bluuebunny/crossref_metadata_2025 huggingface dataset:
HINDLIMB MUSCLE FIBER POPULATIONS OF FIVE MAMMALS
Authors: ARIANO M. A. , EDGERTON V. R. , ARMSTRONG R. B.
Published: April 2011

The fiber type profiles of hindlimb muscles in the Hartley guinea pig, Sprague-Dawley rat, cat, Galago senegalensis (lesser bushbaby) and Nycticebus coucang (slow loris) were estimated histochemically. Fibers were classified as fast oxidative glycolytic, fast glycolytic or slow oxidative according to their myosin adenosine triphosphatase, α-glycerophosphate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. It was found that the soleus and vastus intermedius muscles had the highest proportion of slow oxidative fibers in all five species, demonstrating the constancy of muscle fiber profiles dependent upon anatomical position and functional utilization. The tensor fascia latae and white vastus lateralis of the guinea pig were mostly fast glycolytic, while the red vastus lateralis of the guinea pig consisted of predominantly fast oxidative glycolytic fibers. The majority of muscles investigated in these five mammals were heterogeneous, having a wide range of percentages of the three fiber types.
THE AXOPLASMIC TRANSPORT OF THIAMINE IN RAT SCIATIC NERVE
Authors: TANAKA CHIKAKO , ITOKAWA YOSHINORI , TANAKA SEISUKE
Published: April 2011

Localization of thiamine in the ligated and unligated sciatic nerve of rats was studied histochemically and biochemically. Thiamine content increased, up to 200% of the unligated nerve, in the nerve just above the ligation 48 hr after surgery. This accumulation of thiamine was observed in the axoplasm just above the ligation. Thiamine in the unligated sciatic nerve exhibited a proximodistal gradient of concentration. The data suggest that accumulation of thiamine is due to the proximodistal streaming of axoplasm.
CYTOCHEMICAL AND PHYSICAL PROPERTIES OF MYOFIBER EXTERNAL LAMINA
Authors: ZACKS S. I. , VANDENBURGH H. , SHEFF M. F.
Published: April 2011

The structure and staining characteristics of isolated rat muscle sarcolemmal tubes were examined in the optical and electron microscopes with and without prior staining with ruthenium red. The sarcolemmal tubes, which chiefly consisted of a layer of external lamina with a few collagen microfilaments embedded in the surface and segments of cell membrane, were little affected by incubation with neuraminidase, hyaluronidase and collagenase. However, approximately 50% of the external lamina (EL) was extracted by 0.1-0.3 M lithium diiodosalicylate. Dialysis of the lithium diiodosalicylate extract yielded a fluffy, poorly soluble, thixotropic, acidic (pK 9.9) gel (external lamina substance) which had staining properties similar to the EL in tissue sections. External lamina substance has properties consistent with an acidic glycoprotein or proteoglycan.
QUANTITATIVE MICROFLUORIMETRIC AND NEUROCHEMICAL STUDIES ON DEGENERATING ADRENERGIC NERVES
Authors: SACHS CHARLOTTE , JONSSON GÖSTA
Published: April 2011

The short term effects on noradrenaline (NA) uptake-storage mechanisms and nerve density of the adrenergic nerves in mouse and rat iris following ganglionectomy have been investigated. Uptake-storage mechanisms were studied by measuring 3H-NA uptake-retention and endogenous NA content and nerve density (number of nerve terminals) by using formaldehyde fluorescence histochemical technique in combination with scanning microfluorimetry. 3H-NA uptake-retention, endogenous NA and nerve density in mouse iris were almost unaffected up to 10 hr after ganglionectomy, whereas between 10 and 16 hr there was a drastic and an approximately similar reduction of all of the parameters studies down to about 20-30% of control. The same pattern of changes were observed in rat iris, except that the onset of the changes in NA uptake-storage were delayed about 2 hr, possibly related to the length of the neuron. Although all of the parameters determined showed approximately the same time-course, the results obtained indicate that the function of the storage mechanism is lost somewhat prior to that of the axonal "membrane pump" uptake mechanism. The very close correlation between the nerve density and the 3H-NA uptake shows that 3H-NA uptake is a good index for the relative number of adrenergic nerve terminals. The results presented support the view that the NA uptake-storage mechanisms are intact up to a certain time-point after ganglionectomy, when these functions are rapidly lost with a concomitant disappearance of the transmitter stores. The onset of these changes are somewhat varying for different terminal systems. The ganglionectomy does not have any acute effects on the uptake-storage mechanisms except for those related to the disruption of the nervous impulse flow. The present results also indicate that axotomy and isolation of the iris for in vitro incubation do not have any effects on the NA uptake-storage mechanisms.
THE LOCALIZATION OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE IN THE HEART, CARDIAC GANGLION AND THE LATERAL AND DORSAL NERVES OF LIMULUS POLYPHEMUS
Authors: STEPHENS LEE B. , GREENBERG MICHAEL J.
Published: April 2011

Histochemical localization has confirmed that both the heart muscle and the cardiac ganglion along with its branches have cholinesterase activity. Acetylcholinesterase (AChE) is localized in the nerve fibers of the cardiac ganglion and its side (segmental branch) nerves and in the lateral nerves, but AChE could not be demonstrated in the cytoplasm of the ganglionic cell bodies. The dorsal cardioregulatory nerves (segmental cardiac nerves), arising in the central nervous system, also stain intensely for AChE. Butyrylcholinesterase is localized in the cardiac muscle tissue. The presence of intensely staining AChE in the cardiac ganglion and its branches, the lateral nerves and the cardioregulatory nerves sustains the notion that a cholinergic system is involved in the regulation of the Limulus heart.
STUDIES ON MICROPEROXISOMES IV. INTERRELATIONS OF MICROPEROXISOMES, ENDOPLASMIC RETICULUM AND LIPOFUSCIN GRANULES
Authors: NOVIKOFF ALEX B. , NOVIKOFF PHYLLIS M. , QUINTANA NELSON , DAVIS CLEVELAND
Published: April 2011

