MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : II. Metabolic Activity of Collagen Associated with Subcellular Fractions of Guinea Pig Granulomata

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-¹⁴C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-¹⁴C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-¹⁴C-valine into TCA-insoluble material by various combinations of subcellular fractions.     

CHEMICAL AND MORPHOLOGICAL STUDIES OF BACTERIAL SPORE FORMATION : IV. The Development of Spore Refractility

From the stage of a completed membranous forespore to that of a fully ripened free spore, synchronously sporulating cells of a variant Bacillus cereus were studied by cytological and chemical methods. Particular attention was paid to the development of the three spore layers—cortex, coat, and exosporium—in relation to the forespore membrane. First, the cortex is laid down between the recently described (5) double layers of the forespore membrane. Then when the cortex is ⅓ fully formed, the spore coat and exosporium are laid down peripheral to the outer membrane layer covering the cortex. As these latter layers appear, the spores, previously dense by dark phase contrast, gradually "whiten" or show an increase in refractive index. With this whitening, calcium uptake commences, closely followed by the synthesis of dipicolinic acid and the process is terminated, an hour later, with the formation of a fully refractile spore. In calcium-deficient media, final refractility is lessened and dipicolinic acid is formed only in amounts proportional to the available calcium. If calcium is withheld during the period of uptake beyond a critical point, sporulating cells lose the ability to assimilate calcium and to form normal amounts of dipicolinic acid. The resulting deficient spores are liberated from the sporangia but are unstable in water suspensions. Unlike ripe spores, they do not react violently to acid hydrolysis and, in thin sections, their cytoplasmic granules continue to stain with lead solutions.     

WOUND HEALING AND COLLAGEN FORMATION : II. Fine Structure in Experimental Scurvy

The sequence encountered in healing skin wounds in scorbutic guinea pigs has been examined by methods of light and electron microscopy. Linear incisions in the skin of female guinea pigs fed a scorbutigenic diet were allowed to heal. The wounds were removed for examination at 1, 3, 5, 9, and 14 days after wounding. The fibroblasts of the scorbutic wounds differ from those of the controls in three major aspects. First, little collagen is present within the intercellular spaces, although small groups of individual collagen fibrils can be found adjacent to some of the fibroblasts; in addition, large amounts of somewhat fibrillar, poorly structured, dense matter are present throughout the extracellular regions. The second alteration consists of large aggregates of intracytoplasmic lipid deposits present within the majority of the fibroblasts. Third, the endoplasmic reticulum of the fibroblasts is altered in form from that of the controls. The profiles of the cisternae are round, non-continuous within the plane of section, and are less extensively developed than in the controls. An increased macrophagic activity of the histiocytes is also described. These changes are discussed in light of the available biochemical data associated with this abnormality of protein synthesis.     

STUDIES ON THE BIOSYNTHESIS OF COLLAGEN : I. THE GROWTH OF FOWL OSTEOBLASTS AND THE FORMATION OF COLLAGEN IN TISSUE CULTURE

1. A tissue culture method was devised in which suspensions of osteoblasts, obtained directly from frontal bones of fowl embryos, were grown in a fluid, fibrin-free medium. 2. Maximum growth of the tissue, as measured by dry weight, with the formation of collagen protein, based on the estimation of hydroxyproline, was obtained in periods of up to 6 days. 3. Appreciable amounts of protein-bound hydroxyproline were formed during the first 24 hour growth period, but electron microscopy of portions of the same cultures failed to demonstrate the presence of any typical collagen fibrils. 4. The subsequent formation of many characteristic collagen fibrils was not associated with a significant rise in the mean hydroxyproline content of the tissue. 5. The cytoplasmic granules of the osteoblasts stained intensely with the P.A.S. technique when the collagen fibrils were being formed. 6. It is suggested that collagen-forming cells synthesise and secrete a hydroxyproline-rich precursor of protein or large peptide nature, which subsequently becomes directly transformed into typical collagen fibrils.     

WOUND HEALING AND COLLAGEN FORMATION : I. Sequential Changes in Components of Guinea Pig Skin Wounds Observed in the Electron Microscope

