Sulfation of fucoidan in Fucus embryos. I. Possible role in localization

Zygotes of the brown alga Fucus distichus L. Powell divide into two cells which are structurally and biochemically different from each other. Cytochemical staining and autoradiography indicate that a sulfated polysaccharide is localized in only one of the two cells. Up to 10 hr after fertilization, no localization of sulfated polysaccharides is detectable in zygotes, and little ³⁵S (Na₂³⁵SO₄) is incorporated into an acid-soluble carbohydrate fraction. Between 10 and 16 hr, during rhizoid initiation and several hours before the first cell division, there is a large increase in the amount of ³⁵S incorporated into this fraction. The label is found associated with the sulfated fucose polymer fucoidan. Various extraction techniques and labeling experiments demonstrate that fucoidan is unsulfated at fertilization and undergoes little metabolic activity or turnover during the first 24 hr. Thus, the incorporation of sulfate into this carbohydrate fraction appears to involve a sulfation of a preexisting, unsulfated fucan polymer. The degree of sulfation achieved at this time in vivo is sufficient for migration of fucoidan through an electric field in agarose or acrylamide gels. The possible role of sulfation as a mechanism for the localization of fucoidan in the rhizoid cell by means of an intracellular electrical gradient is discussed.     

Glycosaminoglycan biosynthesis in arterial wall. Sulfation of heparan sulfate in cell membrane of aortic media-intima

Incubation of microsomal fractions with labelled 3'-phosphoadenylyl sulfate results in incorporation of [35S]sulfate into endogenous glycosaminoglycans. Specific radioactivity observed incorporated into heparan sulfate chains is 10-fold greater than that incorporated into chondro?tin sulfate chains. This is in agreement with the results obtained for glycosylation of glycosaminoglycans in arterial wall membrane fractions. Sulfation of heparan sulfate was studied since it contains N- and O-sulfate groups in contrast with the other sulfated glycosaminoglycans which contain only O-sulfate groups. Sulfation of heparan sulfate occurs rapidly, since sulfate incorporation is detected after exposure for only 0.5 min. Heparan sulfate was identified on the basis of its resistance to hyaluronidase and chondro?tin ABC lyase, its susceptibility to heparitinase, its sensitivity to nitrous acid and the presence of glucosamine as the only hexosamine. The chemical composition of the purified heparan sulfate fractions provides evidence for the high degree of sulfation of its chains. Studies into the distribution of sulfate residues on heparan sulfate at different times of sulfation indicate that N-sulfate groups are not randomly introduced into the polymer. The relationship between the processes of N- and O-sulfation was studied. The present results demonstrate that preferential N-sulfation is obtained for incorporation of labelled precursor over a short period, the O-sulfation occurring on previously N-sulfated heparan sulfate.     

Identification of genes required for cytoplasmic localization in early C. elegans embryos

We have isolated and analyzed eight strict maternal effect mutations identifying four genes, par-1, par-2, par-3, and par-4, required for cytoplasmic localization in early embryos of the nematode C. elegans. Mutations in these genes lead to defects in cleavage patterns, timing of cleavages, and localization of germ line-specific P granules. Four mutations in par-1 and par-4 are fully expressed maternal effect lethal mutations; all embryos from mothers homozygous for these mutations arrest as amorphous masses of differentiated cells but are specifically lacking intestinal cells. Four mutations in par-2, par-3, and par-4 are incompletely expressed maternal effect lethal mutations and are also grandchildless; some embryos from homozygous mothers survive and grow to become infertile adults due to absence of functional germ cells. We propose that all of these defects result from the failure of a maternally encoded system for intracellular localization in early embryos.     

Ultrastructural localization of cytoplasmic phosphatases in preimplantation mouse embryos.

The appearance and localization of the cytoplasmic phosphatases [acid phosphatase (AcPase) as a marker of lysosomes, TPPase as a marker of the Golgi apparatus, and NDPase (IDPase) as enzymatic marker of the endoplasmic reticulum (ER)] were cytochemically studied on the ultrastructural level in secondary oocytes and in preimplantation mouse embryos. The detectable AcPase activity, located on the inner surface of the membrane delimiting some cytoplasmic vacuoles (lysosomes and autophagic vacuoles), appears at the eight-cell stage and grows pregressively stronger up to the blastocyst stage. Golgi-associated reaction for TPPase was detectable in oocytes, dropped in one-cell embryos and became negative in the two-cell embryos. The reaction for TPPase and IDPase was present in plasma membranes of oocytes and early embryos and appeared in the delimiting membrane of some cytoplasmic vesicles in eight-cell embryos. Some activity of IDPase was found in small segments of the ER at the morula and blastocyst stage. The observed results suggest that the lysosomes are the first organelles in early embryos showing activity of the marker enzymes of the phosphatase type, while the activity of other marker enzymes is mainly concentrated in the plasma membrane of blastomeres. It cannot be excluded, however, that positive reaction for TPPase and IDPase in the plasma membrane results from nonspecific action of other phosphatases.     

