bluuebunny/crossref_metadata_2025 huggingface dataset:
Observations on the Fine Structure of the Gastric Parietal Cell of the Rat
Authors: Lawn A. M.
Published: May 2004

The structure of the rat parietal cell was examined by electron microscopy. The intercellular and intracellular canalculi are lined by microvilli which are more numerous and larger than those of other gastric cells. The numerous mitochondria have closely packed cristae and a dense matrix containing opaque particles. The cytoplasmic vacuoles typical of parietal cells are part of a network of smooth surfaced tubules and vacuoles (the endoplasmic reticulum) which is intimately associated with the mitochondria and probably connected with the lumen of the canaliculi. Only a few dense particles are found attached to the surface of these tubules. The structure of the parietal cell is compared with that of other cells whose function also is transport of inorganic ions and water. Evidence is presented supporting the hypothesis that parietal cells differentiate from a less structurally specialized cell in the neck region of the gastric gland.
The Effect of Partial Protein Extraction on the Structure of the Eggs of the Sea Urchin, Arbacia punctulata
Authors: Kane R. E.
Published: May 2004

The structure of the eggs of the sea urchin, Arbacia punctulata, has been investigated after the removal of one-half of the cellular protein. The procedure involves treatment of the eggs with 30 per cent ethanol at -10°C. followed by extraction of the soluble proteins with water. The eggs remain intact, although all of the cytoplasmic matrix is removed. Most cell structures can still be identified, although only the membranes of most remain. The mitochondria lose all of their matrix but retain the inner membranes or cristae.

The annulate lamellae appear unaffected by this extraction procedure, remaining intact and apparently undamaged. The nuclear envelope is also retained, although it often undergoes a curious disorganization, apparently as the result of the separation of its two layers. The significance of these observations with respect to the structure of the envelope is discussed.
Epoxy Resins in Electron Microscopy
Authors: Finck Henry
Published: May 2004

A method of embedding biological specimens in araldite 502 (Ciba) has been developed for materials available in the United States. Araldite-embedded tissues are suitable for electron microscopy, but the cutting qualities of the resin necessitates more than routine attention during microtomy. The rather high viscosity of araldite 502 also seems to be an unnecessary handicap.

The less viscous epoxy epon 812 (Shell) produces specimens with improved cutting qualities, and has several features—low shrinkage and absence of specimen damage during cure, minimal compression of sections, relative absence of electron beam-induced section damage, etc.—which recommends it as a routine embedding material. The hardness of the cured resin can be easily adjusted by several methods to suit the materials embedded in it.

Several problems and advantages of working with sections of epoxy resins are also discussed.
On the Significance of the Extractable Collagens
Authors: Jackson David S. , Bentley John P.
Published: May 2004

This investigation has sought to determine the significance of the wide range of extractable collagen fractions which appear to exist in growing connective tissues and to determine their position in the process of fibrogenesis.

Carrageenin granulomata were induced in guinea pigs and, after injection of 14C-glycine, this tissue and skin from the same animal were subjected to successive extractions with neutral salt solutions of increasing ionic strength, citrate buffer pH 3.6, and to gelatinization. The specific activity of these fractions was determined at various time intervals. At 8 hours it was found that the specific activity decreased with increasing ionic strength of the neutral salts and was still lower in the citrate extracts and gelatin. At 36 hours the situation was almost completely reversed except that the citrate extract and gelatin still had the lowest activities. The data from skin were more clear cut than that from the granuloma and the reasons for this are discussed.

It is concluded that at any given time in developing connective tissue, there is a continuous spectrum of collagen aggregates of varying degrees of strength of cross-linkage, dependent upon the time that has elapsed since their constituent molecules were synthesized. The various extraction media used remove a particular cross-section of these aggregates depending upon their disaggregating power. These extracts will thus be biologically heterogeneous. The fraction extracted with 0.14 M NaCl will contain the collagen molecules most recently synthesized and in this respect can be considered the earliest form of extracellular collagen.
Effects of 5-Fluorodeoxyuridine and Related Halogenated Pyrimidines on the Sand-Dollar Embryo
Authors: Karnofsky David A. , Basch Ross S.
Published: May 2004

The embryo of the sand-dollar (Echinarachnius parma) was exposed to various concentrations of fluorinated pyrimidines immediately after fertilization. FUDR (5-fluorodeoxyuridine) was most active, and a concentration of 2 to 4 mγ/10 cc. (0.8 to 1.6 x 10-6 m.eq./liter) blocked development at the early blastula stage. Larger doses interrupted development at the same stage. This effect was prevented by thymidine (TDR) and thymine (T); and these pyrimidines protected against many times the minimal lethal concentration of FUDR. TDR was active as a protective agent if added just before early blastula formation.

The other fluorinated pyrimidines, 5-fluorouracil (FU), 5-fluorouridine (FUR), 5-fluorocytidine (FCR), 5-fluorodeoxycytidine (FCDR), and 5-fluoroorotic acid (FO), were also studied. These drugs produced effects on embryonic development similar to those seen with FUDR. The effective concentrations, however, varied greatly. T and TDR provided protection against these drugs, but in most cases they were not so effective as against FUDR.

