Onboarding of siponimod in secondary progressive multiple sclerosis patients in Australia: Novel, real-world evidence from the MSGo digital support programme.

Background: Siponimod is approved for use in people with secondary progressive multiple sclerosis (pwSPMS). An integrated digital platform, MSGo, was developed for pwSPMS and clinicians to help navigate the multiple steps of the pre-siponimod work-up. Objective: To explore real-world onboarding experiences of siponimod amongst pwSPMS in Australia. Methods: Retrospective, non-interventional, longitudinal, secondary analysis of data extracted from MSGo (20 April 2022). The primary endpoint was the average time for siponimod onboarding; secondary endpoints were adherence and sub-group analyses of variables influencing onboarding. Results: Mixed-cure modelling estimated that 58% of participants (N = 368, females 71%, median age of 59 years) registered in MSGo would ever initiate siponimod. The median time to initiation was 56 days (95% CI [47-59] days). Half of the participants cited 'waiting for vaccination' as the reason for initiation delay. Cox regression analyses found participants with a nominated care partner had faster onboarding (HR 2.1, 95% CI [1.5-3.0]) and were more likely to continue self-reporting daily siponimod dosing than were those without a care partner (HR 2.2, 95% CI [1.3-3.7]). Conclusions: Despite the limitations of self-reported data and the challenges of the COVID-19 pandemic, this study provides insights into siponimod onboarding in Australia and demonstrates the positive impact of care partner support.

The evolution of "No Evidence of Disease Activity" in multiple sclerosis.

The availability of effective therapies for patients with relapsing-remitting multiple sclerosis (RRMS) has prompted a re-evaluation of the most appropriate way to measure treatment response, both in clinical trials and clinical practice. Traditional parameters of treatment efficacy such as annualized relapse rate, magnetic resonance imaging (MRI) activity, and disability progression have an important place, but their relative merit is uncertain, and the role of other factors such as brain atrophy is still under study. More recently, composite measures such as "no evidence of disease activity" (NEDA) have emerged as new potential treatment targets, but NEDA itself has variable definitions, is not well validated, and may be hard to implement as a treatment goal in a clinical setting. We describe the development of NEDA as an outcome measure in MS, discuss definitions including NEDA-3 and NEDA-4, and review the strengths and limitations of NEDA, indicating where further research is needed.

Impact of olanzapine or risperidone treatment on insulin sensitivity in schizophrenia or schizoaffective disorder.

AIM: To assess changes in insulin sensitivity in non-diabetic adults with schizophrenia or schizoaffective disorder treated with olanzapine or risperidone. METHODS: One hundred and thirty patients were randomly assigned to 12 weeks double-blind treatment with olanzapine or risperidone. Insulin sensitivity was measured using a two-step euglycaemic, hyperinsulinaemic clamp procedure. Whole-body adiposity was measured using dual-energy X-ray absorptiometry. The primary endpoint was the within-group change from baseline in insulin sensitivity normalized to fat-free mass (M(ffm) /I) during the clamp procedure's low-insulin phase, using an analysis of covariance model including the covariate weight change. RESULTS: Forty-one olanzapine-treated and 33 risperidone-treated patients completed baseline and endpoint clamp measurements. Mean M(ffm) /I during the low-insulin phase declined 9.0% (p = 0.226) in olanzapine-treated patients and 13.2% (p = 0.047) in risperidone-treated patients (between-group difference p = 0.354). During the high-insulin phase, M(ffm) /I declined 10.4% (p = 0.036) in olanzapine-treated patients and 2.1% (p = 0.698) in risperidone-treated patients (between-group difference p = 0.664). Changes in M(ffm) /I correlated inversely with changes in body weight and adiposity, which were generally higher in olanzapine-treated patients. Significant within-group increases in fasting glucose, but not haemoglobin A1c (HbA1c), were observed during olanzapine treatment. The fasting glucose change was not correlated with M(ffm) /I changes. CONCLUSIONS: Small, but statistically significant, decrements in insulin sensitivity were observed in olanzapine- and risperidone-treated patients at 1 of 2 insulin doses tested. Significant increases in fasting glucose and insulin and total fat mass were observed only in olanzapine-treated patients. Changes in insulin sensitivity correlated significantly with changes in weight or adiposity, but not with changes in glucose.

