N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Oligo-riboprobes. Tools for in situ hybridisation

In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. Moreover, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes ("oligo-riboprobes"). These probes can be labelled to very high (10(9) cpm/micrograms) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise beta (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from beta preprotachykinin cDNA.     

Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.     

Prostaglandin I2 and the constricted maternal ovine placenta: effects of local infusion and placental age

We have tested the hypotheses that systemic responses to the infusion of prostaglandin I2 may have masked the ability of this substance to dilate the maternal placenta and that the inability of prostaglandin I2 to dilate the maternal near-term placenta may be a function of placental age. Regional blood flows were measured with radioactive microspheres. In 8 near-term sheep the control flows were measured and angiotensin II (AII) infusion was begun at 5 micrograms/min and continued for the duration of the experiments. At t = 15 min, regional blood flows were again measured. Prostaglandin I2 was then infused via a retrograde uterine arterial catheter at 10 micrograms/min. At t = 30 min, the flows were again measured. At this time the infusion of prostaglandin I2 was stopped and at t = 45 min the blood flows were measured for the last time. AII increased the resistance of all tissues examined. The blood pressure increased with AII and did not change thereafter. The non-placental uterine tissue served by the retrograde catheter dilated with prostaglandin I2. The placental tissue had an initial resistance of 59 +/- 6 mmHg.ml-1.min.g which increased to 98 +/- 22 mmHg.ml-1.min.g with the infusion of AII (P less than 0.05). This resistance remained constant at 82 +/- 19 mmHg.ml-1.min.g with the administration of prostaglandin I2 and did not change after prostaglandin I2 was removed. The local application of prostaglandin I2 in the presence of AII induced vasoconstriction caused vasodilatation in the nonplacental vessels but could not change the AII induced constriction in the placental vasculature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Zero-stress states of arteries

The no-load configuration of a living organ is, in general, not the zero-stress state. The difference can be revealed by cutting up an unloaded organ to such an extent that the stress becomes zero in the tissue everywhere. For the aorta, it is shown that the configuration of the zero-stress state differs considerably from being a cylindrical tube. It is, in fact, an open sector with opening angles varying along the arterial tree. This article presents data on the zero-stress state in the arteries of the rat in normal condition.     

Interactions of porphyrins with purified DNA and more highly organized structures

Studies of the solution properties of gold(III)tetrakis(4-N-methylpyridyl) porphine and its DNA binding characteristics have been conducted utilizing uv/vis absorption spectroscopy, circular dichroism (CD), Mossbauer spectroscopy, and temperature-jump relaxation techniques. These studies indicate that over the concentration range considered this water soluble gold(III) porphyrin does not aggregate, binds axial ligands only weakly with a preference for soft Lewis bases, and is capable of intercalation into nucleic acids of appropriate base pair content. The interaction of this and several other porphyrins with the synthetic polynucleotide poly(dA-dC).poly(dT-dG) has been studied. Spectroscopic signatures for intercalation were found for those derivatives not having axial ligands. Intercalation into chromatin in vitro can also occur with those porphyrins and metalloporphyrins which do not have axial ligands. Finally, studies utilizing microinjection techniques indicate that once within the cell, tetrakis(4-N-methylpyridyl)porphine tends to localize in the nucleus.     

Bacterial cellulases and saccharification of lignocellulosic materials

Five locally isolated bacterial strains produced extracellular cellulase enzymes, primarily CMCase, when grown on different natural and commercial cellulosic substrates. Extracellular CMCase and avicelase activity was higher with the strain CLS-32, a Cytophaga sp., compared to four other strains. The whole-cell preparations of these isolates were found to saccharify cellulosic substrates to reducing sugars. Maximum release of reducing sugar (5.75 mg ml⁻¹) was obtained with CLS-32 using sugar cane bagasse as growth and hydrolysis substrates.