5-Bromodeoxyurdine (BrUDR), beginning at the early blastula stage, caused a random pattern of embryonic death up to the pluteus stage. This drug has been shown to be incorporated into bacterial DNA. BrUDR protected embryos against the early lethal effects of FUDR presumably acting as a thymidine substitute, but the embryos died subsequently in a pattern similar to that seen with BrUDR alone.

FUDR and BrUDR appear to inhibit the formation and alter the structure of DNA, respectively, distinctive effects whch may provide a means for studying the role of DNA in embryonic development.
Spermatogenesis in Animals as Revealed by Electron Microscopy
Authors: Yasuzumi G.
Published: May 2004

Testes of Eriocheir japonicus were fixed in buffered (pH 7.2) 1 per cent osmium tetroxide and thin sections of the methacrylate-embedded tissue were studied with the electron microscope. Spermatozoa from the vasa deferentia and spermatids from the testes were examined in smear preparations and sections. The most useful stainings proved to be the Feulgen, Unna-Pappenheim, and PAS reactions.

The present paper covers one of the late stages of spermatogenesis. At the late stage of differentiation of the spermatid, the nucleus shows a concave disc-like contour in longitudinal sections and the karyoplasm is finely alveolar in appearance. A vesicle with a fibrillar or granular content appears situated between the cytoplasm and the nucleus. In an advanced spermatid, a large vacuole develops between the nucleus and this vesicle. As development of the spermatid proceeds, the cytoplasm is sloughed off. Thus, the advanced spermatid consists of an ovoid vesicle surrounded by a flattened nuclear disc. The former corresponds to the "capsule" or the "head," and the latter to the "pseudopodia." Concurrently with the sloughing off of the cytoplasm, a lamellar structure appears at the periphery of the head. It is composed of thin plates imbricated like the leaves of an onion. Each plate consists of triple layers, two dense layers separated by a lighter space. Each layer measures about 7 mµ in diameter.

At the distal pole of the head, a depression develops into a tubule which reaches the proximal pole of this structure. The lumen of the tubule is occupied by a dense substance part of which arises from the limiting membrane of the vacuole. The dense material begins to fill the tubule from the distal towards the proximal end. It finds its way till the middle of the tubule, leaving its proximal half unfiled. Thus, the proximal portion of the mature sperm head has a straight tubule whlich opens on its surface. The latter is covered by a dense membrane about 60 mµ in width.

Cytochemical analysis reveals that the pseudopodia contain DNA, and the head a carbohydrate component as well as PNA. The classical concept that the head becomes the male pronucleus after fertilization can no longer be accepted on the basis of the cytochemical analysis. The present study came to no definite conclusions concerning the functional significance of the head.
Improvement in the Histochemical Localization of Leucine Aminopeptidase with a New Substrate, L-Leucyl-4-Methoxy-2-Naphthylamide
Authors: Nachlas Marvin M. , Monis Benito , Rosenblatt David , Seligman Arnold M.
Published: May 2004

A new method for the histochemical demonstration of leucine aminopeptidase in fresh frozen sections was developed with the substrate L-leucyl-4-methoxy-2-naphthylamide. The superior enzyme localization is due to the more rapid rate of coupling of the hydrolysis product, 4-methoxy-2-naphthylamine as compared to 2-naphthylamine itself, and to the low lipid solubility and high substantivity for protein of the copper chelate of the dye formed on coupling with tetrazotized diorthoanisidine. A comparison of the old and the new method is illustrated, and a description is given of the localization of leucine aminopeptidase in the tissues of the rat and man.
Succinic Dehydrogenase and Cytochrome Oxidase Activities in Cell Cultures
Authors: Leslie I. , Yarnell Margaret
Published: May 2004

Succinic dehydrogenase and cytochrome oxidase have been assayed in permanent cell lines (HEP 1, HEP 2, and HLM), in short-term cultures of chick embryo heart cells, and in various tissues. Their activities in different cells are compared by relating them to deoxyribonucleic acid. They are very low in HEP 1, HEP 2, and HLM cells by comparison with the activities in any normal tissues examined. All the succinic dehydrogenase was shown to be located in the mitochondria of the permanent cell lines by staining with tetrazolium derivatives. Both enzymes were more active in tissues of 19-day chick embryos than in those of 11- or 14-day embryos. The increasing activities found during normal development were quickly curtailed or reversed when heart cells were grown as monolayer cultures.

