Histochemical analysis of newly synthesized and accumulated sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg

The leg musculature from 11, 14, and 17 day chick embryos was analyzed histochemically to investigate the temporal and spatial distribution of various types of sulfated glycosaminoglycans present during skeletal muscle development. Types of glycans were identified by selective degradation with specific glycosidases and nitrous acid coupled with Alcian blue staining procedures for sulfated polyanions and with [35S]sulfate autoradiography. On day 11, radiolabeled chondroitin sulfate glycosaminoglycans are localized extracellularly in both the myogenic and connective tissue cell populations. By day 17, incorporation of [35S]sulfate into chondroitin sulfate is substantially reduced, although Alcian blue-stained chondroitin sulfate molecules are still detectable. With increasing age and developmental state of the tissues, radiolabeled and stained dermatan sulfate and heparan sulfate progressively increase in relative quantity compared to chondroitin sulfate both in muscle and in associated connective tissue elements. These changes in glycosaminoglycans correlate well with similar changes previously determined biochemically and further document the alterations in extracellular matrix components during embryonic skeletal myogenesis.     

Protein Body Composition in Cuburbita maxima Cotyledons as Determined by Energy Dispersive X-Ray Analysis

Energy dispersive x-ray analysis was used to study the composition of certain protein body components in Cucurbita maxima cotyledons. The globoid crystal was rich in phosphorus, potassium, and magnesium. This elemental composition provides further evidence that the globoid crystal in squash cotyledon protein bodies is composed of phytin, a myoinositol hexaphosphoric acid salt of potassium and magnesium. Calcium, a common component of phytin in many species, was absent or present in only trace amounts in the globoid crystals of squash. Results of analyses of globoid crystals from seeds produced in different parts of North America suggest that there is definite specificity for the cations used in phytin deposition. Variations in soil types and other environmental factors seem not to have influenced the type of cation stored. Energy dispersive x-ray analysis of the proteinaceous regions revealed the presence of phosphorus, sulfur, and a trace of chlorine. Sulfur was expected, due to the presence of some sulfur containing amino acids in the protein.     

Two Acidic Proteins Associated with Brain Development in Chick Embryo

The developmental changes in protein composition of the chick optic tectum were analyzed by two-dimensional gel electrophoresis. Staining with Coomassie Brilliant Blue R revealed 54 major proteins, eight of which remarkably changed their abundance during development: Four of these proteins (S8, S14, S30, and S54) increased and two of them (S7 and S37) decreased in the course of the brain development. The other two proteins (S5 and S6) appeared at specific embryonic stages and were not detected in the adult. The abundance of S5 protein was highest at day 7, and that of S6 protein at days 9-18. The two proteins were present in other regions of the embryonic brain but were not detected in the embryonic liver. The proteins were purified from the soluble fraction of embryonic chick brains by pH 5.5 precipitation, DEAE-Sepharose column chromatography, ammonium sulfate precipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of S5 and S6 proteins were 95,000 and 100,000, respectively, and their isoelectric points were about 4.5. They were compared by peptide mapping using V8 protease and found to share 11 common peptides out of 17 distinct ones. This indicates a strong degree of structural homology between these two proteins.     

Molecular species of liver triglycerides during development of Gallusgallus

Triglycerides from embryonic chick livers at various developmental stages (13th, 17th and 21st day) were resolved into twelve groups of molecular species differing in their unsaturation degree and fatty acid composition. The relative abundance of each fraction was determined. SM₂ fractions underwent a nearly three-fold increase between 13th and 21st day of embryonic development, whereas S₂H+SMH species decreased from 32.7% to 11.2% during the same period. Nevertheless, on a wet tissue basis only the SM₂ group showed a marked increase throughout that interval. Fatty acid composition of fractions remained practically constant from one stage to another, the only exception being the group SM₂ for which some significant changes were noticed.     