Previous light microscopic studies have shown that the lipofuscin granules of human hepatocytes oxidize 3,3'-diaminobenzidine (DAB). This DAB reactivity has been examined by electron microscopy. Incubation in an alkaline DAB medium demonstrates that the acid phosphatase-rich areas, containing ferritin-like grains, are DAB-positive. Close spatial relations of the lipofuscin granules with smooth endoplasmic reticulum and with microperoxisomes are demonstrated. These interrelations probably have functional significance for lipid storage in the lipofuscin granules. The relations also form the basis for speculations regarding microautophagy and the accumulation of ferritin and other cytosol constituents within the lipofuscin granules.
THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOHISTOCHEMISTRY MOLECULAR IMMUNOCYTOCHEMISTRY OF ANTIBODIES ON THE ERYTHROCYTE SURFACE
Authors: HINTON DENNIS M. , PETRALI JOHN P. , MEYER HOWARD G. , STERNBERGER LUDWIG A.
Published: April 2011

The number of discrete unit stains on the surface of sheep erythrocytes enumerated by electron microscopy after preembedding staining using 125I antierythrocyte antibody (anti-E) and the unlabeled antibody enzyme method was correlated with enzyme activity and radioactivity of lysed cell suspensions. Purified anti-E was applied in a concentration range resulting in the binding of 10-2,600,000 molecules/cell. Sheep serum antirabbit immunoglobulin and peroxidase-antiperoxidase complex were used in excess. With a new light-scattering assay for peroxidase the sensitivity of measurement of enzyme activity on suspensions of cell stromata exceeded that of radioactivity assay allowing measurement of as little as 5 x 10–13 mmole antibody/ml. Discrete unit stains on cell sections were visualized to 1.5 x 104 anti-E/cell. Above this number, staining occurred in patches of increasing size but was still discontinuous to ∼3 x 104 anti-E bound/cell. Above 2-3 x 104 anti-E bound/cell continuous staining of the membrane was observed. Assuming random distribution of bound anti-E, the number of discrete unit stains visualized per cell section was formulated as a probability function. The observed numbers of unit stains per section agreed well with the values so formulated to ∼8,000 anti-E/cell. Thus, enzyme activity of peroxidase-antiperoxidase, in suspensions or by enumeration of unit stains by electron microscopy, served as a sensitive quantitative measure of specific antibody bound per cell. The data permit the interpolation that staining of sparsely distributed cell surface antigens by the unlabeled antibody enzyme method with excess antibody permits the enumeration of antigen sites. In addition, mobility of surface antigens may be quantitated by comparison of observed and predicted numbers of discrete unit stains per section.
PYROANTIMONATE PRECIPITATES IN FROG SKELETAL MUSCLE CHANGES PRODUCED BY ALTERATIONS IN COMPOSITION OF BATHING FLUID
Authors: YAROM R. , MEIRI U.
Published: April 2011

When sartorius muscle of a frog was fixed with potassium pyroantimonate in osmium tetroxide, localized electron-dense deposits occurred mainly in the lateral cisternae of the triads and in the N-lines of the thin filaments. The nature of these deposits was studied by soaking the muscles, prior to fixation, in solutions of varying ionic compositions. These included fluids devoid of Na+ and others containing calcium-chelating agents. The results obtained suggested that the precipitate in the myofilaments was due to Ca2+ as it was absent after chelation with ethylene glycol bis (β-aminoethyl ether)-N,N'-tetraacetate (EGTA) in an excess of Mg2+, but still occurred after removal of Na+. The deposits in other locations were probably caused by several cations capable of precipitating pyroantimonate. It was also observed that: the fixative caused contraction of the muscles; after extended soaking in NaCl-free media, severe contractions and dispersal of the precipitate occurred; after EGTA pretreatment there was dilation of the lateral cisternae until the formation of microcysts. Thus, conditions before, during and after fixation could affect the presence and localization of the pyroantimonate deposits.
DEPLETION AND REPLETION OF ADRENAL DOPAMINE-β-HYDROXYLASE AFTER RESERPINE IMMUNOHISTOCHEMICAL AND FINE STRUCTURAL CORRELATES
Authors: FRYDMAN ROSLYN , GEFFEN LAURENCE B.
Published: April 2011

Immunohistochemical staining of dopamine-β-hydroxylase (DβH) in the lamb adrenal medulla, using antibodies conjugated to horseradish peroxidase, has provided morphologic evidence that reserpine initially causes a depletion of DβH protein followed by recovery within a week of the enzyme content. This corroborates biochemical evidence that increased reflex stimulation of the medulla by reserpine produces a loss of DβH through exocytosis of chromaffin vesicles and that recovery is due to synthesis rather than activation of the enzyme. The ultrastructural changes observed were consistent with recent findings that chromogranins are synthesized by ribosomes in the endoplasmic reticulum and incorporated into vesicles in the Golgi apparatus. The changes in catecholamine and DβH staining were not uniform throughout the medulla but were most marked in the epinephrine-containing cells near the corticomedullary junction where the cholinesterase staining was densest. This is consistent with biochemical evidence that synthesis and release of the enzyme are under both neural and hormonal control.
THE QUANTITATIVE HISTOCHEMISTRY OF THE SYMPATHOADRENAL SYSTEM I. ENZYMES OF GLYCOLYSIS
Authors: NELSON JAMES S. , WAKEFIELD P. LYNN
Published: April 2011