Digitonin-treated mitochondria produced preparations with much higher activities of cytochrome oxidase than untreated samples. Activities measured in this way were again very much lower in HEP 1, HEP 2, and HLM cells than in the normal tissues. From the derived ratio of cytochrome oxidase:succinic dehydrogenase, it was apparent that cytochrome oxidase is diminished to a greater extent than succinic dehydrogenase in both permanent cell lines and short-term cultures, by comparison with the corresponding activities in embryonic and adult tissues. The features common to the metabolism of proliferating cells in vitro and malignant cells are discussed.
Some Characteristics of DNA Synthesis and the Mitotic Cycle in Ehrlich Ascites Tumor Cells
Authors: Edwards Joshua L. , Koch Arthur L. , Youcis Pauline , Freese Herbert L. , Laite Melville B. , Donalson J. Thomas
Published: May 2004

In vivo studies of Ehrlich ascites tumor cells during the first 5 days of growth in peritoneal cavities of mice consisted of the following:

1. Determination of growth curves by direct enumeration of cells.

2. Estimation of the duration of each phase of the mitotic cycle based on incidence of cells in different phases.

3. Radioautographic studies to determine the proportion of cells in different phases of the mitotic cycle that incorporate tritiated thymidine during a single brief exposure to this precursor of DNA.

4. Estimation of the rate of incorporation of tritiated thymidine at different times during the period of DNA synthesis by comparison of mean grain counts over nuclei in radioautographs at different times following exposure to tritiated thymidine.

The assumptions underlying these experiments and our observations concerning the duration of the period of DNA synthesis and its relation to the mitotic cycle are discussed. It is concluded that DNA synthesis is continuous, occupying a period of 8.5 hours during the interphase and that the average rate of synthesis is approximately constant.
Incorporation and Rate of Loss of S35-Methionine by Adult Rat Trachea in Organ Cultures and in Living Animals
Authors: Crocker T. Timothy , Pelc S. R.
Published: May 2004

Full-thickness pieces of adult rat trachea were supported on rayon on the surface of clotted medium in watch glasses. Differentiated epithelium was reduced in height during 25 days of cultivation because basal cells and some columnar cells migrated to cover exposed parts of the explants and because some differentiated cells died and were shed.

S35-methionine was (a) placed on explants in vitro and (b) injected intraperitoneally in living rats. Cultured tissues and tissues of living rats were examined by autoradiography at 4 and 24 hours and 4, 7, and 11 days after labeling.

Although migratory undifferentiated epithelial cells appeared in cultured trachea, all living epithelial cells in vitro incorporated and subsequently lost S35-methionine to the same extent as did epithelium of intact rats. The biologic half-life of methionine in rat tracheal epithelium in vivo and in vitro was about 5 days.
Segregation of Ferritin in Glomerular Protein Absorption Droplets
Authors: Farquhar Marilyn G. , Palade George E.
Published: May 2004

Ferritin was used as a tracer to study the mechanism by which proteins are segregated into droplets by the visceral epithelium of glomerular capillaries.

In glomeruli from both normal and aminonucleoside-nephrotic rats ferritin molecules introduced into the general circulation penetrated the endothelial openings and were seen at various levels in the basement membrane. Striking differences between nephrotic and controls were seen only in the amount of ferritin incorporated into the epithelium. In normal animals, a few ferritin molecules were seen in small invaginations of the cell membrane limiting the foot processes, within minute vesicles in the epithelium, or within occasional large vacuoles and dense bodies. In nephrotics, epithelial pinocytosis was marked, and numerous ferritin molecules were seen within membrane invaginations and in small cytoplasmic vesicles at all time points. After longer intervals, the concentration of ferritin increased in vacuoles and particularly within the dense bodies or within structures with a morphology intermediate between that of vacuoles and dense bodies. In nephrotic animals cleft-like cavities or sinuses were frequently encountered along the epithelial cell surface facing the urinary spaces. Some of these sinuses contained material resembling that filling the dense bodies except that it appeared less compact.

The findings suggest that ferritin molecules—and presumably other proteins which penetrate the basement membrane—are picked up by the epithelium in pinocytotic vesicles and transported via the small vesicles to larger vacuoles which are subsequently transformed into dense bodies by progressive condensation. The content of the dense bodies may then undergo partial digestion and be extruded into the urinary spaces where it disperses.

The activity of the glomerular epithelium in the incorporation and segregation of protein is similar in normal and nephrotic animals, except that the rate is considerably higher in nephrosis where the permeability of the glomerular basement membrane is greatly increased.
Formation of Sperm Entry Holes in the Vitelline Membrane of Hydroides hexagonus (Annelida) and Evidence of their Lytic Origin
Authors: Colwin Laura Hunter , Colwin Arthur L.
Published: May 2004

Electron micrographs of inseminated eggs of Hydroides hexagonus previously had shown that in the immediate vicinity of the penetrating spermatozoön a small portion of the vitelline membrane regularly was absent, and it had been suggested that this area was a hole made by lytic activity of the individual spermatozoön during the course of its passage through the membrane. This deduction would receive support if it could be established that a sperm entry hole does form in living material.

During the present study a hole repeatedly observed and photographed in the membrane of living eggs was found to arise as the spermatozoön penetrated the membrane. Gently compressed eggs formed exovates only through this hole. The holes, and exovates, were not found except at sperm entry sites. It was concluded that this hole is the counterpart of the area from which the membrane is absent in the electron micrographs cited above, and that the spermatozoön makes this hole.