BIOSYNTHESIS OF COLLAGEN AND NON-COLLAGEN PROTEIN DURING DEVELOPMENT OF THE CHICK CORNEA

The relationship between the rates of increase of corneal protein fractions and incorporation of labeled precursors has been examined during embryonic and early posthatching development of the chick corneal stroma. Non-collagen protein increased gradually from 9 through 20 days of incubation. Collagen accumulated approximately logarithmically through the 19th day, the most rapid rate occurring between 13 and 20 days of incubation. The rates at which labeled amino acids are incorporated into collagen in vivo and in vitro undergo marked changes during the last week of embryonic development, corresponding closely to the rate of collagen accumulation in vivo; whereas incorporation into non-collagen protein changes much less markedly. Changes in the rate of incorporation of precursors into collagen are not due to changes in the rate of conversion of collagen from the soluble to insoluble form, or to changes in the endogenous amino acid pool size. Chick embryo corneal stroma collagen turns over very slowly, if at all. Non-collagen protein turns over more rapidly. An increase in cell number, as indicated by DNA content, does not account for the increased rate of collagen synthesis between the 9th and 16th day of incubation. It is concluded that the observed changes in collagen synthesis reflect changing activities in the individual cornea fibroblasts. These activities are comparable in the intact tissue in vivo and in isolated corneas in vitro.     

An X-Ray Scattering Study into the Structural Basis of Corneal Refractive Function in an Avian Model

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3–4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.     

An X-Ray Scattering Study into the Structural Basis of Corneal Refractive Function in an Avian Model

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3–4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.     

Developmental changes in glycosaminoglycans,collagen and collagenase activity in embryonic chick skin

In order to study remodelling of connective tissue during development, changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin at various stages have been studied.Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20).At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis.Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Direct and indirect influence of sulfur availability on phytoplankton evolutionary trajectories

The sulfate facilitation hypothesis suggests that changes in ocean sulfate concentration influenced the rise to dominance of phytoplankton species of the red lineage. The mechanistic reasons for this phenomenon are not yet understood. We started to address this question by investigating the differences in S utilization by algae of the green and red lineages and in cyanobacteria cultured in the presence of either 5 mmol · L^?1 (approximately equivalent to Paleozoic ocean concentrations) or 30 mmol · L^?1 (corresponding to post‐Mesozoic/extant concentrations) sulfate. The activities of the main enzymes involved in SO₄^2? assimilation changed in response to changes in growth sulfate concentration. ATP sulfurylase showed different kinetics in the various taxa, with an especially odd behavior for the dinoflagellate. Sulfate availability had a modest effect on cell organic composition. Species‐specific differences in the use of some elements were instead obvious in algae grown in the presence of different sulfate concentrations, overall confirming that algae of the red lineage do better at high sulfate than algae of the green lineage. The increase in sulfate concentration may thus have had an impact on phytoplankton radiation both through changes in their enzymatic machinery and through indirect repercussion on elemental usage.     

Developmental changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin.

In order to study remodeling of connective tissue during development, changes in glycosaminoglycan, collagen and collagenase activity in embryonic chick skin at various stages have been studied. Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20). At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum, indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis. Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Detection of genetically altered copper levels in Drosophila tissues by synchrotron x-ray fluorescence microscopy

Tissue-specific manipulation of known copper transport genes in Drosophila tissues results in phenotypes that are presumably due to an alteration in copper levels in the targeted cells. However direct confirmation of this has to date been technically challenging. Measures of cellular copper content such as expression levels of copper-responsive genes or cuproenzyme activity levels, while useful, are indirect. First-generation copper-sensitive fluorophores show promise but currently lack the sensitivity required to detect subtle changes in copper levels. Moreover such techniques do not provide information regarding other relevant biometals such as zinc or iron. Traditional techniques for measuring elemental composition such as inductively coupled plasma mass spectroscopy are not sensitive enough for use with the small tissue amounts available in Drosophila research. Here we present synchrotron x-ray fluorescence microscopy analysis of two different Drosophila tissues, the larval wing imaginal disc, and sectioned adult fly heads and show that this technique can be used to detect changes in tissue copper levels caused by targeted manipulation of known copper homeostasis genes.     

