Journal of Histochemistry & Cytochemistry

WELCH, ROBERT M.; RESCH, KATHLEEN

doi: 10.1177/11.6.675pmid: N/A

A modification of the Deitch "standard" naphthol yellow S technique is described and validated for use in deoxyribonucleic acid and protein determinations in drosophila larval material. By the use of this technique in conjunction with cytophotometry relative amounts of protein have been determined in 436 salivary gland nuclei from 37 individuals of wild-type, lgl lethal, and experimentally treated larvae of D. melanogaster and in 294 nuclei from 44 individuals by photometric interferometry. Altogether, 8 comparisons were made by comparing the ratio between two kinds of material obtained by naphthol yellow S cytophotometry with that obtained on the same kind of material by photometric interferometry. In 6 of the first 7 comparisons the cytophotometric ratio could be reconciled with the interferometric ratio by an adjustment well within statistical probability. In the seventh comparison, the ratios could not be reconciled by any adjustment within statistical probability but could be so reconciled when repeated on additional material of the same kind. Inconsistency of naphthol yellow results between the original and repeat comparisons indicated the probability of cytophotometric error due to nonproportionality of staining in the material used. Extensive additional tests revealed no further instance of inconsistency and implicated the material rather than the method in the one aberrancy discovered. It is concluded that the cytophotometric and interferometric data are in mutual support and indicate that either technique is a reliable method for protein determination; but that the naphthol yellow S technique may result in an occasional aberrancy in drosophila material that must be guarded against. Advantages and disadvantages of each technique are discussed.

JERVIS, HELEN ROLAND

doi: 10.1177/11.6.692pmid: N/A

The topographical distribution of alkaline phosphatase, acid phosphatase, and reduced diphosphopyridine nucleotide (DPNH) diaphorase in the small intestine of the normal rat, guinea pig, and rabbit has been studied by histochemical methods. Activity of these enzymes is pronounced in the mucosal epithelium. In the rat, alkaline and acid phosphatase are distributed along a gradient decreasing from duodenum to ileum, and in creasing from the neck of the crypts to the tip of the villi. In the guinea pig and the rabbit, maximal activity of both enzymes is observed in the middle third of the small intestine and at the base of the villi. DPNH diaphorase activity exhibits a gradient decreasing from the duodenum to the ileum in all three species examined. An incomplete survey indicates that monoamine oxidase (MAO) is also distributed along a gradient decreasing from duodenum to ileum in the guinea pig and rabbit; with the method used only traces of it have been demonstrated in the rat.

ROGERS, G. E.

doi: 10.1177/11.6.700pmid: N/A

The Sakaguchi reaction for arginine and the Ehrlich reagent for citrulline, which was modified to lessen tissue swelling, have been used to localize these amino acids in the inner sheath and the medulla layers in sections of actively growing hair follicles of the rat. The trichohyalin droplets are rich in arginine but gave no detectable reaction for citrulline. Conversely, the hardened substance of the inner sheath and of the medulla gave a relatively weak reaction for arginine but an intense one for citrulline. The presence of high concentrations of citrulline in the proteins of the hardened inner sheath and medulla has only recently been reported from chemical studies.The results support biochemical and electron microscope studies indicating a conversion of arginine residues into citrulline residues without protein breakdown, concomitant with the morphological transformation of trichohyalin droplets into the hardened fibrous protein of the inner sheath or the hardened amorphous material of the medulla. Other possible biochemical functions for the inner sheath and the medulla are also discussed.

ALLEN, SALLY LYMAN; MISCH, MARGARET SEGUR; MORRISON, BARBARA M.

doi: 10.1177/11.6.706pmid: N/A

The acid phosphatases of variety 1 of Tetrahymena pyriformis can be separated into 17 zones by electrophoresis in starch gels of pH 7.5. All of these acid phosphatases have an optimal pH of about 5.0 and are inhibited by 10 mM sodium fluoride on d-tartaric acid. With one exception, all hydrolyze sodium α-naphthyl acid phosphate. Differences between the acid phosphatases are observed in their ability to hydrolyze other substrates using either the coupling technique or a modification of the Gomori-lead method. Inhibition of 2 of the acid phosphatases occurs in the presence of 1 mM of Mn++ or Zn++; 0.1 mM of p-chloromercurobenzoic acid inhibits these and others. Variations between the acid phosphatases were observed under different growth conditions and in their distribution in various cell fractions. A major variation in the acid phosphatases that is under genetic control occurs in extracts of different genotypes.The results suggest that the electrophoretically separated acid phosphatases are a family of enzymes that vary in their degree of relationship. Some are different enzymes. Others are more closely related and represent variations of a single enzyme; i.e., mutant forms produced by different alleles, hybrid forms produced by interaction of alleles, and isozymes produced by a single gene.Most of the acid phosphatases are probably associated with lysosomes, although one appears to be associated with microsomes. This acid phosphatase is much more active in synthetically grown cells than in cells grown in proteose-peptone or in bacterized medium. It does not hydrolyze sodium β-glycerophosphate.