In an electron micrograph two spermatozoa which had penetrated the membrane at separate but closely neighboring points now occupy a single hole. It is argued that if each spermatozoön had displaced the membrane mechanically to make its hole, then there should be two holes, with a partition of membrane between them, but if each had eroded the membrane by applying lysin, a single hole should have formed as the eroded areas expanded and finally merged into one. The latter view agrees with the facts of the electron micrograph. It is concluded that lysis is the most probable means by which the individual spermatozoön makes its hole.
Electron Microscopic Study of the Phagocytosis Process in Lung
Authors: Karrer H. E.
Published: May 2004

Diluted India ink was instilled into the nasal cavity of mice and the lungs of some animals were fixed with osmium tetroxide at various intervals after one instillation. The lungs of other animals were fixed after 4, 7, 9, 16, or 18 daily instillations.

The India ink was found to be phagocytized almost exclusively by the free alveolar macrophages. A few particles are occasionally seen within thin portions of alveolar epithelium, within the "small" alveolar epithelial cells, or within occasional leukocytes in the lumina of alveoli. The particles are ingested by an invagination process of the plasma membrane resulting in the formation of intracellular vesicles and vacuoles. Ultimately large amounts of India ink accumulate in the cell, occupying substantial portions of the cytoplasm.

The surfaces of phagocytizing macrophages show signs of intense motility. Their cytoplasm contains numerous particles, resembling Palade particles, and a large amount of rough surfaced endoplasmic reticulum. These structures are interpreted as indicative of protein synthesis. At the level of resolution achieved in this study the membranes of this reticulum appear as single dense "lines." On the other hand, the plasma membrane and the limiting membranes of vesicles and of vacuoles often exhibit the double-line structure typical of unit membranes (Robertson, 37).

The inclusion bodies appear to be the product of phagocytosis. It is believed that some of them derive from the vacuoles mentioned above, and that they correspond to similar structures seen in phase contrast cinemicrographs of culture cells. Their matrix represents phagocytized material. Certain structures within this matrix are considered as secondary and some of these structures possess an ordered form probably indicative of the presence of lipid.

The possible origin and the fate of alveolar macrophages are briefly discussed.
Electron Microscope Observations on the Fine Structure of Parietal Cells
Authors: Vial Juan D. , Orrego Héctor
Published: May 2004

An electron microscope study has been made of the structure of parietal cells in cats, dogs, and rats and of the cells lining the gastric glands of Bufo spinulosus. It is characteristic of all these cells to contain numerous vesicles about 0.05 to 0.3 µ in size. In the mammalian parietal cells an intracellular system of canaliculi is also observed, which is much more complex in the rat than in the cat or dog. Stimulation with histamine causes in the cat a very marked hypertrophy of the canalicular system with development of a large number of villi and a decrease in the number of vesicles. In Bufo, histamine induces the formation of a very complex system of membrane infoldings which circumscribe finger-like processes that entirely fill the glandular lumen. The cytoplasmic vesicles diminish or disappear.

These experiments show that under histamine stimulation all these cells undergo a great increase in the cell membrane area. These findings provide additional circumstantial evidence that parietal cells play a role in the secretion of hydrochloric acid.
The Anatomy of Secretion in the Follicular Cells of the Thyroid Gland
Authors: Wissig Steven L.
Published: May 2004

The paper contains a description of the fine structure of the thyroid gland of the normal rat.

The follicular colloid, a homogeneous substance of faintly granular texture, is bounded by cuboidal or low columnar epithelial cells. Numerous pleomorphic microvilli, often permeated by small vesicles extend from the apices of the epithelial cells into the colloid. Many small, membrane-limited vesicles lie in the superficial cytoplasmic layer just below the apical plasmalemma. The ergastoplasmic sacs of the follicular cells are dilated and contain a substance resembling colloid. They are of irregular outline, and the larger sacs tend to be located in the base of the cells. The Golgi apparatus lies in the vicinity of the nucleus and consists primarily of numerous small, membrane-bound droplets with a homogeneous content. Droplets, similar to the Golgi vesicles but larger, lie in the same vicinity and are tentatively identified as colloid droplets. The colloid droplets contain an extremely fine, dense particulate material. Other droplets with a denser, more heterogenous content are also present.

Both the follicular cells and the perifollicular capillaries are bounded by a continuous basement membrane. The capillary endothelium is in certain regions extremely attenuated and is pierced by numerous patent pores, 450 A in diameter.

