A CHEMICAL AND HISTOCHEMICAL STUDY OF THE LIPIDES OF THE PYLORIC CECUM OF THE STARFISH, ASTERIAS FORBESI

The lipides of the diverticula of Asterias forbesi have been studied by histochemical and biochemical means. Correlations between results obtained by histochemical examination of sections, and chemical analysis of isolated lipide have been made, particularly with respect to phosphatides, steroids, and aldehyde lipides. The results of the histochemical study were in good agreement with the chemical data as to the nature of the phosphatide fraction, the presence of acetone-soluble aldehyde lipides, and the composition of the free droplet fat. Homogenized diverticula were differentially centrifuged in order to establish the distribution of types of lipides in the various cellular components. In addition, data have been presented which demonstrate a direct correlation between the titer of α-glycerol ethers and that of acetone-soluble lipide acetals in the unsaponifiable fraction.     

Partition of cytoplasmic lipides

By means of differential centrifugation three fractions: large granules (L₂), microsomes (M₁), and supernatant fluid (MS), have been isolated from the cytoplasm of mouse liver cells and the contained lipides have been extracted and characterized.The particulate fractions, large granules and microsomes, make up approximately 38% of the total solids of the cytoplasm, but they contain 62% of the total lipide and 85% of the phospholipide. The phospholipide of the particulates has an N:P ratio of 1 while the supernatant phospholipide has an N:P ratio of 1.3, suggesting that the supernatant fluid contains lipides of higher nitrogen content than that of either lecithins or cephalins.The fatty acids of the large granule fraction are highly unsaturated and contain 20% of fatty acids with four double bonds. The unsaturation of the fatty acids of microsomes and the supernatant fluid is comparable, as indicated by the iodine values, but the supernatant fluid contains much more of the fatty acids with one double bond and less of the fatty acids with four double bonds than do the microsomes.     

Qualitative analysis of products formed during the acid catalyzed liquefaction of bagasse in ethylene glycol

Bagasse was liquefied in ethylene glycol (EG) catalyzed by sulfuric acid at 190 degrees C under atmospheric pressure. The compositions of the crude products obtained were analyzed after separating them into three fractions: a water-soluble fraction, an acetone-soluble fraction and a residue. With infrared, gel permeation chromatography and elemental analyses, the residue mainly included undissolved cellulose and lignin derivatives and the acetone-soluble fraction mainly contained lignin degradation products with high molecular weights. The water-soluble fraction, after further analyzed by GC-MS and HPLC, showed EG, diethylene glycol, EG derivatives, saccharides, alcohols, aldehydes, ketones, phenols, especially some acids such as formic acid, levulinic acid, acetic acid, oxalic acid and 2-hydroxy-butyric acid and their esters. The Higher Heating Value (HHV) of the residue and the acetone-soluble fractions were higher than that of bagasse. The results showed that some useful chemicals and biofuels could be obtained by this process.     

Studies on the proteins of Poecilocera picta. I-changes taking place in the haemolymph protein during moulting

The haemolymph proteins of the 6th nymph of P. picta were fractioned by the polyacrylamide gel disc electrophoresis. A total of seven proteins fractions have been detected from the haemolymph. The chemical nature of different protein fractions have been examined by histochemical methods. The changes taking place in the cuticle and epidermal cells have been examined during the transformation of 6th nymph into adult. The fat body proteins have been electrophoretically fractioned and the changes in the concentration of different protein fractions have been examined. It is suggested that the protein fraction 3 of the haemolymph is utilized in the formation of new cuticle. It is concluded by the histochemical observations that proteins of the band 3 are synthesized in the fat body.     

Biological Activity of Fractions Obtained by Differential Centrifugation of the Bovine Adrenal Cortex

This study is an attempt to correlate adrenocortical hormonal activity with various cell fractions obtained by homogenization and differential centrifugation of bovine adrenocortical tissue. Bioassays involving the decrease in circulating eosinophils and sodium retention were employed for the estimation of the glucocorticoids and mineralocorticoids respectively. The supernatant was the most active fraction in both bioassays, while the microsome and mitochondrial fractions demonstrated varying degrees of activity. The total nitrogen, lipide phosphorus, and cholesterol contents of the fractions were determined. The microsome fraction contained a relatively higher amount of lipide phosphorus and a marked amount of cholesterol.     