Quantitative histochemical methods were used to measure the activities of four glycolytic enzymes, hexokinase (HE), phosphoglucoisomerase (PGI), phosphofructokinase (PFK) and lactic dehydrogenase (LDH), in sympathetic ganglion, adrenal medulla, dorsal root ganglion and cerebellar granular layer. Based upon HIK activity the capacity of the glycolytic pathway in sympathetic ganglion and cerebellar granular layer is 2-2.5 times greater than in adrenal medulla or dorsal root ganglion. Direct regulation of glycolysis probably occurs at the HK and PFK steps in all four tissues. Indirect regulation may possibly be mediated through LDH in sympathetic ganglion.
ANTIBODY ENZYME CONJUGATES THE PREPARATION OF INTERMOLECULAR CONJUGATES OF HORSERADISH PEROXIDASE AND ANTIBODY AND THEIR USE IN IMMUNOHISTOLOGY OF RENAL CORTEX
Authors: CLYNE DAVID H. , NORRIS STEPHEN H. , MODESTO ROSARIO R. , PESCE AMADEO J. , POLLAK VICTOR E.
Published: April 2011

The studies reported were done with the objectives of preparing several different peroxidase-labeled antibody conjugates and of testing their usefulness for the detection of soluble and insoluble tissue antigens. Glutaraldehyde, toluene diisocyanate and N,N'-dicyclohexyl carbodiimide were used to cross-link horseradish peroxidase to goat anti-rabbit immunoglobulin G; the resulting conjugates were characterized by molecular size and enzymatic and immunologic activity. They were then tested for their properties as immunohistologic reagents using 0.5-µ sections of freeze-substituted paraffin-embedded renal cortical tissue. Excellent results were obtained with a highly polymerized conjugate made with glutaraldehyde and with an unpolymerized conjugate made with toluene diisocyanate. With the use of these conjugates tissue localization of both soluble and insoluble antigens was achieved after subsequent fixation of tissue and counterstaining with periodic acid-Schiff-hematoxylin.
FLUORESCENCE HISTOCHEMICAL DEMONSTRATION OF PEPTIDES WITH NH2- OR COOH-TERMINAL TRYPTOPHAN OR DOPA BY CONDENSATION WITH GLYOXYLIC ACID
Authors: BJÖRKLUND ANDERS , HAKANSON ROLF , LINDVALL OLLE , SUNDLER FRANK
Published: April 2011

Condensation with glyoxylic acid vapor, recently introduced as a highly sensitive method for fluorescence histochemical visualization of biogenic monamines, has been found to allow also the demonstration of certain tryptophan- or dopa-containing peptides. Thus, in model experiments, treatment with glyoxylic acid vapor induced fluorescence from di- and tetrapeptides with tryptophan or dopa in NH2-terminal or COOH-terminal position. Peptides without tryptophan or dopa in terminal positions gave no observable fluorescence. Differences were recorded between peptides with NH2-terminal and COOH-terminal tryptophan with respect to their fluorescence yields under different reaction conditions and the spectral characteristics of the fluorophores (excitation/emission maxima: 375-380/500-520 nm and 340-370/435 nm, respectively). In addition, the tryptophan-containing peptides could be distinguished microspectrofluorometrically from the dopa-containing peptides. Thus, the fluorophore of the NH2-terminal dopa peptide had excitation/emission maxima at 330 and 380/495 nm, and the corresponding values for the fluorophore of the COOH-terminal dopa peptide were 330 and 370/460 nm. The glyoxylic acid treatment produced intense fluorescence in certain endocrine cell systems in pituitary, thyroid and gastric mucosa, previously suggested to store peptides with NH2-terminal tryptophan. The present findings support this hypothesis.
STIMULATION OF GLUCOSE-6-PHOSPHATASE ACTIVITY IN THE MUCOSAL CELLS OF THE MOUSE INTESTINE
Authors: HUGON J. S. , MAESTRACCI D. , MÉNARD D.
Published: April 2011

Adult Swiss ICR male mice were fed fructose or glucose for several days. Glucose-6-phosphatase activity in the duodenal and jejunal epithelium was measured by biochemical and cytophotometric means, and it was also localized cytochemically. The fructose diet stimulated glucose-6-phosphatase activity within 6 hr of feeding. The enzyme was stimulated more in the absorptive cells of the basal third than in those of the median and apical third of the villi. After 3 days of feeding of fructose, the smooth endoplasmic reticulum hypertrophied and occupied large areas in the absorptive cells. Lipoprotein spherules and polymorphic dense bodies were also observed. No such modifications were seen in glucose-fed animals.
THE pH RANGE OF THE DIAZOSAFRANIN REACTION OF RAT AND OTHER MAST CELLS
Authors: LILLIE R. D. , PIZZOLATO PHILIP , VACCA LINDA L. , CATALANO ROY A. , DONALDSON PATRICIA T.
Published: April 2011

5-Hydroxytryptophan and 5-hydroxytryptamine (5-HT) give strong red azo coupling colors with fresh p-nitrodiazobenzene (fast red GG) at coupling pH levels of 3-9. Tyrosine, histidine, histamine, adrenaline, noradrenaline, dopa, dopamine and tryptophan give strong red colors at pH 7-9, weakening to orange and yellow at pH 5-6 and negative below. Tryptophan gives weak orange-yellow to about pH 4. Rat mast cells color deep red with diazosafranin at pH 3-8. Protein colors pink to red at pH 7-8, weaker at 5-6, and remains almost uncolored below that. Preoxidation with 10 min 1% H5IO6, 2 hr 0.1 M FeCl3, 3 hr 5% K2Cr2O7 or 1 hr 3°C 10% I2/CH3OH does not prevent the diazosafranin reaction of rat mast cells. Periodic acid does not inhibit in vitro azo coupling of 5-HT or 1-naphthol, that of noradrenaline is prevented, and can be restored by reduction with Na2S2O5 or Na2S2O3. Azo coupling of enterochromaffin and adrenal medulla is prevented by these oxidations and restored by Na2S2O4 reduction. Diazosafranin staining of mast cells is not extracted by 24 hr 0.24 N HCl/70% alcohol. Safranin and other cationic dye staining of rat mast cells resists aqueous 0.1 N HCl some hours, largely disappearing at 24 hr, and is removed by 5-15 min in 0.12 N HCl/7O% alcohol. At pH 1 0.1% toluidine blue colors rat mast cells deep violet; when superimposed after acid diazosafranin the red mast cells assume a deep purple, intermediate color. Since in extended use of the method pH 3 diazosafranin colors only bilirubin casts, hematoidin and dog, rat, mouse and gerbil mast cells, and not monkey, most human or lead and mercury fixed guinea pig mast cells, it is suggested that the method is showing 5-HT in rat mast cells. The occasional reaction of human mast cells may be due to pathologic presence of that substance in these cells.
HISTOCHEMICAL AZO COUPLING REACTIONS A CATECHOLAMINE IN ENTEROCHROMAFFIN CELLS IN PLACE OF OR IN ADDITION TO 5-HYDROXYTRYPTAMINE
Authors: LILLIE R. D. , PIZZOLATO P. , VACCA L. L. , CATALANO R. A. , DONALDSON P. T.
Published: April 2011