Sulfation of the N-linked oligosaccharides of influenza virus hemagglutinin: temporal relationships and localization of sulfotransferases

The occurrence of sulfate substituents on several positions of glycoprotein N-linked oligosaccharides prompted us to determine the subcellular localization and temporal relationships of the addition of these anionic groups employing as a model system the hemagglutinin (HA) produced by influenza virus-infected Madin-Darby canine kidney (MDCK) cells. It became apparent from a study of the HA glycoprotein in subcellular fractions resolved by Nycodenz gradient centrifugation following pulse-chase radiolabeling that sulfation of the complex N-linked oligosaccharides occurs only after they have been processed to an endo-beta-N-acetylglucosaminidase-resistant state and have reached the medial/trans Golgi and the trans Golgi network (TGN), with the former carrying out most of the sulfation activity. Hydrazine/nitrous acid/NaBH(4) treatment of the HA from the subcellular fractions indicated that C-3 of the galactose as well as C-6 of the N-acetylglucosamine residues of the N-acetyllactosamine chains became sulfated in these post ER fractions, as did the C-6 of the outer N-acetylglucosamine of the di-N-acetylchitobiose core. Consistent with the specificities of the stepwise assembly of the N-acetyllactosamine branches, we observed that the 3'-phosphoadenosine 5'-phosphosulfate (PAPS):GlcNAc-6-O-sulfotransferase migrated in the gradient to a medial/trans Golgi position while in contrast the PAPS:Gal-3-O-sulfotransferase was found in both Golgi and TGN locations. In accordance with the concept that beta-galactosylation must precede the sulfation catalyzed by the latter enzyme, we observed the presence of UDP-Gal:GlcNAc galactosyltransferase in both these sites in the MDCK cells. The presence of the Gal-3-O-sulfotransferase in the TGN is particularly important in the influenza virus-infected cells, as it makes possible the addition of terminal anionic groups after removal of the sialic acid residues by the viral neuraminidase.     

Ectonucleotidases in the digestive system: focus on NTPDase3 localization

Extracellular nucleotides and adenosine are biologically active molecules that bind members of the P2 and P1 receptor families, respectively. In the digestive system, these receptors modulate various functions, including salivary, gastric, and intestinal epithelial secretion and enteric neurotransmission. The availability of P1 and P2 ligands is modulated by ectonucleotidases, enzymes that hydrolyze extracellular nucleotides into nucleosides. Nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase are the dominant ectonucleotidases at physiological pH. While there is some information about the localization of ecto-5'-nucleotidase and NTPDase1 and -2, the distribution of NTPDase3 in the digestive system is unknown. We examined the localization of these ectonucleotidases, with a focus on NTPDase3, in the gastrointestinal tract and salivary glands. NTPDase1, -2, and -3 are responsible for ecto-ATPase activity in these tissues. Semiquantitative RT-PCR, immunohistochemistry, and in situ enzyme activity revealed the presence of NTPDase3 in some epithelial cells in serous acini of salivary glands and mucous acini and duct cells of sublingual salivary glands, in cells from the stratified esophageal and forestomach epithelia, and in some enteroendocrine cells of the gastric antrum. Interestingly, NTPDase2 and ecto-5'-nucleotidase are coexpressed with NTPDase3 in salivary gland cells and stratified epithelia. In the colon, neurons express NTPDase3 and glial cells express NTPDase2. Ca(2+) imaging experiments demonstrate that NTPDases regulate P2 receptor ligand availability in the enteric nervous system. In summary, the specific localization of NTPDase3 in the digestive system suggests functional roles of the enzyme, in association with NTPDase2 and ecto-5'-nucleotidase, in epithelial functions such as secretion and in enteric neurotransmission.     

RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain.     

Immunocytochemical localization of binding 'sites' for LH and hCG in preimplantation mouse embryos.

By using an immunoperoxidase antibody method, mouse blastocysts were found to bind specifically hCG and ovine LH but not FSH or the beta unit of hCG. Brown peroxidase reaction products were present in the morula and increased with the formation of the blastocoele. The LH/hCG binding 'sites' may be related to the initiation of steroidogenesis in the embryo.     

Cellulose in algal cell wall: an "in situ" localization.

In order to ascertain the presence and the in muro localization of cellulose, the enzyme-gold affinity test was applied to algal cell walls. The high specificity of affinity cytochemistry allowed us, by using the enzyme cellulase, to confirm the available biochemical data and to give a map of the cellulose localization in different algal groups. Taking into account the complex skeletal polysaccharide structure and composition of the algal cell walls, this method proved to be a reliable tool in this field.     

Bacterial cell wall synthesis: new insights from localization studies.

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

The mitochondrial localization of coproporphyrinogen III oxidase.

The location of coproporphyrinogen III oxidase in mitochondria was studied in rat liver by using the digitonin method or hypo-osmotic media for fractionation. The enzyme was found in the intermembrane space with a fraction loosely bound to the inner membrane. This fraction was released by washing the inner-membrane-matrix complex with alkaline solutions or solutions of high ionic strength. The enzyme in both fractions had the same Km (0.16 micrometer) for coproporphyrinogen III. When incubation was performed in a medium that avoided destruction of enzyme membrane binding, a dramatic increase in activity was observed after sonication of whole mitochondria or of the inner-membrane-matrix complex.     

Rhizoid differentiation in Spirogyra I. Basic features of rhizoid formation

Several types of rhizoids occurring in the process of differentiationin Spirogyra sp. are described and their interrelation was elucidated.There are two differentiation sequences: P_pPRh_ros or PpPRh_rodRh_ros(for explanation of abbreviations see p. 533), although undersome conditions the sequences ceased halfway through. The initiationtime for rhizoid formation had no relation to the cell cyclestage. The difference in growth patterns between the rhizoidand ordinary filament cells was demonstrated with Calcofluor-stainingand centrifugation. The optimum temperature and pH of the culture medium for rhizoiddifferentiation were 20?C and pH 7, respectively. A contactstimulus was not necessary for induction. Of the several environmental factors examined, light was themost important, for rhizoid formation, since a rhizoid was inducedonly when light was given after cutting the filament. (Received December 14, 1972; )