Quantitative Studies of Total Lipids of Pathogenic Fungi

Ten species of dermatophytes and four of the systemic fungi were assayed for total lipids, acetone-soluble fraction, and phospholipid content in different types of cultures. The yeast phase of each of the systemic fungi grown on solid medium exhibited a higher total lipid content than did the mycelial growth in liquid medium, either shake or still. Shake cultures, in all the fungi tested, produced the least lipids. The yeasts were consistently higher also in the acetone-soluble fraction. Histoplasma duboisii in the yeast phase and Microsporum gypseum produced the greatest amount of phospholipid, and Blastomyces dermatitidis in the yeast phase and M. canis produced the largest acetone-soluble fraction among the systemic fungi and dermatophytes, respectively.     

A Histochemical Study of Normal and Denervated Red and White Muscles of the Rat

The distribution and characterization of the fibers of normal and denervated red and white muscles of the albino rat are reported in this study. Histochemical procedures for succinic dehydrogenase, lipides, adenosinetriphosphatase, esterase, and glycogen were utilized to differentiate muscle fibers, and these methods facilitated the study of the distribution of fiber types within whole muscle. Muscle fibers of the granular type (dark or red fibers) can be clearly distinguished from those with clearer sarcoplasm (light or white fibers) by methods for demonstrating succinic dehydrogenase, lipides, and esterase. The method for adenosine-triphosphatase reveals differences only under the special conditions described in the text. Additional fiber types are described in the cat's diaphragm and in the extrinsic ocular muscles of the rat. Succinic dehydrogenase and adenosinetriphosphatase activities of the soleus and biceps femoris were studied 14 days after denervation of these muscles. The histochemical findings are discussed principally in the light of current biochemical knowledge of these enzymes.     

Variation in Lipid Content of Strains of Histoplasma capsulatum Exhibiting Different Virulence Properties for Mice

Nielsen, H. S., Jr. (Duke University Medical Center, Durham, N.C.). Variation in lipid content of strains of Histoplasma capsulatum exhibiting different virulence properties for mice. J. Bacteriol. 91:273-277. 1966.-Lipid content and virulence were studied in six isolates of Histoplasma capsulatum in an attempt to determine whether or not the two factors could be correlated in this fungus. Virulence was evaluated by injecting dba line 1 male mice intracerebrally with 2.8 x 10(4) infective yeast-phase units and recording organ involvement and spontaneous deaths occurring in a 20-day period. Yeast cells were extracted with mixtures of ethyl alcohol-diethyl ether (3:1, v/v), and the total extractable lipid, as determined by solubility in petroleum ether, was separated into acetone-soluble and phospholipid fractions by acetone precipitation. Neutral lipids were measured directly by weighing, whereas total phospholipids were calculated after the colorimetric determination of phosphorus. The mixed phosphatides of two isolates, differing in virulence, were separated into five fractions by use of a column of silicic acid and Hyflo Super-Cel. In the six isolates studied, neither total extractable lipid, acetone-soluble lipid, nor phospholipid showed a quantitative correlation with virulence. Phosphatidylserine, cephalin, phosphoinositides, and sphingolipids were present in essentially the same amounts in the two strains investigated; however, a lecithin fraction was absent in the less virulent form. These data suggest that the quantity of phosphatidylcholine demonstrated for a given isolate of H. capsulatum may provide some insight as to its virulence, although such a relationship is lacking for total lipid, the acetone-soluble fraction, and the combined phospholipids of yeast-phase growth.     

Studies on Delayed Hypersensitivity of Antigens Isolated from Mycoplasma pneumoniae Cells

Cells of Mycoplasma pneumoniae Mac strain were fractionated into acetone-soluble and insoluble fractions. Acetone-insoluble fractions were digested with pronase and further purified by chromatography on Sephadex G-75, yielding three water-soluble fractions which were free from lipid and consisted mainly of polysaccharide-protein complex. All these water-soluble fractions possessed eliciting antigenicity to delayed hypersensitivity for M. pneumoniae as measured by skin reactions and macrophage migration inhibition tests, but not to complement-fixing antibodies. In contrast, the acetone-soluble fraction was reactive with the complement-fixing antibodies but not for the delayed hypersensitivity.     