Appreciable azo coupling of enterochromaffin cells (EC) and of adrenal medulla (AM) is restricted to the alkaline range, fading out around pH 6, negative at 3-5. Low pH (3.0) azo coupling of rat and mouse mast cells (MC) with diazosafranin correlates with their known content of 5-hydroxytryptamine (5-HT) and with the pH 3-9 range of deep red azo coupling color reaction of p-nitrodiazobenzene with 5-hydroxytryptophan and 5-HT. The MC reaction is covalent azo bonding and not cationic dye salt bonding. The MC reaction is not influenced by prior oxidation by HIO4, FeCl3, K2Cr2O7 or I2/CH3OH exposures which prevent azo coupling of EC and of AM. The oxidation blockades of the azo coupling of EC and AM are reversed by Na2S2O5, N2S2O4 or Na2S2O3 reductions. These reductions after oxidation do not affect the MC diazosafranin reaction. In vitro the azo coupling of 5-HT is only slightly retarded but not weakened by HIO4 oxidation and then slightly enhanced by Na2S2O3, while noradrenaline gives red before oxidation, light yellow after, and after Na2S2O3, again deep red, the control α-naphthol giving deep red with p-nitrodiazobenzine at all three phases, before HIO4, between HIO4 and after Na2S2O3. Indole reactions for EC have been reported on glutaraldehyde (G), acetaldehyde and acrolein fixed but not formaldehyde (F) fixed guinea pig duodenum by Solcia and Sampietro (63), Geyer (27) (G), Barter and Pearse (5, 6) and Lillie and Greco-Henson (44). Repetition of the Lillie and Greco-Henson test of combining in the same preparation a blue indole reaction (postcoupled benzylidene) and a red azo coupling reaction with p-nitrodiazobenzene in both sequences disclosed numerous red EC and blue Paneth cells and eosinophil leukocytes, alike on F and G tissue. Direct observation during the second reaction and color photography of the same field after each of the two reactions showed that no red stained cell was replaced by a blue or purple one, and that no blue stained cell altered its color to purple or red. Moreover, azo positive cells were seen in the epithelium of the tips of the villi and in the sides of the gastric glands of the guinea pig pylorus and fundus. Indole positive cells did not occur in villus tip or pyloric gland epithelium and were restricted to fundus chief cells.

It is concluded that the azo reactive substance in EC is a catechol and not 5-HT. The fluorescence studies have indicated the presence of 5-HT in EC, but the quantity present is insufficient for the acid azo coupling reaction shown by rat and mouse MC with diazosafranin.
PROBLEMS ASSOCIATED WITH THE DEMONSTRATION BY LEAD METHODS OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN RESIDENT PERITONEAL MACROPHAGES AND EXUDATE MONOCYTES OF THE GUINEA PIG
Authors: POELMANN R. E. , DAEMS W. T. , VAN LOHUIZEN E. J.
Published: April 2011

This cytochemical and electron microscopic study on peritoneal macrophages of the guinea pig has raised doubts concerning the validity of lead methods for the demonstration of plasma membrane-bound adenosine triphosphatase activity. The problems encountered are inherent in the use of lead ions as a capture reagent. The nonenzymatically formed precipitates reflect sites of heterogeneous nucleation specific for certain kinds of cells, e.g., resident peritoneal macrophages, eosinophilic granulocytes and, to a lesser degree, exudate monocytes. This type of precipitation is also catalyzed on the surface of nonbiologic matrices such as latex particles. Enzymatic processes may well occur, but they cannot be distinguished from nonenzymatic processes.
STUDIES ON MICROPEROXISOMES III. OBSERVATIONS ON HUMAN AND RAT HEPATOCYTES
Authors: NOVIKOFF PHYLLIS M. , NOVIKOFF ALEX B. , QUINTANA NELSON , DAVIS CLEVELAND
Published: April 2011

Normal human and rat liver, unincubated or following incubation in a 3,3'-diaminobenzidine (DAB), pH 9.7, procedure which visualizes catalase-containing peroxisomes, were studied by electron microscopy. In human hepatocytes the DAB-positive organelles are microperoxisomes, i.e., anucleoid cytoplasmic organelles characterized by numerous slender communications with smooth endoplasmic reticulum (ER) and a moderately electron-opaque matrix. These connections are best seen by viewing the specimen at different tilt angles. Rat hepatocytes possess both DAB-positive anucleoid (verified by serial sectioning) microperoxisomes and DAB-positive nucleoid-containing peroxisomes. The numerous slender connections with smooth ER are present in both. The peroxisomes may also possess wide type continuities with the ER. The significance of smooth ER cisternae adjacent to peroxisomes, noted by numerous electron microscopists, is clarified by the frequent finding of slender communications between these two organelles. A possible sequence of development of microperoxisomes into peroxisomes in rat liver is presented. Close spatial relations to lipid droplets of three organelles—ER, mitochondria and microperoxisomes—are described.
LOCALIZATION OF A PRIMATE-SPECIFIC ESTERASE USING IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE TECHNIQUES
Authors: VLADUTIU GEORGIRENE D. , BIGAZZI PIERLUIGI E. , ROSE NOEL R.
Published: April 2011