A 5-week study of the pharmacokinetics and pharmacodynamics of LY2189265, a novel, long-acting glucagon-like peptide-1 analogue, in patients with type 2 diabetes.

AIM: To investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of LY2189265 (LY), a novel, long-acting glucagen-like peptide-1 analogue, administered once weekly to subjects with type 2 diabetes. METHODS: This was a placebo-controlled, parallel-group, subject- and investigator-blind study of LY in subjects (N = 43) with type 2 diabetes mellitus controlled with diet and exercise alone or with a single oral antidiabetic medication. Subjects taking metformin or thiazolidinediones continued on their therapy. Subjects receiving sulfonylurea, acarbose, repaglinide or nateglinide were switched to metformin prior to enrollment. Subjects received five once-weekly doses of 0.05, 0.3, 1, 3, 5 or 8 mg. Effects on glucose, insulin and C-peptide concentrations were determined during fasting and following standard test meals. The pharmacokinetics of LY and its effects on HBA1c, glucagon, body weight, gastric emptying and safety parameters were assessed. RESULTS: Once-weekly administration of LY significantly reduced (p < 0.01) fasting plasma glucose, 2-h post-test meal postprandial glucose and area under the curve (AUC) of glucose after test meals at doses ≥1 mg. These effects were seen after the first dose and were sustained through the weekly dosing cycle. Most doses produced statistically significant increases in insulin and C-peptide AUC when normalized for glucose AUC. Statistically significant reductions in HBA1c were observed for all dose groups except 0.3 mg. The most commonly reported adverse effects (AEs) were nausea (35 events), headache (20 events), vomiting (18 events) and diarrhoea (8 events). CONCLUSIONS: LY showed improvement in fasting and postprandial glycaemic parameters when administered once weekly in subjects with type 2 diabetes. The pharmacokinetics and safety profiles also support further investigation of this novel agent.

Tumour necrosis factor (TNF) inhibitor therapy in Susac's syndrome.

Susac's syndrome is the clinical triad of encephalopathy, branch retinal artery occlusions and sensorineural hearing loss (Susac 1994) [1]. It occurs predominantly in young females and is believed to be an immune-mediated endotheliopathy of small vessels of the brain, retina and cochlea (Neumayer et al. 2009) [2]. Early, aggressive, and sustained immunosuppressive therapy has been recommended for Susac's syndrome and anecdotal evidence has suggested a therapeutic role for monoclonal antibodies (Rennebohm et al. 2008, Lee and Amezcua 2009) [3,4]. We report a case of Susac's syndrome in which the patient improved immediately after tumour necrosis factor (TNF) inhibition with the monoclonal antibody, infliximab.

Electrophysiological effects of activating the peptidergic primary afferent innervation of rat mesenteric arteries.

1. Intracellular recording was used to investigate the electrophysiological effects of activating peptidergic primary afferent axons with capsaicin in the smooth muscle of rat mesenteric arteries in vitro. In addition, continuous amperometry was used to monitor the effects of capsaicin on noradrenaline release from the sympathetic nerves. 2. Capsaicin (1 microm) produced a hyperpolarization (-11+/-2 mV) and a reduction in the time constant of decay of excitatory junction potentials (e.j.p.'s) evoked by electrical stimulation of the perivascular sympathetic nerves. These effects of capsaicin were mimicked by calcitonin gene-related peptide (CGRP; 1 and 10 nm) but not by substance P (50 nm), which produced a small hyperpolarization (maximum -3+/-1 mV) but did not change excitatory junction potential (e.j.p.) time course. 3. The hyperpolarization produced by capsaicin and CGRP was blocked by glibenclamide (10 microm) but was not changed by the CGRP antagonist, CGRP8-37 (0.5 microm). Mechanical denudation of the endothelium also did not reduce the effect of capsaicin on membrane potential. 4. Capsaicin (1 microm) increased the amplitude of e.j.p.'s. This effect was not mimicked by CGRP or substance P nor blocked by glibenclamide or CGRP8-37. 5. All effects of capsaicin desensitized. 6. Capsaicin (1 microm) had no effect on noradrenaline-induced oxidation currents evoked by electrical stimulation, indicating that noradrenaline release was unchanged. 7. These results suggest that CGRP released from primary afferent axons hyperpolarizes vascular smooth muscle by activating glibenclamide-sensitive K+ channels. The findings also indicate that an unknown factor released by the primary afferent axons increases e.j.p. amplitude.