The energy dispersive X-ray microanalysis technique in the study of fluids and tissues of the brain and the inner ear

Summary The temporal bone from the fetal and adult mouse and brain from fetal and adult rat were cryosectioned in a conventional cryostat at –30° C using 16 m thick sections. The elemental composition was analyzed in the fluid-filled spaces of the membraneous labyrinth and ventricular cavities of the brain. After drying these spaces contained small crystal-like aggregations of material which morphologically differed depending on their elemental composition. This changed during embryonic maturation. The qualitative X-ray analytical microscopy seems to be a reliable method to follow changes in the elemental composition of fluids.Supported by grant no 12X-720 from the Swedish Medical Research Council. Doctor Wroblewski was during the course of this investigation supported by a research fellowship from the Muscular Dystrophy Association, USA     

Elemental Sulfur and Thiol Accumulation in Tomato and Defense against a Fungal Vascular Pathogen

The occurrence of fungicidal, elemental S is well documented in certain specialized prokaryotes, but has rarely been detected in eukaryotes. Elemental S was first identified in this laboratory as a novel phytoalexin in the xylem of resistant genotypes of Theobroma cacao, after infection by the vascular, fungal pathogen Verticillium dahliae. In the current work, this phenomenon is demonstrated in a resistant line of tomato, Lycopersicon esculentum, in response to V. dahliae. A novel gas chromatography-mass spectroscopy method using isotope dilution analysis with 34S internal standard was developed to identify unambiguously and quantify 32S in samples of excised xylem. Accumulation of S in vascular tissue was more rapid and much greater in the disease-resistant than in the disease-susceptible line. Levels of S detected in the resistant variety (approximately 10 microg g-1 fresh weight excised xylem) were fungitoxic to V. dahliae (spore germination was inhibited >90% at approximately 3 microg mL-1). Scanning electron microscopy-energy dispersive x-ray microanalysis confirmed accumulation of S in vascular but not in pith cells and in greater amounts and frequency in the Verticillium spp.-resistant genotype. More intensive localizations of S were occasionally detected in xylem parenchyma cells, vessel walls, vascular gels, and tyloses, structures in potential contact with and linked with defense to V. dahliae. Transient increases in concentrations of sulfate, glutathione, and Cys of vascular tissues from resistant but not susceptible lines after infection may indicate a perturbation of S metabolism induced by elemental S formation; this is discussed in terms of possible S biogenesis.     

Connective tissue metabolism in muscular dystrophy. Levels of collagen and mucopolysaccharides in embryonic chickens with genetic muscular dystrophy

There were marked differences between the levels of collagen (measured as hydroxyproline) and mucopolysaccharides (measured as hexosamine) found in embryonic chicks with genetic muscular dystrophy and their normal controls. The chief differences were that the dystrophic tissues (gastrocnemius muscle and tendon, pectoralis major and skin) had: (a) greater amounts of hexosamine early in embryonic development; (b) hydroxyproline levels that rose at a faster rate, yielding different slopes than their normal controls; (c) relatively greater amounts of hydroxyproline than hexosamine later in embryonic life (day 20). Connective tissue systems in muscles were preferentially affected. The connective tissue system associated with dystrophic tissues appeared to lag behind the normal rhythm pattern of embryological development. The changes in connective tissue metabolism observed in dystrophic chicks suggested that the collagen from dystrophic embryonic chicks may be of a different structure or composition than that found in the normals.     

Lectin histochemistry of the embryonic heart: expression of terminal and penultimate galactose residues in developing rats and chicks

Rat embryos at days 10-18 of gestation and chicken embryos at days 3-6 of incubation were fixed and processed for lectin histochemistry. The distribution of binding sites for a lectin from the peanut Arachis hypogaea (PNA) conjugated to horseradish peroxidase (HRP) was determined on tissue sections both before and after enzymatic cleavage of sialic acid with neuraminidase (sialidase). Endocardial cushion tissue in the rat, but not in the chick, reacted with PNA-HRP prior to digestion with sialidase. Endocardium of both species (12 and 13 days in rat, 5 and 6 days in chick), particularly at the level of endocardial cushions, reacted strongly with the sialidase-PNA sequence; this staining decreased markedly after day 14 of gestation in the rat. PNA binding sites capped by sialic acid were most abundant in the developing rat heart during the critical period of endocardial cushion formation and decreased as development proceeded. The marked changes in the appearance and distribution of cardiac cell and tissue glycoconjugates during cardiogenesis support the concept that rapid changes occur in the structure of complex carbohydrates during embryonic and fetal development. The findings also suggest that such glycosylation-related events may be species specific.     