The techniques of mixed antibody immunoperoxidase and indirect immunofluorescence were used for the localization of an esterase isoenzyme in human and monkey tissue culture cells and in monkey kidney frozen sections. In cultured cells, the esterase was found to be cytoplasmic in close apposition to the nucleus with a distribution resembling that of the Golgi region. The esterase in monkey kidney was predominantly found in the cytoplasm of the proximal convoluted tubules. There was slight staining in the Bowman's capsule of the glomerulus and in the collecting tubules of the medulla. It is suggested that the kidney is actually producing the esterase in its proximal tubule cells.
FLUORIMETRIC DETERMINATION OF CREATINE KINASE ACTIVITY IN ISOLATED SINGLE NEURONS
Authors: KRASNOV IGOR B.
Published: April 2011

A quantitative fluorimetric method for determination of creatine kinase activity in isolated single nerve cells (2-10 ng dry weight) has been developed. Enzyme activity was measured in the direction of adenosine triphosphate and creatine formation. The creatine generated was measured by the fluorescent compound formed with ninhydrin in alkali. The coefficient of variation for the assay of enzyme activity in brain homogenate (47 ng wet tissue/sample) was 3.17%. The coefficient of variation for single neurons of the lateral vestibular nucleus (nucleus Deitersi) in rabbit was about 15%. The creatine kinase activity in these cell bodies was 342.0 ± 14.2 moles/kg dry tissue/hr, at 38°C.
FLUORESCENT IDENTIFICATION OF Y AND X CHROMATIN BODIES IN HUMAN TISSUES
Authors: TISHLER PETER V. , JAVIER CARLOS
Published: April 2011

Quinacrine mustard interphase fluorescence may be exploited to study Y and X chromosomal ploidy of many human tissues. Acetic alcohol-fixed cryostat tissue sections are superior, but formalin-fixed sections may also be used after the formalin is removed by vigorous washing in water. The percentage of cells of presumptively euploid female tissues which exhibit Y-like bodies is low, with means of less than l0% of all tissues except brain. In contrast, the mean percentages for euploid male tissues range between 50 and 63% (except for heart) and 30 and 40% when fixed in acetic alcohol and formalin, respectively. The differences between male and female tissues with either method of fixation were statistically significant. Nuclei with two fluorescent bodies were rare. The Barr body could also be recognized in all female tissues, and its presence could be quantitated in nuclei of skeletal and smooth muscle. This method permits the prospective and retrospective assessment of sex chromosomal ploidy, and the correlation of localized tissue karyotype with tissue function in individuals mosaic for aneuploidy of the Y.
THE RIBONUCLEIC ACID AND DEOXYRIBONUCLEIC ACID CONTENT OF MONONUCLEAR LEUKOCYTES OF DIFFERENT SIZES STUDIED BY ULTRAVIOLET CYTOPHOTOMETRY
Authors: GARCIA ALFREDO MARIANO , SULLIVAN PATRICIA A. N.
Published: April 2011

Rat mononuclears (lymphocytes and monocytes) were studied for total nucleic acid content by means of ultraviolet cytophotometry. Another set was treated with ribonuclease, and deoxyribonucleic acid (DNA) was measured using the same technique. It was found that total nucleic acid content (DNA and RNA) increases linearly with cell size from about 20 units in lymphocytes having 5 µ in diameter up to around 30 units in cells having 12-14 µ in diameter; this is to say, an almost 50% increase for a 6-7-fold enlargement. After ribonuclease treatment, however, the value of the integrated extinction (DNA) tends to remain constant for different cell sizes. A 650% variation in area is accompanied by a DNA change of less than 6%. The differences between treated and nontreated cells are nonsignificant for populations having up to 7.0-7.5 µ in diameter, which implies that small lymphocytes either have a negligible amount of RNA or that the instrument is not sensitive enough to detect it (less than 7% of the DNA content, this figure being the random error of our technique). These differences become highly significant for mononuclears having 8 µ or more in diameter. Therefore, while DNA tends to be constant and independent from cell size, RNA content tends to be harmoniously inconstant, since it is correlated with cell (and nuclear) size and degree of chromatin diffusion.
DUAL ULTRASTRUCTURAL LOCALIZATION OF ACID PHOSPHATASE IN MOUSE KIDNEY TUBULE CELLS
Authors: SASAKI MITSUO , FISHMAN WILLIAM H.
Published: April 2011

Localization of a membrane-associated acid phosphatase of the tubule epithelial cells of mouse kidney was demonstrated at the fine structural level using a postdiazo coupling technique designed by Smith and Fishman (1969). Enzyme activity appeared in the plasma and infolding membranes and in the lysosomes of epithelial cells of both proximal and distal tubules. Basal infolding membranes were stained most intensely by dense deposits of the enzyme product, which were localized in the outer leaflets. Basement membranes underlying the tubule cells also revealed dense enzyme product. These results were compared with those obtained by Gomori's technique on the same mouse kidney. In this case, lysosomes were positive and infolding and basement membranes were negative. The plasma membrane acid phosphatase is considered to be the morphologic counterpart of microsomal acid phosphatase observed in earlier biochemical studies in our laboratory. Finally, these findings can be interpreted as suggesting separate kidney microsomal and lysosomal isoenzymes of acid phosphatase, each possessing a separate ultrastructural identity.
CELLULAR LOCALIZATION OF CARBONIC ANHYDRASE IN AVIAN TISSUES BY LABELED INHIBITOR AUTORADIOGRAPHY
Authors: GAY CAROL V. , MUELLER WERNER J.
Published: April 2011