Subcellular distribution and characterization of human liver aldehyde dehydrogenase fractions.

The subcellular distribution of human liver aldehyde dehydrogenases (E.C. 1.2.1.3) have been studied and the different types have been separated by ion exchange chromatography. The cytoplasmic fraction contained at least two chromatographically separable aldehyde dehydrogenases, which accounted for about 30% of the total activity. One of the cytoplasmic aldehyde dehydrogenases had a high K_m for aldehydes (in the millimolar range). A considerable part of the activity found in this fraction was due to an enzyme with a low K_m for aldehydes (in the micromolar range). It had properties similar to those of the mitochondrial main enzyme fraction, from where it may have originated as a contamination during subcellular fractionation. Specific betaine aldehyde and formaldehyde dehydrogenases were separated from these unspecific activities in the cytoplasmic fraction. In mitochondria, where more than 50% of the total aldehyde dehydrogenase activity was found, there was also evidence for slight high-K_m activity. The microsomal fraction contained only a high-K_m aldehyde dehydrogenase, which accounted for about 10% of the total activity.     

Aldehyde-fuchsin: historical and chemical considerations.

The staining mechanisms of Gomori's aldehyde-fuchsin are not yet fully understood. It seemed therefore timely to review the history of this dye class in context with current dye and aldehyde chemistry. In 1861 Lauth treated basic fuchsin with acetaldehyde. This dye became known as Aldehyde Blue, but consisted of violet and blue dyes. Schiff (1866) studied several aldehyde-fuchsins; these compounds contained two molecules of dye and three molecules of aldehyde. Acetaldehyde-fuchsin prepared according to Schiff's directions showed staining properties similar to those of Gomori's aldehyde-fuchsin. This dye class was soon superseded by new dyes more suitable for textile dyeing, and chemical investigations of aldehyde-fuchsins ceased around the turn of the century. Gomori's aldehyde-fuchsin has been regarded as a Schiff base. However, according to chemical data, low molecular aliphatic aldehydes and aromatic amines tend to form condensation products. Correlations of chemical and histochemical observations suggest such processes during aging of dye solutions. Models of dimers and polymers of aldehyde-fuchsin could be built without steric hindrance. The nature of the bonds formed by various components of aldehyde-fuchsin solutions is not clear. However, cystine in proteins, e.g. in basement membranes, apparently does not play a role in the binding of aldehyde-fuchsin by unoxidized Carnoy- or methacarn-fixed sections.     

Alterations in biochemical composition and ribonucleic acid metabolism induced in rat liver by cortisone

An investigation of the effect of cortisone administration upon the chemical composition of intracellular particulates of rat liver has been made. Livers were homogenized in 0.25 M sucrose solutions and submitted to differential centrifugation. Five fractions were prepared: mitochondria (Mit), microsomes (Mi), ultracentrifugable (U), non-sedimentable (S), and nuclear (Nuc). Measurement was made of total and polymerized RNA, nitrogen, lipide P, and uptake of P(32) by the RNA of each fraction. The following observations were made:- Cortisone administration caused a fall in concentration in all measured constituents except glycogen. On a per liver basis, however, total liver RNA was unchanged in amount; nitrogen content of Mi fell and that of S increased; the lipide P of Mit and Mi also decreased. The biochemical composition of a statistical mitochondrion was significantly altered; in contrast, the microsomal fraction decreased in amount, but the relationship between the chemical constituents was unchanged. When polymerized RNA was sought by a process involving precipitation from ethanol at 20 degrees C., none was found in the Mit of cortisone livers and the amount in Mi was much less than found in the normal. When, however, precipitation was conducted at 4 degrees C., yields of polymerized RNA in all fractions after cortisone were equal to or greater than those found in the normal. Furthermore, incubation of mixtures of homogenates from normal and cortisone livers resulted in loss of warm precipitable RNA. These data strongly suggest the presence of an enzyme in cortisone livers which upon incubation with normal livers made preparation of polymerized RNA virtually impossible by use of the warm method. This agent, thought to operate in vivo and in vitro, was not present in significant amounts in normal livers, since incubation in this instance had no effect upon the amount of polymerized RNA. Mit from cortisone livers obtained by the cold technique had a significantly decreased rate of incorporation of P(32) even though the yield of RNA from this fraction was increased. To reconcile these observations, it was proposed that under the influence of cortisone a variant of normal RNA is synthesized or normal RNA is converted to this variant. This "new" RNA has new solubility properties, a new rate of incorporation of P(32), and conceivably it cannot act as a template for normal protein synthesis.     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Silver methenamine and phosphotungstic acid staining of the acrosome of Mytilus edulis.

