. No influence of the label on the specificity and stability of the hybrids was found. Maximum specific fluorescence was found after hybridization at 23 degrees C in 70% formamide/3 x SSC. These results made possible the successful use of fluorochrome-labeled RNA to perform cytochemical hybridization followed by detection of the hybrids with fluorescence microscopy. This will be described in an accompanying article.
Immunocytochemical localization of luteinizing hormone-releasing hormone (LHRH) in Vibratome-sectioned brain.
Authors: Joseph S A , Piekut D T , Knigge K M
Published: April 2011

Immunocytochemical localization of neuropeptides such as luteinizing hormone-releasing hormone (LHRH) is generally performed on Bouin's fixed tissue sections, following tissue dehydration in alcohols and embedment in paraffin. When the final reaction product accurately reflects content and distribution of the neuropeptide has not been examined carefully. Our data indicate a decrease in radioimmunoassayable LHRH content of brain fixed in Bouin's solution and a further significant reduction following dehydration with alcohol. In order to circumvent this loss of hormone, sections of fixed brain were cut on a Vibratome at 30 micron and collected in phosphate-saline buffer. These Vibratome sections revealed a significantly greater amount of overall immunoreactivity and fiber distribution as compared to paraffin-embedded sectioned tissue. These results suggest that certain neuropeptides, soluble in alcohol, can be extracted during dehydration processes necessary for paraffin embedment.
Immunohistochemical localization of pancreatic exocrine enzymes in normal and neoplastic pancreatic acinar epithelium of rat.
Authors: Hansen L J , Mangkornkanok/Mark M , Reddy J K
Published: April 2011

The transplantable pancreatic acinar carcinomas of rat established recently provide useful model systems to examine the composition of secretory proteins as well as the secretory process in transformed pancreatic exocrine epithelium. The neoplastic acinar cells exhibit considerable variation in the extent of cytodifferentiation. In the present study the enzymatic profile of this heterogeneous tumor cell population has been investigated by the indirect immunofluorescent technique using antibodies against six pancreatic enzymes. By immunofluorescence, all neoplastic cells stained positively for the six enzymes tested: amylase, lipase, carboxypeptidase A, chymotrypsinogen, trypsinogen, and ribonuclease. Some variability in the intensity of immunofluorescence was noted, suggesting possible quantitative differences in the content of a given enzyme among tumor cells. These observations suggest that neoplastic acinar cells with or without secretory granules contain secretory proteins, but to a variable extent.
Cationic ferritin binding sites and surface charge densities of transformed cells.
Authors: Walker D G
Published: April 2011

Bioelectric surface properties of the high and low tumor-producing cell lines, NCTC 2472 and NCTC 2555, respectively, were determined by cationic ferritin binding and the electrophoretic mobility of intact cells. Measurements of anionic sites were bases on the number of cationic ferritin particles per 0.01 mu 2 that were electronically tagged and counted by an image analyzer. The average particle count was 45 for the control "high" cells and 34 for the control "low" cells. The surface charge densities, expressed as electrostatic units per cm-2 x 10(-13) were 2.34 and 1.18 at 50 V (2 mA) for the "high" and "low" control cells, respectively. Enzymic cleavage of sialic acid and other carbohydrate moieties resulted in up to an 81% reduction in the charge densities and a 57% reduction of the anionic sites of the "high" cells. The electrophoretic mobility of cells with bound cationic ferritin showed that up to 50% of the exposed anionic sites fail to bind cationic ferritin. Preliminary findings on the particle size/distribution by image analysis showed wide ranges in both particle size and interparticle distances that may limit cationic ferritin binding.
Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents.
Authors: Nygren H , Hansson H A
Published: April 2011

Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.
New fluorochromes for thiols: maleimide and iodoacetamide derivatives of a 3-phenylcoumarin fluorophore.
Authors: Sippel T O
Published: April 2011

N-(4-(7 - Diethylamino - methylcoumarin - 3 yl)phenyl)maleimide (CPM) and the corresponding iodoacetamide are described. When applied to sections at pH 6 and 9, respectively, the two fluorogens are very similar in their reactivity and selectivity toward thiols with which they form adducts having much the same bright blue fluorescence. However, difference in other properties favor CPM for further study.
Investigation of proteinases in the digestive tract using 4-methoxy-2-naphthylamine (MNA) substrates
Authors: Gossrau R
Published: April 2011