Carbonic anhydrase was localized at the light microscope level using labeled inhibitor autoradiography. Laying hens, 10-day-old chicks and adult female Japanese quail received injections of 1-25 µC 3H-acetazolamide/g body weight. After 3 hr (quail) and 24 hr (hen and chick), 2-mm3 pieces of tissue were removed under anesthesia, frozen at –160°C, freeze-dried, fixed with OsO4 vapor, embedded in Epon, sectioned at 1 µ and covered with NTB-2 emulsion. The following results validate the method: (a) the concentration of acetazolamide was 100 times greater in red blood cells than in plasma 24 hr after injection in the hen, indicating that free and loosely bound acetazolamide was rapidly cleared from the body; (b) no significant amounts of label were found in muscle, cartilage, adipose tissue and nuclei, which have no carbonic anhydrase activity; (c) except for the pancreas, there was good agreement among the grain counts for the different classes of birds; and (d) the autoradiographic localizations were in general agreement with concepts of tissue function. Carbonic anhydrase was localized in: the proximal, distal, collecting tubules and the mesangium of the kidney; the acinar, but not the centroacinar, cells of the pancreas; the tubular glands and the columnar cells of the shell gland; the lining cells, but not the zymogenic cells, of the proventriculus; erythrocytes; erythroblasts; heterophils; and lymphoid nodules, but not in fat and interstitial tissue of bone marrow.
APPLICATION OF THE METHOD OF KINETIC ANALYSIS OF STAINING AND DESTAINING PROCESSES TO THE COMPLEX FORMED BETWEEN HYDROLYZED DEOXYRIBONUCLEOPROTEIN AND SCHIFF'S REAGENT IN MODEL FILMS
Authors: DUIJNDAM W. A. L. , HERMANS J. , VAN DUIJN P.
Published: April 2011

Recently, we described a method for the analysis of the kinetics of the formation and decomposition of complexes formed between macromolecular substances and covalently bound dyes (Duijndam, Hermans and van Duijn, 1973). Application of this method to the complex formed between the hydrolyzed deoxyribonucleoprotein present in polyacrylamide model films and Schiff's reagent revealed that this complex is formed via a colorless but ultraviolet-absorbing intermediate. Analysis of the process of destaining with 1 N HCl showed that two components are present in the complex, these components differing only slightly in spectral characteristics but significantly in stability toward acid. The main component, making a contribution of about 90% to the total absorbance, was less stable toward acid than the minor (10%) component. The molecular absorbance of the two separate components was found to be 8.2 x 104 and 10.8 x 104 cm2/mmole at 543 nm for the main and minor components, respectively. The conditions that must be checked before conclusions of this kind may be drawn are discussed and controlled.
STUDIES ON MICROPEROXISOMES V. ARE MICROPEROXISOMES UBIQUITOUS IN MAMMALIAN CELLS?
Authors: NOVIKOFF ALEX B. , NOVIKOFF PHYLLIS M. , DAVIS CLEVELAND , QUINTANA NELSON
Published: April 2011

A variety of mammalian cell types has been studied by electron microscopy following incubation in a 3,3'-diaminobenzidine medium at pH 9.7 and containing a high H2O2 concentration. This medium visualizes the recently described anucleoid microperoxisomes as well as the nucleoid-containing peroxisomes. All 24 cell types contain 3,3'-diaminobenzidine-positive microperoxisomes but none shows nucleoid-containing peroxisomes. The number of microperoxisomes within a cell varies greatly among different cell types. There are huge numbers in some cell types; in others microperoxisomes are common, few or rare. Such differences imply varying functional significance of these organelles in the metabolism of different cell types. Whether the endoplasmic reticulum (ER) is abundant or scarce or whether its membrane is studded with numerous ribosomes or not, ribosomes are lacking where the ER is connected to the microperoxisomes by slender channels. It may be presumed that molecular interchange occurs between these two organelles. Such interchange may occur between microperoxisomes, ER and lipid droplets, as previously suggested, and between zymogen granules of guinea pig pancreas and ER and microperoxisomes. Two rapidly growing malignant cell types were studied (HeLa and Novikoff hepatoma) and both show moderate numbers of microperoxisomes.
ASSAY OF ANTIHORSERADISH PEROXIDASE ANTIBODY IN AN IMMUNOCYTOCHEMICAL MODEL SYSTEM OF POLYACRYLAMIDE GEL FILMS
Authors: YOKOTA SADAKI
Published: April 2011

The effects of fixatives on the antibody and horseradish peroxidase (HRP) activities in the immunocytochemical system were investigated quantitatively by using a model of polyacrylamide gel films containing proteins such as goat γ-globulin, anti-HRP guinea pig antiserum or HRP. The activity of the anti-HRP antibody was measured indirectly by staining the HRP coupled with its antibody in the gel films. About 40% of the antibody activity was lost by fixation in 2.5% glutaraldehyde and approximately 35% in 4% formalin. The inactivation of HRP by the refixation in glutaraldehyde or formalin was also examined. The loss of HRP activity was inversely related to the concentration of the fixatives. The fixation in 2.5% glutaraldehyde and 4% formalin inhibited HRP activity 35% and 40%, respectively. The inactivation occurred generally within 40 min of the refixation.
HISTOCHEMISTRY OF CULTURED, EMBRYONIC AND REGENERATING RAT MUSCLE
Authors: ASKANAS VALERIE , HEE DICKSON
Published: April 2011

Histochemical features of cultured rat muscle were compared to those of cultured chicken muscle, as well as of young embryonic and regenerating rat muscle. Primary cultures were established from trypsin-dissociated myoblasts of thigh muscle from 17- to 19-day-old rat embryos and of breast muscle from 10- to 12-day-old chicken embryos. Histochemical reactions for myofibrillar adenosine triphosphatase (ATPase) performed at pH 9.4 and for ATPase after acid pH 4.35 preincubation, phosphorylase, succinic dehydrogenase, reduced diphosphopyridine nucleotide dehydrogenase and myoglobin were applied to examine cultured muscle, gastrocnemius muscle of 15-17 day-old embryos and regenerating muscle fibers in cold-injured gastrocnemius muscle of adult rats. Cultured rat and chicken muscles could be best distinguished by the ATPase reaction following acid preincubation (positive in cultured rat muscle, negative in cultured chicken muscle) and by a different pattern of phosphorylase staining. However, the histochemical features characteristic for cultured aneural rat muscle were also present in young regenerating and embryonic rat muscles before innervation and differentiation of fiber types have occurred in these muscles. It seems, that young, aneural myotubes, regardless of the environment in which they develop, maintain their histochemical features, probably characteristic for the species.
IMMUNOHISTOCHEMICAL LOCALIZATION OF LACTOPEROXIDASE IN BOVINE TISSUES
Authors: HARADA TAKAYUKI , BABA MITSUO , MORIKAWA SHIGERU
Published: April 2011