Developmental changes in glycosaminoglycan sulfotransferase activities in animal sera

Glycosaminoglycan sulfotransferase activities in sera during the prenatal and postnatal development of the ox, rat, and chicken were systematically measured with chemically desulfated cartilage chondroitin 4-sulfate, cornea keratan sulfate, and kidney heparan sulfate as exogenous sulfate acceptors and with [35S]sulfate-labeled 3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor. The results of specificity studies and product analyses indicated that these enzymes introduce sulfates at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 6 of the galactose units of keratan sulfate, and position 2 (an amino group) of the glucosamine units of heparan sulfate, respectively. The results of the enzyme assays indicated that (1) the three activities change in a development-associated manner in each animal species, (2) generally, the activities of the former two enzymes decrease with embryonic development and aging after birth, although in chicken serum they increase transiently at the late prenatal stage and decrease thereafter, and (3) the pattern of the changes in heparan sulfate sulfotransferase activity is species-dependent: the activity increases in the rat, decreases in the ox, and does not significantly change in the chicken during prenatal or postnatal development. These alterations may reflect development-associated biosynthesis of the corresponding glycosaminoglycans or maturation of the proteoglycans in some tissues.     

MORPHOGENESIS OF THE COLLAGENOUS STROMA IN THE CHICK CORNEA

The embryonic chick corneal epithelium produces a highly structured acellular matrix beneath its basal surface during early development. This matrix, which contains collagen, serves as a morphogenetic template for subsequent stromal development in that the three-dimensional architecture of the adult corneal stroma is initially established, in miniature, in this epithelially derived connective tissue. Examination of the early corneal epithelium and matrix in both the light and electron microscope suggests that self assembly of the matrix may be one of several important factors in the morphogenesis of this early connective tissue.     

Evaluating the Potential of Halothiobacillus Bacteria for Sulfur Oxidation and Biomass Production under Saline Soil

Abstract

Salinity negatively affects growth of sulfur-oxidizing bacteria (SOB) and their sulfate production ability, meanwhile decreases the available sulfate for plants in soil. The aim of this study was to isolate and characterize the bacteria of genus Halothiobacillus, as a salt-tolerant SOB, from saline and sulfidic habitats of Iran for the first time and evaluating the effect of salinity on their biomass and sulfate production during the oxidation of different sulfur sources. Isolation process and surveying the morphological, biochemical and 16S rRNA gene analysis resulted into identification of three species (eight strains) of Halothiobacillus genus including H. neapolitanus, H. hydrothermalis and H. halophilus. Salinity (0, 0.5, 1, 2 and 4?M NaCl) had a significant impact (p?≤?0.01) on bacterial biomass and sulfate production during the oxidation of thiosulfate and elemental sulfur. Biomass and sulfate production by strains was accompanied by a decrease in residual content of thiosulfate (RCT) in medium. The amount of produced biomass and sulfate in medium supplemented by thiosulfate was much higher than elemental sulfur. The highest amount of biomass and sulfate was produced by H. neapolitanus strain I19 at 0.5 and 1?M NaCl concentration. The results of this study could be the first step to focus on the application of these bacteria to increase sulfate storage of saline soils and crop production.     

Ocular distribution of keratan sulfates during pre- and postnatal development in rabbits

Twenty-six monoclonal antibodies (MAbs) developed against rabbit corneal proteokeratan sulfate (PKS), were used to evaluate immunohistochemically the ocular distribution of PKS during prenatal and early postnatal development in rabbits. These MAbs were directed against epitopes located in the keratan sulfate (KS) chains of the proteoglycan (SundarRaj et al., 1985). Staining of cryostat sections of the eyes was carried out using an indirect peroxidase-conjugated technique. Only one of the MAbs reacted with the presumptive corneal region at day 13 or 16 of fetal development. By day 20, more MAbs reacted with the corneal stroma. There were distinct differences, however, in the distribution of the epitopes recognized by the various MAbs. A few of them stained only the posterior region of the cornea, whereas others showed a decreasing staining gradient from the posterior to the anterior region. By day 24, all of the MAbs reacted with the corneal stroma, but some reacted also with the limbal region and with the conjunctival stromal matrix. One MAb also reacted with the conjunctival epithelial layer, but only at this stage of development. Conjunctival staining was more intense at day 28 of fetal development and at day 2 postnatally. KS was not detectable in the conjunctiva of adult rabbits with any of the MABs. These results suggest that although KS synthesis starts at very early stages of fetal development, there are progressive changes in its antigenic structure in specific regions of the cornea and conjunctiva during corneal development.