Aminopeptidase (AP) A, B, and M, gamma-glutamyltranspeptidase (GGT), endopeptidase I and II, membrane-associated endopeptidase I and II, dipeptidylaminopeptidase (DAP) I, II, and IV, trypsin and chymotrypsin were investigated with 4-methoxy-2-naphthylamine (MNA) substrates and ester proteinases with n-acetyl-L-methionine-1-naphthylester as substrate in the digestive tract of laboratory rodents. Biochemically, proteinases and ester proteinases show different activities in the salivary glands, esophagus, stomach, liver, pancreas, duodenum jejunum, ileum, and colon; sex differences in proteinase and ester proteinase activity were measured, especially in the submandibular gland of rats and mice. Histochemically these enzymes are preferentially localized in surface membranes, lysosomes, secretion granules, and Golgi apparatus of cells of the endocrine and exocrine secretory system, resorptive system and immune system of the digestive tract. Besides the general occurrence of lysosomal (DAP I and II, single cell types and functional units of these systems possess their own individual proteinase and ester proteinase equipment. The cells of the granulated tubules of rat and mouse submandibular gland contain endopeptidase I and ester proteinases, its acinar cells DAP IV, the chief cells of the stomach APA, enteroendocrine cells APA, APM, and DAP II, hepatocytes DAP IV or GGT and DAP IV, lymphocytes GGT and DAP IV, and enterocytes trypsin, chymotrypsin, and membrane-associated endopeptidase I and II. Sex differences in proteinase activity are most conspicuous in the granulated tubule cells of the rat and mouse submandibular gland. The data suggest that proteinases and ester proteinases are involved in specific functions of the cells of the digestive tract. Furthermore, myoepithelial cells, smooth muscle cells of the muscular layer of the stomach and intestine, connective tissue cells (including mast cells) and fibers, nerve cells of the myenteric plexus and the capillary bed of the digestive organs are equipped with some of these proteinases and with ester proteinases and show organ differences.
Proteinases in pathology. Usefulness of histochemical methods
Authors: Lojda Z
Published: April 2011

The usefulness of optimized and newly elaborated histochemical methods for proteinases is illustrated on two selected substances. DAP IV (Gly-Pro-MNA,FBB,pH 7.2) was discovered in 39% and DAP II (Lys-Ala-MNA,FBB,pH 5.5) in 60% of the lymphocytes of human peripheral blood (ly). The reaction product of such ly differs in quality and quantity. On the ultrastructural level, the reaction product of DAP IV (Gly-Pro-MNA,HNF) was found in cell membranes and lysosomes. Enzyme activity in other areas was probably suppressed during the preparation procedure. Although the number of ly revealed with Lys-Pro-MNA and Phe-Pro-MNA at pH 5.5 and with Lys-Pro-MNA at pH 7.2 is high, these substrates do not distinctly discriminate DAP IV and DAP II. DAP IV occurs exclusively in T lymphocytes. The number of DAP IV-positive ly was not decreased in patients with myelofibrosis, plasmacytoma, chronic granulocytic leukemia, or tricholeukemia. It was, however, greatly reduced in chronic lymphatic leukemia (CLL). In patients with malignant lymphomas other than CLL, ly presence is related to the stage of the disease. Decreased values indicate a more severe stage or a relapse. In the majority of patients with gastric cancer DAP IV-positive ly were decreased. They were normal or increased in patients with peptic ulcer. The assessment of the number of DAP IV-positive ly is a simple method that provides information regarding the condition of patients with malignant lymphomas and gastric carcinoma. Neutrophilic leukocytes and their precursors, and to a lesser extent monocytes, are revealed when N-acetyl-Met-I-naphthyl ester (Ac-Met-N) is used as substrate. Membrane-bound lysosomal and cytosol proteinases were investigated together with disaccharidases in jejunal biopsies of patients with malabsorption syndrome. Activities of all enzymes were affected in patients with celiac disease. According to their impairment enzymes could be arranged: Lactase(L). trehalase (T), brush border endopeptidase (BBEP), gamma-glutamyl transferase (GGT), DAP IV, enzyme(s) cleaving Ac-Mer-N, aminopeptidase A, cytosol peptidases and aminopeptidase M. In the propria, DAP IV is decreased or absent, while GGT and, particularly, DAP II are increased. After a gluten-free diet, activities are restored in a reverse order. BBEP and GGT are useful as auxiliary parameters in the assessment of the damage or differentiation degree of enterocytes. DAP IV is a sensitive indicator of the involvement of the propria.
Biochemical and immunological studies of lysosomal and related proteinases in health and disease
Authors: Poole AR , Mort JS
Published: April 2011