The regular sequence encountered in healing guinea pig skin wounds has been examined by methods of light and electron microscopy. Observations on cell populations, their fine structure, and fibril formation in the connective tissue have been made. Linear incisions in the skin of normal female guinea pigs weighing 300 to 350 grams were allowed to heal. The wounds were then excised, fixed with buffered 2 per cent osmium tetroxide, and postfixed in neutral buffered formalin, at 16 and 24 hours and at 3, 5, 9, and 14 days after wounding. They were then embedded in epoxy resin. In the inflammatory phase the exudate observed in the early wounds consists largely of polymorphonuclear neutrophilic leukocytes, macrophages, fibrin, and free extracellular organelles from the disrupted inflammatory cells. These organelles later appear in vacuoles in the cytoplasm of the macrophages. Fibroblasts first appear at 24 hours, and show extensive development and dilatation of the endoplasmic reticulum, which sometimes contains moderately dense flocculent material. In addition, these fibroblasts have enlarged mitochondria and condensations of filamentous material within the cytoplasm near the cell surface. Occasional myelin figures and moderately dense, 0.5 to 1.0 micron bodies are found within the cytoplasm of the early fibroblasts. Collagen fibrils are first seen at 3 days extracellularly near the cell surfaces. They appear at the later times in two populations of sizes. With increasing wound age the fibroblasts retain their morphology and the wounds decrease in cellularity concomitantly with the formation of increasing amounts of collagen. Several proposed mechanisms of collagen fibril formation are discussed in relation to the observed phenomena. The problem of correlating fibril diameter with the appearance of the periodic structure of collagen in relation to the minimal size fibril which would be anticipated to display this appearance is discussed.     

CORRELATED MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES ON ISOLATED SINGLE MUSCLE FIBERS : I. Fine Structure of the Crayfish Muscle Fiber

Single fibers isolated from walking leg muscles of crayfish have 8- to 10-µ sarcomeres which are divided into A, I, and Z bands. The H zone is poorly defined and no M band is distinguishable. Changes in the width of the I band, accompanied by change in the overlap between thick and thin myofilaments, occur when the length of the sarcomere is changed by stretching or by shortening the fiber. The thick myofilaments (ca. 200 A in diameter) are confined to the A band. The thin myofilaments (ca. 50 A in diameter) are difficult to resolve except in swollen fibers, when they clearly lie between the thick filaments and run to the Z disc. The sarcolemma invaginates at 50 to 200 sites in each sarcomere. The sarcolemmal invaginations (SI) form tubes about 0.2 µ in diameter which run radially into the fiber and have longitudinal side branches. Tubules about 150 A in diameter arise from the SI and from the sarcolemma. The invaginations and tubules are all derived from and are continuous with the plasma membrane, forming the transverse tubular system (TTS), which is analogous with the T system of vertebrate muscle. In the A band region each myofibril is enveloped by a fenestrated membranous covering of sarcoplasmic reticulum (SR). Sacculations of the SR extend over the A-I junctions of the myofibrils, where they make specialized contacts (diads) with the TTS. At the diads the opposing membranes of the TTS and SR are spaced 150 A apart, with a 35-A plate centrally located in the gap. It appears likely that the anion-permselective membrane of the TTS which was described previously is located at the diads, and that this property of the diadic structures therefore may function in excitation-contraction coupling.     

CHEMICAL AND MORPHOLOGICAL STUDIES ON THE IN VITRO CALCIFICATION OF AORTA

After a lag period, rat aortas incubated in rat serum in vitro accumulated substantial quantities of calcium and phosphate. Examination of the tissue by x-ray diffraction, microradiography, electron diffraction, and electron microscopy indicated that the calcium-phosphate phase which formed was hydroxyapatite and that the crystals were localized almost exclusively in elastin. Selective elimination of various components of the aorta with proteolytic enzymes indicated that the presence of elastin was required for mineralization. Collagen fibers did not appear to be required for the initiation of calcification, nor did they seem to undergo appreciable calcification in the time periods studied. Analysis of the initial lag period suggested that at least two changes occurred in serum prior to the mineralization of this tissue. Inhibitors of the reaction were destroyed, and the level of dialyzable calcium was elevated owing to its release from serum protein.     

MORPHOLOGICAL STUDIES IN ZYGOPHYLLACEAE. I. THE FLORAL DEVELOPMENT AND VASCULAR ANATOMY OF NITRARIA RETUSA

The floral development and vascular anatomy of Nitraria retusa were investigated in order to understand its characteristic androecium of 15 stamens and to clarify the systematic position of the genus relative to Zygophyllaceae. Sepals arise in a helical sequence and are relatively small at maturity. Petals are initiated almost simultaneously or in a rapid helical sequence. Five stamen primordia arise opposite the sepals. Next, two other antesepalous primordia are incepted centrifugally to the first primordia on the remaining receptacular surface. The outer stamens tend to be squeezed between the petals and upper stamens and appear to make up an antepetalous whorl of stamens. Three carpels arise from a low ringwall and grow into a hairy trilocular pistil. In each locule a single pendulous ovule is present. Disclike nectarial tissue is initiated in pits between the stamens and petals. Long trichomes develop on its surface. It is concluded that the androecium is linked with a haplostemonous condition because the stamens of each triplet develop on strictly localized sectors. The distinction between stamens arising on complex primordia and the inception of three independent units is explained by the “principle of variable proportions.” The vasculature also tends to confirm that the outer stamen pairs belong to antesepalous triplets.     