TONNA, EDGAR A.; CRONKITE, EUGENE P.; PAVELEC, MILDRED

doi: 10.1177/11.6.720pmid: N/A

The localization, distribution and cellular turnover of tritium labeled-glycine were followed and quantitatively assessed autoradiographically in the femora of young mice. Initially the labeled amnino acid was taken up by the cellular phase of the skeletal system, and was observed within cells 15 minutes after isotope administration. Thirty minutes after H3-glycine injection represented the peak of cellular uptake (assessed by autoradiographic grain density over cells). Shortly after cells incorporated H3-gycine, silver grains appeared over newly deposited bone and cartilage matrices. At the surfaces of bone, silver grains were arranged in bands which were followed up to 14 days after H3-glycine administration. These growth bands were found over deeper layers of bone with increasing time. The movement of these bands was illustrated and discussed. Labeling of cartilage matrix was diffuse.

BÉLANGER, LEONARD F.; MIGICOVSKY, B. B.

doi: 10.1177/11.6.734pmid: N/A

Proteolytic activity has been revealed at the level of the large osteocytes of young chick and rat bone by digestion of gelatin in a photographic emulsion substrate.The reaction was enhanced by pretreatment of the animals with parathyroid extract.The reaction was inhibited by addition to the incubating medium of sodium fluoride or potassium ferrocyanide. It was also inhibited by fixation in ethanol-formaldehyde acetic acid.The present results seem to confirm and explain further the resorptive potential of the mature osteocytes.

CARMICHAEL, G. G.

doi: 10.1177/11.6.738pmid: N/A

A histochemical method demonstrating lipoprotein, depending on the reaction between choline, ethanolamine and serine, and hydroquinone with consequent reduction of tetrazolium salts is described. In vitro tests supporting the chemistry of the reaction are given. The role of this phenomenon in relation to histochemical aspects of the dehydrogenase reactions is discussed.

ALTMAN, JOSEPH

doi: 10.1177/11.6.741pmid: N/A

Fine-resolution autoradiography was used to study the regional uptake of intraperitoneally administered leucine-H3 in the rat brain. Microdensitometric measurements were made of the differential rates of autoradiographic blackening over different brain regions, and low- and high-power photomicrographs were prepared of some representative sections of the neuraxis. Evidence of satisfactory consistency in regional density scores was obtained by comparing the grain density of several bilateral structures on the right and left side in single sections, and of homologous structures in different sections. It was concluded that fine-resolution autoradiography is a suitable technique for studying alterations in protein metabolism of homologous brain structures as a consequence of pathological changes in the brain and possible shifts due to physiological and behavioral manipulation of experimental animals.

GOLAND, PHILIP; ENGEL, MILTON

doi: 10.1177/11.6.751pmid: N/A

Cyanuric chloride and related water-soluble dichloro-s-triazines (Procion dyes and Lissatan PR) or monochloro-s-triazines (Procion H and Cibacron dyes, were utilized as fixatives for fresh and freeze-dried animal tissues. With these reagents, endogenous and exogenous substances were irreversibly insolubilized and rendered resistant to chemical and enzymatic degradation. Certain components have been preserved heretofore lost by other methods. Fixation may depend mainly on the formation of covalent and chelate bonds with the tissue colloids.These chloro-s-triazines can be applied in various ways to meet specific fixation needs. In order to avoid the effects of solvents, cells and thin segments of tissues can be cyanurated in the vapors of cyanuric chloride. The water-soluble Procion dyes and Lissatan PR are effective fixatives for some constituents in fresh tissues; enamel matrix is especially well preserved. The most effective of these cyanurating agents for general use appears to be cyanuric chloride in nonpolar solvents when applied to freeze-dried tissues.Following some cyanurations, unreacted striazine halogen appears available for further condensation with active hydrogen in reactants containing certain functional groups (i.e.,—NH2, —SH, —OH, etc.) By condensations with suitable reactants it appears possible to establish crosslinked polymers in tissues. There is evidence that reaction of fresh hydrated tissues with dichloro-s-triazines imparted some water-repellency to the tissues. Through unreacted halogen in the previously condensed s-triazinyl rings, it appears possible at ambient temperature to effect additional condensations with water-soluble agents utilized in hydrophobing treatments.

TORACK, RICHARD M.; BARRNETT, RUSSELL J.

doi: 10.1177/11.6.763pmid: N/A

The reaction product resulting from the hydrolysis of various nucleoside phosphate esters in the Wachstein-Meisel medium at a neutral pH has been localized by means of electron microscopy to various glial and neuronal membranous fine structures. Enzyme activity following the use of adenosine triphosphate as a substrate was localized to the interspace between the plasma membranes of neurons and glial dendrites adjacent to the neuronal cell body, the proximal axon and synaptic terminals. The relationship of this enzymatic activity to the transfer of cations, especially sodium and potassium, as well as to the synthesis of acetylcholine at synapses is discussed. A reaction product was also found in the Golgi apparatus of neurons and although it was most prominent following the use of inosine diphosphate as a substrate, it was also noted with other nucleoside phosphates. In addition, enzymatic activity was noted on the plasma membrane of oligodendroglia, particularly when adenosine triphosphate and cytidine triphosphate were used as substrates.