The discovery of glycogenin and the priming mechanism for glycogen biogenesis

The biogenesis of glycogen in skeletal muscle requires a priming mechanism that has recently been elucidated. The first step is catalysed by a protein tyrosine glucosyltransferase and involves the formation of a novel glycosidic linkage, namely the covalent attachment of glucose to a single tyrosine residue (Tyr194) on a priming protein, termed glycogenin. The next stage is the extension of the glucan chain from Tyr194 and involves the sequential addition of up to seven further glucosyl residues. This reaction is brought about autocatalytically by glycogenin itself, which is a Mn2+/Mg(2+)-dependent UDP-Glc-requiring glucosyltransferase. The glucan primer is elongated by glycogen synthase, but only when glycogenin and glycogen synthase are complexed together. Glycogen synthase dissociates from glycogenin during the synthesis of a glycogen molecule, enabling glycogen molecules to reach their maximum theoretical size. Each mature glycogen beta particle in muscle contains one molecule of glycogenin attached covalently, and an average one glycogen synthase catalytic subunit bound non-covalently. As evidence accumulates that a priming protein may be a fundamental property of polysaccharide synthesis in general, the molecular details of mammalian glycogen biogenesis may serve as a useful model for other systems.     

Specific cytosol binding of diethylstilbestrol in rat skeletal muscle

Rat skeletal muscle cytosol proteins bound ³H-diethylstilbestrol (³H-DES). More than 90% of this binding was high capacity and low affinity. Serum albumin accounted for roughly 50–60% of the binding, as evidenced by its precipitation with anti-rat albumin IgG. About half of the binding was distinguishable from albumin and other serum proteins by its precipitation in 40% saturated ammonium sulfate. This material sedimented at 4–5S in high-salt sucrose gradients, and resolved into two components (8S and 4–5S) in low-salt. Following incubation at 23–27°C for one hour, 2% of the bound ³H-DES in whole cytosol (approximately 2 fmole/mg cytosol protein) was retained by DNA-cellulose, and was eluted with 0.6 M KCl. This small fraction of the total binding was inhibited by estrogens and DES analogues: estradiol-17β, DES, dienestrol, and hexestrol were strong inhibitors; isodienestrol, dimethylstilbestrol, estradiol-17α, estrone, tamoxifen, MER-25, CI-628, and nafoxidine were weak inhibitors; dihydrotestosterone, testosterone, and prednisone did not compete. These observations indicate that specific estrogen-binding sites exist in rat skeletal muscle.     

Detection of the homology among proteins by immunochemical cross-reactivity between denatured antigens. Application to the threonine and methionine regulated aspartokinases-homoserine dehydrogenases from Escherichia coli K 12.

The two isofunctional enzymes aspartokinases-homoserine dehydrogenases I and II from Escherichia coli K 12 are compared using immunochemical techniques. The antibodies raised against one of these two proteins when in its native state can only recognize the homologous antigen, whether it is native or denatured. Contrarily, the antibodies raised against one of these two proteins when in its denatured state can recognize both the homologous and heterologous denatured antigens. The existence of this cross-reaction only between the two denatured aspartokinases-homoserine dehydrogenases suggests that these two enzymes have some similarity since such a reaction is not detected with several other denatured proteins. The regions involved in this similarity are buried inside the native proteins, and become exposed only upon denaturation. The same results, the existence of a cross-reaction between denatured species and none between the native ones, is obtained with proteolytic fragments derived from these two proteins and endowed with homoserine dehydrogenase activity. This resemblance between the two aspartokinases-homoserine dehydrogenases suggests that these proteins derive from a common ancestor. It is also proposed that such a cross-reaction between two denatured proteins is evidence for an homology between their amino acid sequences, and that the use of denatured proteins as both immunogens and antigens could be useful in detecting sequence homologies.     

High-performance liquid chromatographic determination of 1-β- -arabinofuranosyl-E-5-(2-bromovinyl)uracil and its metabolite (E)-5-(2-bromovinyl)uracil in serum

A high-performance liquid chromatographic method was developed to assay 1-β- -arabinofuranosyl-E-5-(2-bromovinyl)uracil and its metabolite (E)-5-(2-bromovinyl)uracil in serum. The chloro analogue of the parent drug is used as internal standard. Human serum samples were assayed to establish the pharmacokinetic parameters. Acetonitrile, used as a protein precipitant, was evaporated to dryness and the residue, containing the analytes and internal reference, was dissolved in mobile phase prior to chromatographic analysis. The minimum quantifiable level was 0.02 μg of each analyte per ml of serum.     

The Influence on Survival of Season of Onset of Childhood Acute Lymphoblastic Leukemia (All)

Rhythmicity has been found to be an important characteristic of leukocytes. We tested, therefore, whether rhythmicity could be found to be reflected in one or more of the developmental stages of childhood acute lymphoblastic leukemia. The patient material comprised 205 children 96 girls and 109 boys) whose disease was diagnosed in Israel during the years 1976-1981. Their mean age was 5.9 years ± 3.94. No seasonal onset of disease was found, either in the whole group or in subgroups divided by sex or lymphocyte surface markers. Survival analyses however, revealed that the season of diagnosis influenced survival. Excluded from the study were 9 cases (4.4%) due to lack of some items. The follow-up period was between 1 and 72 months (median 26 months). It appeared that the life table curves were running in their temporal order, each following curve representing a consecutive 3-month period. The difference between the curves was statistically significant (p = 0.025/0.009). The survival percentages at the end of the follow-up increased steadily, from a trough during November-January (56.5%) to a peak in August-October (80.0%). Analysis by sex and white blood cell (WBC) count showed that the difference between the life table curves was primarily due to girls presenting less than 50,000 cells per cumm (n = 73, p = 0.005/0.0028). The survival percentages in the same order as mentioned earlier were: 47.1%, 68.4%, 89.5% and 94.4%. The steady rise in the different female time series suggests a circannual pattern. In the male series no temporal order could be detected nor did the difference between seasonal survival reach a level of significance. The significance of the participation of natural environmental factors in causing the seasonal variations in malignancy is discussed.     

Muscle activity in rat locomotion: movement analysis and electromyography of the flexors and extensors of the elbow.

Footfall patterns and time sequence of activity are described for white rats conditioned to run freely in an activity wheel (which they drive). Motion is described in terms of soft contact, hard contact, soft contact, and flip phases. Duration of stride decreases and length of stride increases from walk to trot to canter to gallop. Myographic analysis shows that the brachialis has a major tonic function after it fires strongly during the flip phase and during much of the hard contact phase. Animals running at canter or gallop show major asymmetries between forelimb muscles on the first paw and on the lead paw sides.