The Mytilus acrosome was investigated by histochemical methods combined with electron microscopy, using silver methenamine (SM) and phosphotungstic acid (PTA) staining, as well as some chemical and enzymatic pretreatments followed by the staining. As one of two major components in the Mytilus acrosome, the egg-membrane lysin was conspicuously stainable with PTA and susceptible to pronase digestion. The other component, that occupies the space between the acrosomal membrane and the axially located strand containing lysin, was stained with SM very specifically. This staining property was not affected by pronase digestion or treatment that blocked aldehyde and SH groups.     

Light microscopic histochemistry on plastic sections

As compared with conventional paraffin, celloidin, and frozen sections, semithin plastic sections offer a superior quality of the light microscopic image in terms of better resolution, absence of distortion and shrinkage artifacts, and suitability for calcified tissues. Application of histochemical methods to such sections often encounters, however, serious difficulties resulting from a considerably reduced reactivity of plastic-embedded biological material. Factors involved include a poor penetration of reagents into plastic embedding media due to a steric or hydrophobic hindrance, as well as a blockade of the reactive chemical groups in the sample due to interactions with fixatives and plastics. Embedding in polar (hydrophilic) plastics, such as glycol methacrylate, permits carrying out a large number of histochemical reactions, including the demonstration of enzymatic activities, directly on sections, but is less suitable for combined light/electron microscopic studies because of an imperfect ultrastructural preservation of tissues. Embedding in nonpolar epoxy resins, particularly if combined with a double aldehyde-osmium fixation, results in a high quality ultrastructure but almost fully inhibits the histochemical reactivity of the embedded material. In order to restore this reactivity, i.e. to unmask chemical groups bound by the polymerized resin, semithin epoxy sections require the removal of the embedding matrix by alkoxides prior to the histochemical procedure. Additional steps are also often necessary: treatment of osmium-fixed sections with oxidative agents, e.g., hydrogen peroxide or periodate which reoxidize the bound osmium and remove it from tissue, and a controlled proteolytic digestion, especially useful in immunocytochemical studies, which probably cleaves the bonds between the primary aldehyde fixative, and the reactive sites. This article reviews histochemical methods which have been successfully applied to plastic-embedded material. Using polar methacrylates and/or nonpolar epoxy resins as embedding media, it has been possible to demonstrate proteins and aminoacid residues, carbohydrates, lipids, nucleic acids, biogenic amines, inorganic ions, and some enzymes, although the spectrum of methods found as suitable for plastic-embedded material is far narrower than that available for paraffin or frozen sections.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aldehyde-fuchsin: Historical and chemical considerations

Summary The staining mechanisms of Gomori's aldehyde-fuchsin are not yet fully understood. It seemed therefore timely to review the history of this dye class in context with current dye and aldehyde chemistry. In 1861 Lauth treated basic fuchsin with acetaldehyde. This dye became known as Aldehyde Blue, but consisted of violet and blue dyes. Schiff (1866) studied several aldehyde-fuchsins; these compounds contained two molecules of dye and three molecules of aldehyde. Acetaldehyde-fuchsin prepared according to Schiff's directions showed staining properties similar to those of Gomori's aldehyde-fuchsin. This dye class was soon superseded by new dyes more suitable for textile dyeing, and chemical investigations of aldehyde-fuchsins ceased around the turn of the century. Gomori's aldehyde-fuchsin has been regarded as a Schiff base. However, according to chemical data, low molecular aliphatic aldehydes and aromatic amines tend to form condensation products. Correlations of chemical and histochemical observations suggest such processes during aging of dye solutions. Models of dimers and polymers of aldehyde-fuchsin could be built without steric hindrance. The nature of the bonds formed by various components of aldehyde-fuchsin solutions is not clear. However, cystine in proteins, e.g. in basement membranes, apparently does not play a role in the binding of aldehyde-fuchsin by unoxidized Carnoy- or methacarn-fixed sections.     