A review of continuing studies on the physiological and pathological roles of the lysosomal proteinases, cathepsin D and cathepsin B is presented. Intracellular release of cathepsin D from lysosomes has been demonstrated during myocardial ischemia. Both this proteinase and cathepsin B have been found, by organ culture in the presence of the appropriate antiserum, to be released into the extracellular space in rheumatoid synovium, where they may play a part in cartilage destruction. Also reviewed is the finding of a cathepsin B-like proteinase that, in organ culture, is secreted in markedly increased amounts from human malignant breast carcinomas in comparison to amounts secreted from benign tumors or normal tissue.
Immunohistochemical localization of galactosyltransferase in human fibroblasts and HeLa cells.
Authors: Berger E G , Mandel T , Schilt U
Published: April 2011

Anti-human galactosyltransferase (E.C. 2.4.1.22) antibodies were elicited in rabbits and purified on a galactosyltransferase-agarose column. Purified antibodies were used to localize galactosyltransferase in acetone-fixed HeLa cells and human lung fibroblasts. Both protein A-peroxidase developed with 3-amino 9-ethylcarbazole and swine anti-rabbit IgG-fluorescein isothiocyanate served to detect binding of anti-galactosyltransferase antibodies. In cells of confluent cultures, anti-galactosyltransferase staining appeared as a concise triangular structure in the juxtanuclear region with one angle oriented toward the bulk of the cytoplasm. The stained structure appeared as a dense cap on the nucleus in HeLa cells and as a more extended granular structure in fibroblasts. In cells of sparse cultures, specific anti-galactosyltransferase staining appeared in both HeLa cells and fibroblasts as a granular, extended structure, which was occasionally perinuclear. There was no evidence of cell surface localization of galactosyltransferase by light microscopy. The positively stained structures are interpreted to be part of the Golgi complex.
Autoradiography of phosphatidyl choline.
Authors: Saffitz J E , Gross R W , Williamson J R , Sobel B E
Published: April 2011

Saturated choline phosphatides are extracted during conventional tissue processing for electron microscopy. To facilitate autoradiographic subcellular localization of arrhythmogenic myocardial phospholipids, we evaluated tissue processing procedures for preservation of saturated phosphatidyl choline (PC). Suspensions, of a murine plasmacytoma were incubated with negative, unilamellar liposomes containing 14C-choline-labeled PC or 14C-1-palmitate dipalmitoyl PC. Extraction of radioactivity was monitored at each processing step by liquid scintillation spectrometry. Conventional fixation with glutaraldehyde and osmium tetroxide followed by acetone dehydration and Spurr's plastic embedding led to extraction of nearly all radioactivity. However, treatment of cells with 1.5% tannic acid after glutaraldehyde but before osmium tetroxide fixation preserved 93.1 +/- .6% of 14C-choline-labeled PC. Virtually identical results were obtained with dipalmitoyl PC. Autoradiography demonstrated no significant translocation of labeled PC from plasmacytoma cells to unlabeled avian erythrocytes, mixed in equal proportions after fixation but before dehydration and embedding.
Lectin histochemistry of human skeletal muscle.
Authors: Pena S D , Gordon B B , Karpati G , Carpenter S
Published: April 2011