The marked similarity between the presumptive colloid droplets and vesicles of the Golgi apparatus suggests that the droplets arise from this organelle. On morphological grounds alone no relation can be established between any of the organelles of the follicular cell and the process of colloid resorption.
The Lamellar Systems of Cytoplasmic Membranes in Dividing Spermatogenic Cells of Drosophila virilis
Authors: Ito Susumu
Published: May 2004

Spermatogenic cells of Drosophila virilis were studied by light and electron microscopy. The persistence of a "nuclear wall" during the meiotic divisions has been reported by a number of early cytologists, but this interpretation has been a subject of debate. Electron micrographs of dividing spermatocytes reveal the presence of multiple layers of paired membranes surrounding the nuclear region. These lamellar membrane systems are not typical of the nuclear envelope, but were interpreted as such by light microscopists. The membranes constituting a pair are separated by an interspace of ∼ 100 A and successive pairs are 200 to 400 A apart. These spacings are similar but not identical to those found in the lamellar systems of the Golgi complex. The cisternae of the endoplasmic reticulum in this material are devoid of attached ribonucleoprotein particles, are more precisely ordered than in vertebrate cells, and show a uniform, narrow intracisternal space of ∼ 100 A. The conspicuous asters appear to be made up of similar paired membranes radiating from the centriolar region.

The primary spermatocyte has numerous dictyosomes and a well developed endoplasmic reticulum in cisternal form, but no typical Golgi complex or endoplasmic reticulum is found during the meiotic division stages of metaphase to telophase. Evidence is presented that these cytoplasmic organelles contribute to the formation of the extensive lamellar systems that appear during meiosis. The results of the Golgi silver staining methods and staining tests for phospholipids, basophilia, and the PAS reaction, indicate that the lamellar arrays of membranes present during meiosis are indistinguishable from the Golgi complex in their tinctorial properties.
The Fine Structure of the Epithelial Cells of the Mouse Prostate
Authors: Brandes David , Portela Adolfo
Published: May 2004

The fine structure of the epithelial cells of the anterior lobe, or coagulating gland, of the mouse prostate has been investigated by electron microscopy. This organ is composed of small tubules, lined by tall, simple cuboidal epithelium surrounded by connective tissue and smooth muscle. The epithelial cells are limited by a distinct plasma membrane, which covers minute projections of the cytoplasm into the lumen. The cell membranes of adjacent cells are separated by a narrow layer of structureless material of low density. The cavities of the endoplasmic reticulum are greatly dilated, and the cytoplasmic matrix is reduced to narrow strands, in which the various organelles are visible. The content of the cavities of the endoplasmic reticulum appears as structureless material of lesser density than the cytoplasmic matrix. Material which may be interpreted as secretion products can be seen in the lumina of the tubules. The possible nature of the material inside the cisternal spaces and the secretory mechanisms in these cells is discussed.
Electron Microscope Study of the Normal Rat Aorta
Authors: Keech M. K.
Published: May 2004

The fine structure of the normal rat aorta is described. The presence of a sub-endothelial layer, the oblique orientation of the smooth muscle cells with respect to the aortic axis, and the occurrence of desmosomes between these cells and adjacent elastic laminae, are emphasized. Lead-stained collagen presented a characteristic signet-ring appearance on cross-section. The rats examined were the pair-fed controls for the lathyritic series described in a separate communication.
A Biochemical and Morphological Study of Rat Liver Microsomes
Authors: Moulé Y. , Rouiller C. , Chauveau J.
Published: May 2004

Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point of view.

1. Under these experimental conditions, the "total microsome" fraction was obtained by centrifuging the cytoplasmic extract free of nuclei and mitochondria, for 3 hours at 145,000 g. Morphologically, the total microsomes consist mainly of "rough-surfaced membranes" and "smooth" ones.

2. The total microsomes have been divided into 2 subfractions so that the 1st microsomal fraction contains the "rough" vesicles (2 hours centrifugation at 40,000 g) while the 2nd microsomal fraction consists essentially of smooth vesicles, free particles, and ferritin (centrifugation of the supernatant at 145,000 g for 3 hours).

3. By the action of 0.4 per cent sodium deoxycholate in 0.88 M sucrose, it was possible to obtain a pellet for each of the 2 fractions which consisted of dense particles, rich in RNA, poor in lipids, and which represented about 50 to 60 percent of the RNA and 10 to 15 per cent of the proteins.

The results have been discussed taking into consideration the hypothesis of the presence of RNA in the membranes of microsomal vesicles.
Electrical Properties of Mitochondrial Membranes
Authors: Pauly Helmut , Packer Lester , Schwan H. P.
Published: May 2004

The electrical capacity of the membrane of rat liver mitochondria is 0.5 to 0.6 µ./cm2. This membrane capacity is obtained from the analysis of the frequency dependence of the admittance of a suspension of swollen mitochondria.

In potassium chloride media the mitochondrial membrane capacity does not depend on the ion concentration.

The internal conductance of the mitochondria was approximately one-half that of the external medium; the same applies if the mitochondria are equilibrated in a medium with a 10-fold difference in potassium chloride concentration. Hence the swollen mitochondria investigated here appear to be able to adjust their internal ion concentration in proportion with that of the external phase.

The similarity of the membrane capacity of isolated mitochondria with the range of values known for other membranes suggests a common molecular structure.