Distribution of lactoperoxidase in bovine tissues was investigated by using fluorescent antibody techniques, and immunochemical properties of the enzyme were also examined. As a result of immunodiffusion, two antigenic components were found, but no cross-reactivity between lactoperoxidase and hemoproteins such as catalase, cytochrome c, hemoglobin or ferritin was observed. Lactoperoxidase was found mainly in the cytoplasm of alveolar cells of the mammary gland; in those acinar cells, which were morphologically identical to serous cells, of the sublingual gland; in acinar cells of the lacrimal gland; and also in some peripheral leukocytes and in a small number of splenic cells in the red pulp. Lactoperoxidase in the alveolar and ductal spaces and in some of the ductal epithelia of those glands was observed after ethanol or acidified ethanol fixation but not after formalin fixation. It has been demonstrated by both the present and previous work (Morikawa and Harada (1969)) that lactoperoxidase and liver-catalase were distinguishable in bovine tissues when fluorescent antibody techniques were utilized.
ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY WITH UNLABELED ANTIBODIES AND THE PEROXIDASE-ANTIPEROXIDASE COMPLEX A TECHNIQUE MORE SENSITIVE THAN RADIOIMMUNOASSAY
Authors: MORIARTY GWEN C. , MORIARTY C. MICHAEL , STERNBERGER LUDWIG A.
Published: April 2011

Titration curves were developed with antisera to 17-39ACTH (adrenocorticotropin) and 1-39ACTH with the techniques of radioimmunoassay and electron microscopic immunocytochemistry. For the latter method, the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was used to stain normal rat pituitary intermediate lobes. By radioimmunoassay standards, the 17-39ACTH antiserum was of poor quality. Its titration curve exhibited a flat slope and it did not bind a significant amount of labeled antigen beyond a 1: 30 dilution. However, immunocytochemical staining was detected with this antiserum at dilutions as high as 1:1,500. The antiserum to 1-39ACTH was of better quality by radioimmunoassay standards. It bound 45% of the labeled antigen at a dilution of 1:5,000. Immunocytochemical staining intensity was nearly maximal at a 1:5,000 dilution and decreased progressively to a limiting value at 1:16,000. However, when incubation times in the antisera were increased from 3 min to match those of the radioimmunoassay (48 hr) maximal staining was achieved at dilutions as great as 1:512,000 where only trace amounts of the labeled antigen were bound in the radioimmunoassay. It was concluded that the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was sensitive enough to detect antibodies in sera of low titer and/or avidity which are not detected by a radioimmunoassay. The technique holds great promise for a sensitive assay system.
VARIABILITY OF THE SHAPE AND ARGENTAFFINITY OF THE GRANULES IN THE ENTEROENDOCRINE CELLS OF THE MOUSE DUODENUM
Authors: NICHOLS DAVID B. , CHENG HAZEL , LEBLOND C. P.
Published: April 2011

The enteroendocrine cells of the mouse duodenum were examined in the light and electron microscopes by comparing Epon sections stained by Gomori's silver methenamine with adjacent sections stained by Masson's ammoniacal silver nitrate, iron hematoxylin or uranyl acetate and lead citrate. Furthermore, the granules present in the enteroendocrine cells stained with silver methenamine were investigated in the electron microscope: their shape was defined as either spherical or nonspherical and, in either case, their density was measured by cytophotometry. The comparison of adjacent sections of mouse duodenum by different techniques indicates that the granules of all enteroendocrine cells are stained by iron hematoxylin, whereas those of about 94% of the cells are stained by silver methenamine. Since silver methenamine stains exactly the same cells as ammoniacal silver nitrate, it may be used as a test of argentaffinity. It is concluded that about 94% of the enteroendocrine cells are argentaffin and about 6% are nonargentaffin. The argentaffin cells display a wide spectrum of granule shape. Nearly all of the granules of a cell may be spherical or nearly all nonspherical or, more commonly, there is a fair number of both kinds. In the few cells which are not argentaffin, over 70% of the granules are spherical. After silver methenamine staining, the mean density of spherical and nonspherical granules is statistically equal within any given cell. Yet, when cells are compared to one another, the mean density of the granules, and therefore the argentaffinity, increases with the proportion of nonspherical granules. It is concluded that, in the mouse duodenun, argentaffinity is most pronounced in the cells with irregular nonspherical granules, but it is by no means confined to such cells. Moreover, the argentaffinity as well as the shape of the granules varies within wide limits.
PROCION YELLOW M-4RS BINDING TO NEURONAL MEMBRANES
Authors: FLANAGAN M. T. , HESKETH T. R. , CHUNG S-H.
Published: April 2011

Procion Yellow M-4RS, an intracellular neuronal marker, has been shown to bind covalently to the subcellular organelles of rat brain under physiologic conditions. Binding to purified proteins, nucleic acids and lipids also occurs under such conditions, the probable site of attachment being the primary amino group. Lipid membranes were highly impermeable to this dye. The fluorescence intensity of Procion Yellow M-4RS was shown to be viscosity-dependent. These results are consistent with the behavior of the dye when it is used for mapping neurons.
IMMUNOFLUORESCENCE COUNTERSTAINS
Authors: SCHENK ERIC A. , CHURUKIAN CHARLES J.
Published: April 2011

