Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Polyamines and basic proteins stimulate activation by cAMP and catalytic activity of Mucor rouxii cAMP-dependent protein kinase

Partial activation of Mucor rouxii cAMP-dependent protein kinase by cAMP was obtained when kemptide was used as substrate, but complete activation was attained with cAMP plus protamine or histone. Full activation could not be achieved by increasing kemptide or cAMP concentration. Complete activation by cAMP could be obtained by addition of 10 microM polylysine, 10 microM lysine-rich histone or 0.5 mM spermine plus spermidine. The degree of stimulation could be up to 5-fold, depending on the amount of enzyme in the assay. The same concentrations of polycations increased 1.5-2.3-fold the Vmax of kemptide phosphorylation by the free catalytic subunits of both Mucor and bovine heart protein kinases; 10 microM polyarginine inhibited completely the activity of both enzymes.     

Protein kinase from Mucor rouxii

Summary Cyclic AMP binding to Mucor rouxii protein kinase holoenzyme and free regulatory subunit was measured by the classical membrane filtration technique and by equilibrium dialysis. The results obtained demonstrate that the filtration method can be used without loss of any cyclic AMP binding site. Both methods unambiguously demonstrate that the number of molecules of cyclic AMP bound to the holoenzyme are half of those bound to the regulatory subunit. This result suggests that unshielding of new cyclic AMP binding sites occurs upon dissociation of the ternary complex holoenzyme-cyclic AMP.     

Molecular cloning of the dog homologue of the lymphocyte antigen CD28

The nucleotide data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L22178.     

Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis

Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver.     

Comparative cytogenetic studies of Curimatidae (Pisces, Characiformes) from the middle Paraná River (Argentina)

Almost all species of the Curimatidae family have a stable karyotype, with a diploid number of 54 metacentric (M) and submetacentric (SM) chromosomes, and one sole nucleolus organizer pair. This family has considerable specific diversity in Argentinean fluvial basins; however, no cytogenetic data are available. Eight species from the Paraná River (Argentina): Cyphocharax voga, C. spilotus, C. platanus, Steindachnerina brevipinna, S. conspersa, Curimatella dorsalis, Psectrogaster curviventris, and Potamorhina squamoralevis were analyzed cytogenetically. Chromosome preparations were obtained from direct samples and through cell culture, and they were processed for conventional, C- and nucleolar organizer region-banding. Six of the species exhibited the standard family karyotype, with 2n = 54 M-SM and fundamental number of chromosomes (FN) = 108, as well as variations in the chromosome formula, and in heterochromatic and nucleolar organizer regions. Though nucleolar organizer regions were located on only one chromosome pair, they varied in both carrier chromosomes and pairs involved. On the other hand, C. platanus showed a complement of 2n = 58 M-SM and subtelocentric with FN = 116, and P. squamoralevis presented 2n = 102, with some M-SM and a large number of acrocentric chromosomes. Even though the karyotype macrostructure appears to be conserved, the speciation process within the family has been accompanied by micro-structural rearrangements, as evidenced by pattern diversity in the heterochromatin and nucleolar organizer regions. Some changes in chromosome macrostructure have also occurred in this group, primarily in C. platanus and P. squamoralevis, in which there have been centric dissociations and inversions.     

Glucose-induced alterations of intracellular ionized magnesium in human lymphocytes

The intracellular ionic content of human erythrocytes may be altered by hyperglycaemia. Despite this, very little is known about the cellular mechanisms linking glucose and cellular magnesium homeostasis. We measured intracellular ionized magnesium in human lymphocytes, by means of a fluorimetric technique, total intracellular magnesium by means of atomic absorption spectrophotometry and intracellular ATP by means of HPLC. The incubation of lymphocytes with D-glucose in the absence of insulin was followed by a significant decrease in intracellular ionized magnesium; this effect did not occur when the cells were incubated with L-glucose. The effect of glucose on intracellular ionized magnesium was blocked by amphotericin B and the EC(50) of the effect of glucose on intracellular ionized magnesium was about 5 mmol/l of glucose. The increase of intracellular ionized magnesium in cells incubated in the absence of glucose was followed by a decrease in intracellular ATP. In a Na(+)-free medium the decrease of intracellular ionized magnesium in the presence of glucose was still present and the incubation of lymphocytes with glucose did not modify total intralymphocyte magnesium. By selective permeabilization of cell membranes, we established that glucose could not increase compartmentalized intracellular ionized magnesium. Our data supports the hypothesis that glucose per se induces a substantial decrease in intracellular ionized magnesium, which is probably due to an augmented binding of intracellular ionized magnesium to cellular ATP.