Effects of modulating Ca2+ entry and activating prejunctional receptors on facilitation of excitatory junction potentials in the guinea-pig vas deferens in vitro.

This study investigated the effects of changing the extracellular Ca2+ concentration on facilitation of excitatory junction potential (EJP) amplitude during trains of 20 stimuli at 1 Hz at sympathetic neuroeffector junctions in the guinea-pig vas deferens. These effects were compared with those of Ca2+ channel blockers and agents which act at prejunctional receptors to increase or decrease neurotransmitter release. In these experiments, alpha-adrenoceptor-mediated autoinhibition of neurotransmitter release was blocked by the alpha-adrenoceptor antagonist, phentolamine (1 microM). Varying the extracellular Ca2+ concentration (0.75-6 mM) changed the amplitude of EJPs without affecting the maximum level of facilitation during the trains of stimuli. Reductions in Ca2+ concentration (from 2 mM) were associated with a slowing in the rate of development of facilitation. The Ca2+ channel antagonists, Cd2+ (2 microM and 5 microM) and omega-conotoxin GVIA (10 nM), and agents which act at prejunctional receptors to reduce neurotransmitter release, adenosine (100 microM and 1,000 microM) and prostaglandin E2 (PGE2; 0.1 nM and 1 nM), produced similar effects to those of lowering the extracellular Ca2+ concentration. Raising the extracellular Ca2+ concentration (from 2 mM) increased the rate of development of facilitation. Angiotensin II (AII; 0.5 microM) produced similar effects to raising extracellular Ca2+. However, isoprenaline (1 microM), while increasing EJP amplitude, reduced the maximum level of facilitation and was without effect on the rate of development of facilitation. In the guinea-pig vas deferens EJPs are produced by neurally released ATP. Thus, the findings support the idea that adenosine, PGE2 and AII change ATP release by modifying Ca2+ entry into the nerve terminal. However, the effects of isoprenaline may not solely be accounted for by modifications in Ca2+ entry.

Effects of a selective neuropeptide Y Y2 receptor antagonist, BIIE0246, on Y2 receptors at peripheral neuroeffector junctions.

1. This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y2 receptor antagonist, on the inhibition of cholinergic neuroeffector transmission in rat heart and guinea-pig trachea and purinergic neuroeffector transmission in guinea-pig vas deferens produced by the NPY Y2 receptor agonist, N-acetyl [Leu28,31] NPY 24-36. 2. In pentobarbitone anaesthetized rats, supramaximal stimulation every 30 s, of the vagus nerve innervating the heart, increased pulse interval by approximately 100 ms. This response was attenuated by intravenous administration of N-acetyl [Leu28,31] NPY 24-36 (10 nmol x kg(-1)). 3. Transmural stimulation of segments of guinea-pig trachea at 1 min intervals with 5 s trains of stimuli at 0.5, 5, 10, 20 and 40 Hz evoked contractions which were reduced in force by N-acetyl [Leu28,31] NPY 24-36 (2 microM). 4. In guinea-pig vasa deferentia, the amplitude of excitatory junction potentials evoked by trains of 20 stimuli at 1 Hz was reduced in the presence of N-acetyl [Leu28,31] NPY 24-36 (1 microM). 5. In all preparations BIIE0246 attenuated the inhibitory effect of N-acetyl [Leu28,31] NPY 24-36 but had no effect when applied alone. 6. The findings support the view that the nerve terminals of postganglionic parasympathetic and sympathetic neurones possess neuropeptide Y Y2 receptors which, when activated, reduce neurotransmitter release.