Biotinyl derivatives of seven plant lectins-concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA I), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and wheat germ agglutinin (WGA)-were bound to cryostat sections of biopsied normal human muscle and visualized with avidin-horseradish peroxidase conjugates. A distinct staining pattern was observed with each lectin. The most general staining was observed with Con A, RCA I, and WGA, which permitted strong visualization of the plasmalemma-basement membrane unit, tubular profiles in the interior of muscle fibers, blood vessels, and connective tissue. PNA gave virtually no intracellular staining, while SBA and UEA I selectively stained blood vessels. DBA was unique in providing good visualization of myonuclei. In each case, lectin staining could be blocked by appropriate sugar inhibitors. Neuraminidase pretreatment of the cryostat sections altered the pattern of staining by all lectins except UEA I and Con A; staining with RCA I became stronger and that with WGA became less intense, while staining with PNA, SBA and DBA became stronger and more generalized, resembling that of RCA I. These effects of neuraminidase pretreatment are in conformity with the known structure of the oligosaccharide chains of membrane glycoproteins and specificities of the lectins involved.
Immunologic studies of bovine aortic and cartilage proteoglycans.
Authors: Mangkornkanok-Mark M , Eisenstein R , Bahu R M
Published: April 2011

Rabbit antisera prepared against bovine cartilage and aortic proteoglycan monomer (PGM) were employed to identify and localize the distribution and cross-species reactivities of these PGMs. A specific reaction to both cartilage and aortic PGM was obtained as revealed by rocket immunoelectrophoresis of the immunogens against the respective antisera, with no reaction against bovine serum albumin or human fibronectin. These antisera appear to demonstrate tissue specificity. Indirect immunofluorescence studies with antiserum to aortic PGM on bovine aorta tissue revealed intense localization of fluorescence in the intima and superficial media with staining alone collagen fibers, around smooth muscle cells, and on the surface of elastin. When antiserum to cartilage PGM was applied to bovine aorta, staining was much weaker and localized more in the interfibrillar matrix. On the other hand, when antiserum to aortic PGM was used to stain bovine nasal septum cartilage, fluorescent staining was restricted to the pericellular matrix, in contrast to the diffuse, intense staining of both pericellular and extraterritorial staining by antiserum to cartilage PGM. Absorption with eigher bovine aorta or cartilage PGM abolished antibody activity against the absorbing molecules only. The immunologic distinction between the pericellular and extraterritorial cartilage matrix was demonstrated further when both antisera were studied with guanidine chloride (GuHCl)-extracted bovine nasal septum cartilage, when they both demonstrated the same pericellular matrix staining. Despite the tissue specificity, these antisera are not completely species specific, since staining in a similar pattern was observed in both human and rat tissues.
Immunoperoxidase localization of a glycoprotein on plasma membrane of secretory epithelium from human breast.
Authors: Imam A , Tökés Z A
Published: April 2011

A glycoprotein component of human milk-fat globule membrane, which is said to derive form apical plasma membrane of mammary secretory epithelium, has been purified. The purified glycoprotein yields a single band under reduced condition and has an estimated molecular weight of 70,000 daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The glycoprotein was termed "secretory epithelial membrane antigen" (SEMA-70) to indicate its origin and molecular size. Specific antisera to SEMA-70 were raised. Using the peroxidase-antiperoxidase method of staining, the presence of SEMA-70 was demonstrated on the apical plasma membrane of the mammary epithelial cells lining the ducts of both normal and lactating breast. No significant staining of cytoplasm was observed. these observations add further evidence as to the origin of milk-fat globule membrane. Although the antigen continues to be expressed on breast adenocarcinoma cells, its cellular distribution appears to be different. The potential usefulness of specific antisera to SEMA-70 is suggested to identify rearrangements in cellular architecture due to pathological changes.
Binding of glycosomes to endoplasmic reticulum and to intermediate filaments in cardiac conduction fibers.
Authors: Rybicka K
Published: April 2011

Canine cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of glycogen and protein) were found attached to membranes of endoplasmic reticulum (ER) and to intermediate filaments (IF). The bound glycosomes retained their integrity after treatment of tissue en block with uranyl acetate, a procedure that destroyed free glycosomes in the same cell. the coincident distributions of ER and IF and their association with glycosomes suggests a functional relation between there three components.