The analysis of experimental data suggests an anisotropic electrical behavior of the interior of mitochondria. This anisotropy is readily explained by the existence of internal membranes.
A Cytochemical Study on the Pancreas of the Guinea Pig
Authors: Siekevitz Philip , Palade George E.
Published: May 2004

Chymotrypsinogen synthesis in the exocrine cell of the guinea pig pancreas was studied under the following conditions: Animals fed after a fast of ∼48 hours received ∼1 hour after feeding an intravenous injection of DL-leucine-1-C14. At various time intervals (1 to 45 minutes) after the injection, the glands were removed and fractionated into a series of cell fractions of known cytological significance. Ten to twelve animals were used for each time point. From each cell fraction, the chymotrypsinogen was isolated by acid extraction and purified by (NH4)2SO4 fractionation, isoelectric precipitation, and chromatography. Because of the minuteness of the quantities involved, chymotrypsinogen amounts were calculated from enzymatic activity figures, and a carrier method was used to precipitate and count the enzyme. The chymotrypsinogen isolated from the attached ribonucleoprotein particles of the microsomal fraction had the highest specific radioactivity at the early time points (1 to 3 minutes). After long intervals (at 15 to 45 minutes), the specific radioactivity of the enzyme increased in the microsomal contents and finally in the zymogen granules. The results are compatible with the view that the chymotrypsinogen is synthesized in or on the attached RNP particles and subsequently transported to other cell compartments.
A Cytochemical Study on the Pancreas of the Guinea Pig
Authors: Siekevitz Philip , Palade George E.
Published: May 2004

Ribonucleoprotein (RNP)1 particles isolated by DOC treatment from pancreatic microsomes have a RNA content of 35 to 45 per cent of their dry weight. In the analytical ultracentrifuge about 85 per cent of the material has a sedimentation coefficient of ∼85 S. These particles contain amylase, RNase, and trypsin-activatable proteolytic activities which cannot be washed off or detached by incubation in 0.44 M sucrose. The enzymes are released, however, by incubation in the presence of low concentrations of ATP, PP, or EDTA, and high concentrations of IP and AMP. At the same time, and at the same concentrations, ∼80 per cent of the RNA and ∼25 per cent of the protein of the particles becomes also non-sedimentable. The simultaneous addition of Mg++ to the incubation medium prevents these losses. This finding, together with the observation that all the Mg++ of the particles is released by the same agents, makes it likely that Mg++ holds the particles together, and that its removal by the chelators used causes the particles to disintegrate. These findings are discussed in relation to the molecular structure of the RNP particles.
Structural Changes in Mammalian Cells Associated with Cooling to -79°C
Authors: Weiss L. , Armstrong J. A.
Published: May 2004

Suspensions of HeLa and S37 cells, with and without added glycerol, were cooled in stages to -79°C. and held at that temperature for 30 minutes. After warming to room temperature the cells were fixed, sectioned, and compared by phase contrast and electron microscopy with similar specimens kept at room temperature. Correlated viability tests were made.

Abnormal cytological characteristics, visible with the phase contrast microscope, were clearly related to the sequence of freezing and thawing, and the proportion of altered cells was highest in specimens cooled without glycerol. Electron microscopy showed that even in the presence of glycerol all cells were markedly altered, with distinctive vesiculation and disruption of the various intracellular membranes. There is evidence that much cytoplasmic damage is compatible with survival, but it seems likely that separation of the two layers of the nuclear envelope and rearrangement of the nuclear contents are signs of irreversible damage.

The findings lend some support to the belief that cell death on cooling is due largely to denaturation of semipermeable membranes, caused by the increasing concentration of electrolytes.
Electron Microscopic Observations on the Three-Dimensional Morphology of Apatite Crystallites of Human Dentine and Bone'
Authors: Johansen Erling , Parks Harold F.
Published: May 2004

In sections of human dentine (carious and sound) and bone examined with the electron microscope, apatite crystallites were seen to present long thin profiles somewhat suggestive of a cylindrical shape, broad profiles indicative of a plate-like shape, and profiles intermediate between these two extremes. With a special stereoscopic specimen holder allowing the specimen to be tilted through an angle of 30° it was possible to record images of two profiles of the same crystallite from different angles and thus gain information concerning the 3-dimensional morphology of crystallites showing a thin profile. In all fields so examined, the thin-profile crystallites that were properly oriented with respect to the axis of tilt exhibited a different width dimension in each of the two micrographs. From this it is concluded that the thin profiles actually represented edge views of plate-like crystallites.
A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Subcellular localization of the protease in porcine skeletal muscle.
Authors: Reville W J , Goll D E , Stromer M H , Robson R M , Dayton W R
Published: May 2004