Counterstains in the direct and indirect immunofluorescence methods may be used to reduce or eliminate background nonspecific staining or autofluorescence and to increase cellular and tissue detail. The advantages and disadvantages of a number of such counterstains in terms of (a) elimination of background fluorescence, (b) blocking of specific staining, (c) cellular detail and (d) rapidity of fading are discussed, and a new method of counterstaining nuclei with methyl green is presented. Methyl green counterstain is the procedure of choice when maximal cellular and tissue detail is desired. The lissamine-rhodamine-conjugated bovine serum albumin and eriochrome black methods are especially useful in reducing or eliminating nonspecific staining and tissue autofluorescence.
QUANTITATIVE MICROFLUORIMETRIC STUDIES ON FORMALDEHYDE-INDUCED FLUORESCENCE OF 5-HYDROXYTRYPTAMINE IN THE PINEAL GLAND OF THE RAT
Authors: TILDERS F. J. H. , PLOEM J. S. , SMELIK P. G.
Published: April 2011

The formaldehyde-induced fluorescence (FIF) of pineal sections of the rat was measured quantitatively under instrumental conditions causing minimal photodecomposition. Fluorescence measurements on different sections of one pineal gland showed only little variation. Also the difference between animal variation was small, which enabled us to use groups of five to six animals for appropriate determinations. The similarity in emission spectra obtained from the pineal gland and a 5-hydroxytryptamine (5-HT)-containing protein model and the results of pharmacologic specificity tests indicated that pineal FIF is derived from 5-HT. Pineal 5-HT seemed to be stored in two compartments: the pinealocytes and the sympathetic nerve fibers. Reserpine, a drug depleting the neuronal 5-HT pool selectively, induced about 30% decrease in chemically detectable 5-HT content, indicating that 30% of total pineal 5-HT content was stored in the nerve fibers, and 70% was stored in the pinealocytes. FIF was not affected by reserpine, suggesting that FIF measurements were largely independent of the neuronal 5-HT store. The relationship between pineal FIF and chemically detectable 5-HT content was investigated in groups of rats treated with drugs increasing or decreasing pineal 5-HT content. From each group, five pineal glands were prepared for microfluorimetrical studies; the remaining pineal glands were used for chemical determinations of 5-HT and protein content. The linearity of this relationship demonstrated that concentration-dependent quenching did not occur in the fluorescence measured. Therefore it was concluded that FIF might be used as a quantitative parameter for 5-HT stored in the pinealocytes.
EFFECTS OF TWO CHELATING AGENTS, OXINE AND DIETHYLDITHIOCARBAMATE (ANTABUSE), ON STAINABILITY AND MOTILITY OF HUMAN SPERMS
Authors: DANSCHER GORM , REBBE HEINRICH
Published: April 2011

Two different staining methods (the sulfide silver method of Timm and a fluorescence method using 2-methyl-8-hydroxyquinoline) revealed a similar distribution of heavy metals in human spermatozoa. Both methods were modified for the staining of spermatozoa. Diethyldithiocarbamate, a compound thought to be the active metabolite of disulfiram (an antialcoholic drug, Antabuse) prevented subsequent staining by these two methods. Low concentrations (1:10,000) of diethyldithiocarbamate in vitro immobilized spermatozoa within 2-4 hr but subsequent intravital staining with eosin indicated that more than 90% of the immobilized spermatozoa were still alive.
THE ULTRASTRUCTURAL LOCALIZATION OF CYTOCHROME c-OXIDASE COMPLEX IN A LIPID-RETAINING, GLUTARALDEHYDE-UREA EMBEDMENT
Authors: NIR IZHAK , PEASE DANIEL C.
Published: April 2011

Kidney tissue, incubated in a phosphate-sucrose buffer with diaminobenzidine (DAB), subsequently was embedded in polymerized glutaraldehyde-urea (Pease and Peterson, 1972). The highly polar character of this embedment retains lipids in ultrathin sections and thus permits a precise localization of reaction products in relation to cytomembranes. Furthermore, since conventional organic solvents are not used during processing, it is thought that oxidized DAB polymers certainly remain in place. Their density can be enhanced by exposing mounted sections to OsO4 vapor, rather than by en bloc staining. DAB oxidation takes place only in the compartment between the inner and outer mitochondrial membranes. When aldehyde-fixed tissue is incubated, the deposits are largely limited to the intracristal spaces, whereas when fresh tissue is incubated, the entire compartment is uniformly filled. Morphologic features of fresh, unfixed tissue are stabilized by ethylene glycol and so survive incubation best when about 30% of this substance is added to the medium.
A CYTOCHEMICAL APPROACH TO THE LIPID COMPOSITION OF MITOCHONDRIAL MATRIX GRANULES IN BROWN ADIPOSE TISSUE AND LIVER
Authors: WENDEL PER-OLA , BARNARD TUDOR
Published: April 2011

Matrix inclusions in mitochondria of prenatal brown fat and liver are rich in lipids, and it has been suggested that these should mainly be phospholipids. This proposition has been investigated. Pellets of glutaraldehyde-fixed mitochondria were extracted with excess hexane or chloroform-methanol (2:1 v/v); in controls buffer was substituted for organic solvent. Following extraction the pellets were postfixed in OsO4. Matrix granules in buffer-treated mitochondria (controls) were strongly osmiophilic and were bleached by H2O2 treatment. In hexane-extracted mitochondria the inclusions were less intensely osmiophilic, but they showed the same response to H2O2 as did the controls. Extraction with chloroform-methanol resulted in empty spaces in place of the matrix granules and H2O2 treatment did not produce significant changes. The fact that the inclusions were not extracted by hexane suggests that they are not composed mainly of neutral lipids. The solubility and osmiophilia of the material in the matrix granules are compatible with the reactions expected of phospholipids, but the evidence does not exclude the possibility that other classes of organic compounds might also be involved.