UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA

Electrotransformation was used to introduce both plasmid and linear DNA into Streptococcus pyogenes. The method was optimized using strain NZ131, for which transformation frequencies up to 10(7) per micrograms of plasmid DNA were obtained. A linear fragment of DNA, containing the streptokinase gene (ska) in which an internal fragment had been replaced with an erythromycin resistance gene (erm), was transformed into strain NZ131 with a frequency of 10(3) per micrograms DNA. The introduction of linear DNA into S. pyogenes by electrotransformation should be useful for future genetic analyses as well as targeted gene replacement.     

Stress- and Growth Phase-Associated Proteins of Clostridium acetobutylicum

The response of Clostridium acetobutylicum ATCC 4259 to the stresses produced by a temperature upshift from 28°C to 45°C and by exposure of the organisms to 0.1% n-butanol or to air was examined by analysis of pulse-labeled proteins. The stress response was the induction of the synthesis of a number of proteins, some of which were elicited by the three forms of stress. Eleven heat shock proteins were identified by two-dimensional electrophoresis, as were two proteins whose synthesis was heat sensitive. In the absence of applied stress, the synthesis of four proteins was found to be associated with the growth phase in batch culture; three of these proteins had a higher rate of de novo synthesis when the cells entered the solvent production phase. One of the stress-induced proteins, hsp74, was partially purified an found to be immunologically related to Escherichia coli heat shock protein Dnak. The similarities of the proteins induced at the onset of solventogenesis and by stress suggest a relationship between the two processes.     

Effect of stereoconfiguration on ripple phases (Pβ′) of dipalmitoylphosphatidylcholine

Mixtures of sn-1 ( ) and sn-3 ( ) enantiomers of fully hydrated dipalmitoylphosphatidylcholine (DPPC) were studied with differential scanning calorimetry and freeze-fracture microscopy. The pretransition temperature of racemic mixtures of DPPC was 1.8 C° below that of either pure sn-1 or sn-3 enantiomers, which had similar pretransition temperatures. The main transition temperature of racemic mixtures was also depressed, but to a lesser extent, 0.8 C°. Freeze-fracture images of liposomes of sn-1, sn-3, and racemic mixtures of DPPC frozen from the P_β′ phase showed well-defined ripples of wavelength 13 nm. Lipid stereoconfiguration had no effect on ripple wavelength, configuration or amplitude, or on the number and nature of surface defects.     

Chromosome distribution of intracisternal A-particle sequences in the Syrian hamster and mouse

Metaphase chromosomes of Syrian hamster and BALB/c mice were hybridized in situ with radiolabeled probes derived from cloned intracisternal A-particle (IAP) genes of the corresponding species. The DNAs of these species are known to contain about 900 and 1,000 copies, respectively, of the retrovirus-like IAP sequence elements per haploid genome. Multiple IAP sequences were found on all chromosomes of both hamster and mouse. In the hamster, more than half of the IAP sequences were located in regions of non-centromeric constitutive heterochromatin, at an average concentration per unit chromosome length 5 times greater than in the euchromatic regions. The other dispersed sequences showed marked local variations in concentration along the chromosome lengths; both discrete foci and large grain clusters were observed as well as regions apparently lacking IAP sequences. Within the resolution of the techniques, IAP sequences appeared to be more evenly distributed over the mouse chromosomes; however, some prominent variations in concentration were seen. The number of potentially active IAP genes in the Syrian hamster, and by extension in the mouse, may be restricted by the preferential location of IAP sequences in genetically inert regions of the genome.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

End Products of Anaerobic Chitin Degradation by Salt Marsh Bacteria as Substrates for Dissimilatory Sulfate Reduction and Methanogenesis

The anaerobic pathway of chitin decomposition by chitinoclastic bacteria was examined with an emphasis on end product coupling to other salt marsh bacteria. Actively growing chitinoclastic bacterial isolates produced primarily acetate, H₂, and CO₂ in broth culture. No sulfate-reducing or methanogenic isolates grew on chitin as sole carbon source or produced any measurable degradation products. Mixed cultures of chitin degraders with sulfate reducers resulted in positive sulfide production. Mixed cultures of chitin-degrading isolates with methanogens resulted in the production of CH₄ with reductions in headspace CO₂ and H₂. The combination of all three metabolic types resulted in the simultaneous production of methane and sulfide, with more methane being produced in mixed cultures containing CO₂-reducing methanogens and acetoclastic sulfate reducers because of less interspecific H₂ competition.     

Interleukin 1 increases the binding of human B and T lymphocytes to endothelial cell monolayers

Lymphocyte binding to specialized high-endothelial venules (HEV) in lymph nodes and Peyer's patches is the first step in normal lymphocyte emigration and recirculation. The development and maintenance of HEV in these lymphoid organs are thought to be immunologically controlled. Because postcapillary venules in chronic inflammatory tissue often resemble the HEV of lymphoid tissue and may also be a site of lymphocyte emigration, examination of the effects of immunologic and inflammatory mediators on endothelial cells (EC) may provide important information about the physiology of both normal lymphocyte recirculation and chronic inflammation. It is reported here that treatment of human umbilical vein EC monolayers in vitro with affinity-purified human interleukin 1 (IL 1) markedly enhances the binding of both B and T lymphocytes. Increased binding was observed within 1 h of treatment of EC with as little as 0.04 U/ml IL 1. This effect of IL 1 was EC-specific, because pretreatment of T cells or human skin fibroblasts with IL 1 did not increase the binding of lymphocytes. Stimulation of binding required active EC metabolism because incubation of EC with IL 1 at 4 degrees C, or prior fixation of EC, prevented enhanced binding. The action of IL 1 was not associated with EC damage. The secretion of IL 1 by macrophages and perhaps other cells in inflammatory lesions may exert a positive feedback signal on EC to enhance further emigration of lymphocytes into the inflammatory focus.     

Involvement of Calcium in the Regulation of Serotonin N-Acetyltransferase in Retina

The possible involvement of calcium in the regulation of retinal serotonin N-acetyltransferase (NAT) activity was investigated using eye cups of Xenopus laevis cultured in defined medium. Omitting CaCl2 from the culture medium completely inhibited the dark-dependent increase of NAT activity at night. Approximately 10(-4)-10(-3) M free Ca2+ was found to be required for the maximal increase of NAT activity in the dark. Other divalent cations--Ba2+, Sr2+, and Mn2+--did not substitute for Ca2+. Antagonists of voltage-sensitive calcium channels, including nifedipine, methoxyverapamil (D600), Co2+, and Mg2+, were found to be effective inhibitors of the dark-dependent increase of retinal NAT activity. Trifluoperazine also decreased retinal NAT activity. These studies indicate that the increase of retinal NAT activity in the dark is mediated by a specific Ca2+-dependent process and that Ca2+ influx through voltage-sensitive calcium channels is involved.     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.