A study was done to determine whether the Ca2+-activated muscle protease (CAF) that removes Z disks from myofibrils in the presence of Ca2+ is located in a sedimentable subcellular organelle. Porcine skeletal muscle cells were diced finely with a scalpel and were suspended in 0.25 M sucrose, 4 mM EDTA with a VIRTIS homogenizer. Filtration of the suspended muscle through four layers of cheesecloth removed most of the myofibrils and stromal protein. Nuclear (1,000 gavg for 15 min), mitochondrial-microsomal (50,000 gavg for 60 min), and supernatant fractions were assayed for succinic dehydrogenase, acid ribonuclease, cathepsin D, and CAF activities. Approximately 96% of total succinic dehydrogenase activity, 81% of cathepsin D activity, and 45% of acid ribonuclease activity, but only 14% of total CAF activity, were found in the nuclear and mitochondrial-microsomal fractions. Cathepsin D activity in the nuclear and mitochondrial-microsomal fractions was decreased if assays were done without prior treatment to rupture membranous structures; hence, our cell rupture and homogenization procedures preserved some intact lysosomal organelles. The results indicate that the small amount of CAF activity in the nuclear and mitochondrial-microsomal fractions was due to contamination by supernate and that CAF is not located in a membrane-bounded subcellular particle. Because CAF is active at the intracellular pH and temperature of living skeletal muscle cells and is in direct contact with the cytoplasm of muscle cells, its activity must be regulated by intracellular cellular Ca2+ concentration to prevent continuous and indiscriminate degradation of myofibrils.
Teleost chloride cell. I. Response of pupfish Cyprinodon variegatus gill Na,K-ATPase and chloride cell fine structure to various high salinity environments.
Authors: Karnaky K J , Ernst S A , Philpott C W
Published: May 2004

Certain euryhaline teleosts can tolerate media of very high salinity, i.e. greater than that of seawater itself. The osmotic gradient across the integument of these fish is very high and the key to their survival appears to be the enhanced ability of the gill to excrete excess NaCl. These fish provide an opportunity to study morphological and biochemical aspects of transepithelial salt secretion under conditions of vastly different transport rates. Since the cellular site of gill salt excretion is believed to be the "chloride cell" of the branchial epithelium and since the enzyme Na,K-ATPase has been implicated in salt transport in this and other secretory tissues, we have focused our attention on the differences in chloride cell structure and gill ATPase activity in the variegated pupfish Cyprinodon variegatus adapted to half-strength seawater (50% SW), seawater (100% SW), or double-stregth seawater (200% SW). The Na,K-ATPase activity in gill homogenates was 1.6 times greater in 100% SW. When 50% SW gills were compared to 100% SW gills, differences in chloride cell morphology were minimal. However, chloride cells from 200% SW displayed a marked hypertrophy and a striking increase in basal-lateral cell surface area. These results suggest that there are correlations among higher levels of osmotic stress, basal-lateral extensions of the cell surface, and the activity of the enzyme Na,K-ATPase.
Sulfhydryls and the in vitro polymerization of tubulin.
Authors: Mellon M G , Rebhun L I
Published: May 2004

The free sulfhydryls of brain tubulin prepared by cyclic polymerization procedures both with and without glycerol have been examined. The average free sulfhydryl titer of tubulin prepared with glycerol (7.0 sulfhydryls/55,000 mol wt) is greater than that of tubulin prepared without glycerol (4.0 sulfhydryls/55,000 mol wt). Diamide, a sulfhydryl-oxidizing agent, inhibits the polymerization of tubulin. Diamide also disperses the 20S and 30S oligomers of tubulin seen in analytical ultracentrifuge patterns of tubulin solutions and, depending on the temperature at which diamide is added, converts all or part of the oligomeric material to 6S dimers. Electron microscopy demonstrates that diamide also destroys the 450-A ring structures characteristic of tubulin solutions. All diamide effects are reversible by the addition of 10 mM dithioerythreitol, a sulfhydryl-reducing agent. That diamide interacts with sulfhydryls on tubulin is directly demonstrated by a 50% decrease in the free sulfhydryl titer of tubulin measured after diamide treatment. Concentrations of CaCl2 which inhibit polymerization also decrease the free sulfhydryl titer of tubulin.
Incorporation of (3H)uridine by the chromatoid body during rat spermatogenesis.
Authors: Söderström K O , Parvinen M
Published: May 2004

The in vitro incorporation of tritiated uridine into RNA by the spermatogenic cells of the rat has been analyzed by high-resolution autoradiography. Special attention has been focused on the unique cytoplasmic organelle, the chromatoid body. After a short labeling time (2 h), this organelle remains unlabeled in the vast majority of the early spermatids although the nuclei are labeled. When the 2-h incubation with (3H)uridine is followed by a 14-h chase, the chromatoid body is seen distinctly labeled in all spermatids during early spermiogenesis from step 1 to step 8. Very few grains are seen elsewhere in the cytoplasm of these cells. When RNA synthesis in the spermatid ceases, the chromatoid body also remains unlabeled. It is likely that the chromatoid body contains RNA which is synthesized in the nuclei of the spermatids. The function of this RNA as a stable messenger RNA needed for the regulation of late spermiogenesis is discussed.
Metabolism of 125I-labeled lipoproteins by the isolated rat lung.
Authors: Pietra G G , Spagnoli L G , Capuzzi D M , Sparks C E , Fishman A P , Marsh J B
Published: May 2004