Compartmentation of enzymes in the rabbit parotid salivary gland. A study by enzyme histochemical, tissue fractionation and morphometric techniques

Synopsis Rabbit parotid gland was chosen as a suitable model tissue for studying the role of membranes in enzyme secretion by acinar cells. The study was initiated using subcellular fractionation techniques. During these experiments the effects of various tissue disruption conditions such as the medium and the duration and vigour of homogenization were explored and the results assessed by enzyme and chemical assays and both quantitatively and qualitatively by electron microscopy. A series of basic fractions was isolated and marker enzyme or chemical assays selected for each of the relevant membrane types (rough endoplasmic reticulum, Golgi apparatus, zymogen granule, plasmalemma). A parallel study was effected using enzyme histochemical methods applied to frozen sections. Interesting comparisons could then be made between histochemical and biochemical methods of enzyme demonstration. These comparisons are discussed. the basic fractions provide the material from which specimens of purified membranes of the four species can be obtained. The isolation and characterization of such purified membranes is the subject of another report.     

Spatial distribution of human liver aldehyde dehydrogenase isoenzymes

 To elucidate the pattern of lesions in the liver parenchyma after ethanol ingestion, the quantitative distribution profiles of both the cytosolic and the mitochondrial aldehyde dehydrogenase isoenzyme activities were determined by the use of ultrathin-layer electrophoresis. It was found that in human liver parenchyma, both isoforms of aldehyde dehydrogenase are almost homogeneously represented in the liver acinus. These quantitative data are supported by the results of an improved histochemical technique. Moreover, sex differences were not detected either in activity or in the distribution pattern. Consequently, it can be assumed that it is not the activity of total aldehyde dehydrogenase or its isoforms which is responsible for the higher susceptibility of the perivenous zone to alcohol-dependent damage. Accepted: 11 March 1999     

Distribution of calcium ions in the muscle fibers of the rat diaphragm depending upon their state and the method of histochemical treatment

Distribution of calcium ions in the rat diaphragm muscle fibers has been studied electron histochemically using various fixation techniques and chemical treatment of the tissue. When potassium pyroantimonate in water solution is used after a short perfusate fixation with aldehydes, the reaction product granules are revealed in mitochondria, in the disk I, in the center of the disk A, more seldom the precipitate is found in the sarcoplasmic reticulum (SR) and in the T-system. The presence of calcium ions in the precipitate is proved by means of treatment the preparations with ethylenglycol- and ethylen-diamine-tetra-acetic acids. When contracture is resulted from potassium rhodanide administration, in mitochondria the reaction product granules decrease in their number, the precipitate disappears from the central part of the disk A, while the number of the granules increases in the SR terminal cisterns. The data obtained are compared with calcium ions distribution observed at the freezing-substitution method without an additional chemical fixation, as well as the histochemical fixations after Oschman method and at a usual fixation with OsO4. Certain similarity is revealed in distribution of the calcium pyroantimonate granules at aldehyde fixation and when the freezing-substitution method is used.     

EFFECTS OF THE PHYSICAL ENVIRONMENT ON SOME LIPOPROTEIN LAYER SYSTEMS AND OBSERVATIONS ON THEIR MORPHOGENESIS

Lipoprotein membrane systems such as chloroplasts and the endoplasmic reticulum exhibit a generalized swelling response. The initial effect is an increase in interlamellar spacing, but as swelling proceeds, the membranes are transformed into closed thin-walled spherical vesicles. Available evidence suggests that morphogenesis of the endoplasmic reticulum of Nitella and the lamellar system of the Zea chloroplasts involves fusion of small spherical vesicles to yield closed double membrane structures, which subsequently undergo further differentiation. It is suggested that the vesicles comprise a convenient "micellar" form by which lipides may be transported within the cell from the sites of lipide synthesis to regions of lamellar growth. The characteristic formation of vesicles in swelling and the apparent fusion of vesicles in morphogenesis appear to represent two aspects of a fundamental plasticity of lipoprotein layer systems.