MORPHOLOGICAL AND EMBRYOLOGICAL STUDIES ON THE LEMNACEAE. I. THE FLORAL STRUCTURE AND GAMETOPHYTES OF LEMNA PAUCICOSTATA

Maheshwari, Satish C., and R. N. Kapil. (U. Delhi, Delhi, India.) Morphological and embryological studies on the Lemnaceae. I. The floral structure and gametophytes of Lemna paucicostata. Amer. Jour. Bot. 50(7): 677–686. Illus. 1963.—In Lemna paucicostata, a locally occurring member of the Lemnaceae, the plant body is represented by a frond which is devoid of lignified elements. The root shows a winged root sheath but does not have root hairs. There are no distinctive layers like the endodermis and xylem. The male archesporium is hypodermal and differentiates normally as in other angiosperms into parietal and sporogenous layers. The tapetum is single-layered and plasmodial. The partition walls of the anther are not derived by sterilization of the sporogenous cells as believed earlier. The microspore tetrads are isobilateral and decussate, the meiotic divisions being successive. The pollen grains are shed at the 3-celled stage. The ovary contains a single hemianatropous, bitegminal and crassinucellar ovule. The development of the embryo sac conforms to the Allium type.     

STUDIES WITH CYANIDIUM CALDARIUM : I. The Fine Structure and Systematic Position of the Organism

The fine structure of Cyanidium caldarium, as seen in thin sections of KMnO₄-fixed cells examined with the electron microscope, is described. This organism, whose taxonomic position among algae is undetermined, contains a single well defined chloroplast, a nucleus, and mitochondria. Studies, with the electron microscope, of Chlorella pyrenoidosa and Nostoc are also reported. Structural differences within cells of Cyanidium, chlorella, and Nostoc are discussed. It is concluded that if Nostoc can be taken as a typical Cyanophyte and Chlorella as a representative Chlorophyte and if the items of fine structure examined are diagnostic, then Cyanidium is certainly not a Cyanophyte and, while it has numerous features in common with Chlorella, is not a green alga similar to Chlorella. Comparisons are also made between Cyanidium and other algae whose fine structure has been described by others.     

MORPHOLOGICAL STUDIES OF THE ROOT OF RED PINE,PINUS RESINOSA I. GROWTH CHARACTERISTICS AND PATTERNS OF BRANCHING

Observations were made of the seasonal root growth behavior under natural conditions and under controlled conditions in plant observation boxes. Under natural conditions root growth conformed to the commonly reported pattern of a surge of growth in the spring, a mid-summer low, and a renewed burst in the fall. Growth of individual roots was cyclic. Growth patterns ordinarily varied according to root diameter and branching and in the plantations were modified by soil moisture conditions. Observations of roots during periods of constant elongation showed that the distance from the root apex to the first lateral root primordium varied directly with growth rate. Laterals did not arise in strict acropetal succession, and lateral root abortion was common, particularly in large-diameter, fast-growing roots. Observations of root initiation in relation to seasonal growth increments and to dormancy structures showed an increase in numbers of laterals on both the proximal and distal portions of a seasonal increment.     

The Fine Structure of Bamboo Fibres: I. OPTICAL PROPERTIES AND X-RAY DATA

Following the recent work on wood tracheides and sisal fibres,a detailed investigation has been made of the fine structureof a wide variety of bamboo fibres. A combination of X-ray analysis,measurement of refractive indices in longitudinal view and ofphase differences in transverse section, has presented a completepicture of cellulose chain orientation. The wall of bamboo fibresis many layered. An outermost, thin layer is composed of cellulosechains making an angle of 35° to cell length and this isfollowed by other, inner layers where the angle steadily decreasesfrom outer to inner layers, first to about 20° and thento about 10°. These layers are separated by thicker layers,dark in transverse section between crossed nicols, in whichthe angle is seldom more than 5–6°. These are averagefigures. The results show most clearly that each angle, , varieswith the fibre length, L, in such a way that the longer cellshave steeper spirals in harmony with a relation of the typeL=A+Bcot already proposed for wood tracheides.     