The capacity of the isolated perfused rat lung to metabolize the protein moieties of serum lipoproteins was assessed using homologous (rat) and heterologous (human) plasma lipoproteins. The protein and lipid moieties of the plasma lipoproteins were labeled in vivo with Na[125I]. In selected cases the lipoprotein peptides were labeled in vivo with 14C- or 3H-labeled amino acids. Uptake of lipoprotein label during perfusion was monitored by measure of losses in perfusate label and by rises in pulmonary tissue labeling as shown by radioassay and by light and electron microscope radioautography. Lipoprotein degradation was assessed by fractionation of perfusate and lung tissue radioactive material into trichloroacetic acid (TCA)-isoluble, TCA-soluble, and ether-ethanol-soluble fractions. When heparin was included in the perfusion medium, there was selective degradation of the protein portion of very low density lipoprotein (VLDL) in the perfusate and concomitant uptake of radioactive label by the lungs. Low density lipoprotein (LDL)) was neither taken up nor catabolized by the isolated rat lung in the absence or presence of heparin. By light and electron microscopy, the label was localized over the interalveolar septa, predominantly the capillary endothelium. Disappearance of TCA-insoluble radioactivity from the perfusate was associated with the generation of both TCA-soluble iodide and noniodide radioactivity. Greater than 50% of the radioactive label taken up by the lungs was found in the delipidated TCA-insoluble fraction. This study provides in vitro evidence for pulmonary catabolism of VLDL apolipoproteins and uptake of peptide catabolic products of VLDL by the lung.
Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs.
Authors: Sluder G
Published: May 2004

Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.
Dependence of centriole formation on protein synthesis.
Authors: Phillips S G , Rattner J B
Published: May 2004

Centriole formation was studied after inhibition of protein synthesis for various portions of the cell cycle. Synchronous populations of mitotic L929 (mouse) cells were plated into petri dishes and the course of procentriole formation was monitored by electron microscope analysis. The frequency with which procentrioles were seen in association with mature centrioles normally increased steadily in the interval from 4 to 12 h after mitosis. The formation of procentrioles was abruptly inhibited by the addition of cycloheximide at any time from mitosis until 12 h postmitosis (S phase). This suggested that the formation of procentrioles was dependent upon protein synthesis immediately before their appearance. Prophase-accociated elongation of procentrioles appeared to occur normally in cells treated with cycloheximide for up to 4 h before prophase, though the mitotic index in treated cultures decreased somewhat. Thus, protein synthesis did not appear to be essential for procentriolar elongation to the mature length.
Secretion-related uptake of horseradish peroxidase in neurohypophysial axons.
Authors: Theodosis D T , Dreifuss J , Harris M C , Orci L
Published: May 2004

During secretion of the neurohypophysial hormones, oxytocin and vasopressin, secretory granule membrane is added to the plasma membrane of the axon terminals. It is generally assumed that subsequent internalization of this additional membrane occurs by endocytosis. In order to study this process, we have traced the uptake of intravenously injected horseradish peroxidase by neurohypophysial axons in rats and golden hamsters. Peroxidase reaction product within the secretory axons was found mainly in vacuolar and C-shaped structures of a size comparable with or larger than the neurosecretory granules. Our observations suggest that these large horseradish peroxidase (HRP)-impregnated vacuoles arise directly by a form of macropinocytosis. Morphometric analysis indicated that this form of membrane retrieval increased significantly after the two types of stimuli used, reversible hemorrhage and electrical stimulation of the pituitary stalk. Microvesicular uptake of HRP was found to be comparatively less.
Axonal transport of [3H]serotonin in an identified neuron of Aplysia californica.
Authors: Goldman J E , Kim K S , Schwartz J H
Published: May 2004

A population of characteristic ellipsoidal dense-core vesicles was identified in axons of the giant cerebral neuron of the mollusc Aplysia. We injected [3H]serotonin into the cell body of this identified serotonergic neuron in the isolated central nervous system in order to study the subcellular components associated with the neurotransmitter. Subcellular fractionation by differential centrifugation indicated that injected serotonin was rapidly taken up into particulate form. [3H]Serotonin appeared in the axons within 2 h after injection, and export continued at a constant rate of 6% of the total in the neuron/h thereafter. The dependence of the total amounts of [3H]serotonin which appeared in the axons in 6 h (export from the cell body) on the amounts injected was consistent with the idea that export is a saturable process, possibly depending on the capacity of somatic vesicles or of some unidentified carrier for serotonin. [3H]Serotonin moved into both major branches of the axon, where it was translocated rapidly. The transmitter, which was shown by autoradiography to be restricted to the axons of the injected cell, was distributed along axons in accumulations of radioactivity; in contrast, its precursor, [5-3H]hydroxytryptophan, moved slowly along axons in a smooth, declining curve, its kinetics consistent with diffusion. Quantitative electron microscope autoradiography revealed that the dense-core vesicles and the cytosol of axons fixed with glutaraldehyde were labeled with [3H]serotonin.