STEREOLOGICAL ANALYSIS OF MAMMALIAN SKELETAL MUSCLE : I. Soleus Muscle of the Adult Guinea Pig

A quantitative analysis of the volumes, surface areas, and dimensions of the ultrastructural components in the soleus muscle fibers of the guinea pig was made by using point counting methods of stereology. Muscle fibers have structural orientation (anisotropy) and have spatial gradients of the structures within the fiber; therefore the standard stereological methods were modified where necessary. The entire analysis was repeated at two section orientations to test the modifications and identical results obtained from both. The volume of lipid droplets was 0.20 ± 0.06% (mean ± standard error, n = 5 animals) and the nuclei volume was 0.86 ± 0.20% of the fiber volume. The total mitochondrial volume was 4.85 ± 0.66% of the fiber volume with about one-third being found in an annulus within 1 µm of the sarcolemma. The mitochondrial volume in the remaining core of the fiber was 3.6 ± 0.4%. The T system has a volume of 0.14 ± 0.01% and a surface area of 0.064 ± 0.005 µm²/µm³ of the fiber volume. The surface area of the sarcolemma is 0.116 ± 0.013 µm²/µm³ which is twice the T system surface area. The volume of the entire sarcoplasmic reticulum is 3.52 ± 0.33% and the surface area is 0.97 ± 0.09 µm²/µm³. The sarcoplasmic reticulum is composed of the terminal cisternae whose volume is 1.04 ± 0.19% and surface area is 0.24 ± 0.05 µm²/µm³. The tubules of the sarcoplasmic reticulum in the I band and A band have volumes of 1.97 ± 0.24% and 0.51 ± 0.08%, and the surface areas of the I and A band reticulum are 0.56 ± 0.07 µm²/µm³ and 0.16 ± 0.04 µm²/µm³, respectively. The Z line width, myofibril and fiber diameters were measured.     

STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS : I. The Structure of the Cell Wall of Cylindrotheca fusiformis Reimann and Lewin

An electron microscope study on the cell wall of the diatom Cylindrotheca fusiformis was carried out using stereoscopic and sectioning techniques. Material prepared by an enzyme treatment or by a mechanical method showed that the wall consists of two major components: a silica shell and organic material. Vapor of hydrofluoric acid was employed to remove the silica and thereby reveal the arrangement of the organic material. An attempt was made to increase the contrast of the organic component by "staining." Uranylacetate not only increased the electron opacity of the organic material but also apparently decreased the electron opacity of the silica shell. In ultrathin sections of complete cells, the structure as revealed by stereoscopy could be confirmed and extended. Every part of the silica shell is tightly enclosed by organic material. In the valve region the silica enclosed in this way is located between other layers of organic material. The whole cell wall is surrounded by a mucilaginous substance which stains with ruthenium red.     

STUDIES ON THE OXIDATIVE METABOLISM OF SACCHAROMYCES CEREVISIAE : I. Observations on the Fine Structure of the Yeast Cell

Vegetative cells of Saccharomyces cerevisiae were fixed with potassium permanganate followed by uranyl nitrate, embedded in methacrylate, and studied in electron micrographs of thin sections. Details of the structure of the cell wall, cytoplasmic membrane, nucleus, vacuole, and mitochondria are described. Cell membranes, about 70 to 80 A thick, have been resolved into two dense layers, 20 to 25 A thick, separated by a light layer of the same dimensions, which correspond in thickness and appearance to the components of the "unit membrane" as described by Robertson (15). The cell wall is made up of zones of different electron opacity. Underlying the cell wall is the cytoplasmic membrane, a sinuous structure with numerous invaginations. The nucleoplasm, often of uneven electron opacity, is enclosed in a pair of unit membranes in which nuclear pores are apparent. The vacuole, limited by a single unit membrane, is usually irregular in outline and contains some dense material. Rod-shaped mitochondria, 0.4 to 0.6 µ in length and 0.2 to 0.3 µ in diameter, are smaller in size, but similar in structure to some of those described in plant and animal cells. Attempts to use osmium tetroxide as fixative were unsuccessful, a result similar to that obtained by other workers. It is suggested that yeast cells are impermeable to osmium tetroxide, except when grown under specific conditions.     

STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI : I. THE TRANSPORT NUMBERS OF ALKALI CASEINATE SOLUTIONS.

1. The deposition of casein on a platinum anode which takes place on the passage of a direct current through solutions of alkali caseinates was quantitatively studied, and it was found that: (a) the amount of casein which is deposited is directly proportional to the current, i.e. it obeys Faraday''s law; (b) the amount of casein deposited is inversely proportional (within the limits studied) to the amount of alkali which is combined with the casein. 2. A method of determining the transport numbers of proteins insoluble at their isoelectric point has been developed. 3. A titration method for determining the amount of alkali in a casein solution is given. 4. Data from the results of transference experiments on sodium caseinate, potassium caseinate, cesium caseinate, and rubidium caseinate solutions are given. It is shown that the data are best explained on the assumption that in these solutions the carriers of the current are alkali metal cations and casein anions. 5. On the basis of our transference results an explanation is given of the results which were obtained by Robertson and by Haas in their migration experiments.