Effects of agents that modulate intracellular cAMP levels on excitatory junction potentials recorded in the guinea-pig vas deferens in vitro.

This study used intracellular recording of excitatory junction potentials (EJPs) and focal extracellular recording of excitatory junction currents (EJCs) to investigate the effects of agents that modulate intracellular cAMP levels on sympathetic neuroeffector transmission in the guinea-pig vas deferens. In this tissue, postjunctional electrical activity is produced by neurally released ATP. The adenylate cyclase activator, forskolin (0.5-5 microM) increased the amplitude of all EJPs evoked by trains of 20 stimuli at I Hz. Forskolin (5 microM) also increased the probability of recording EJCs without changing the amplitude distributions of spontaneous EJP and EJCs, indicating that this agent does not change the postjunctional sensitivity to spontaneously released quanta of ATP. EJP amplitudes were also increased by 8-bromo-cyclic AMP (10 microM), 8-bromo-cyclic GMP (10 microM), the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (100 and 1,000 microM) and the beta-adrenoceptor agonist, isoprenaline (1 microM). The selective protein kinase A inhibitors, H-89 (10 microM) and the Rp isomer of adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS, 100 microM), and the broad spectrum protein kinase inhibitors, [1-(5-isoquinolinesulphonyl)-3-methylpiperazine-diHCl (H-7, 100 microM) and staurosporine (1 microM), did not block the facilitatory effects of forskolin on EJP amplitude. In addition, the effects of forskolin were not blocked by the cyclic nucleotide-gated ion channel blocker, spermine (50 microM). These results suggest that elevating intracellular cAMP levels increases ATP release in the guinea-pig vas deferens by a mechanism which does not involve activation of protein kinases A or G.

Electrochemical and electrophysiological characterization of neurotransmitter release from sympathetic nerves supplying rat mesenteric arteries.

1. Characteristic features of noradrenaline (NA) and adenosine 5'-triphosphate (ATP) release from postganglionic sympathetic nerves in rat small mesenteric arteries in vitro have been investigated on an impulse-by-impulse basis. NA release was measured using continuous amperometry and ATP release was monitored by intracellular recording of excitatory junction potentials (e.j.ps). 2. Electrical stimuli evoked transient increases in oxidation current. During trains of ten stimuli at 0.5 - 4 Hz there was a depression in the amplitude of oxidation currents evoked following the first stimulus in the train. 3. The neuronal NA uptake inhibitor, desmethylimipramine (1 microM), increased the amplitude of the summed oxidation current evoked by ten stimuli at 1 Hz and slowed the decay of oxidation currents evoked by trains of ten stimuli at 1 and 10 Hz. 4. The alpha2-adrenoceptor antagonist, idazoxan (1 microM), increased the amplitudes of the oxidation currents evoked during trains of ten stimuli at 0.5 - 10 Hz but had no effect on the oxidation currents evoked by the first stimulus in the train. 5. Idazoxan (1 microM) increased the amplitude of all e.j.ps evoked during trains of stimuli at 0.5 and 1 Hz. In addition, the facilitatory effect of idazoxan on e.j.ps was significantly greater than that on oxidation currents. 6. The findings indicate that NA release from sympathetic nerves supplying small mesenteric arteries is regulated by activation of presynaptic alpha2-adrenoceptors and that clearance of released NA in this tissue depends, in part, upon neuronal uptake. The different effects of idazoxan on the oxidation currents and e.j.ps may indicate that the release of NA and ATP is differentially modulated.

Effects of A1-adenosine receptor antagonists on purinergic transmission in the guinea-pig vas deferens in vitro.

1. Intracellularly recorded excitatory junction potentials (ej.ps) were used to study the effects of adenosine receptor antagonists on neurotransmitter release from postganglionic sympathetic nerve terminals in the guinea-pig vas deferens in vitro. 2. The A1 adenosine receptor antagonists, 8-phenyltheophylline (10 microM) and 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM), increased the amplitude of e.j.ps evoked during trains of 20 stimuli at 1 Hz in the presence, but not in the absence, of the alpha2-adrenoceptor antagonist, yohimbine (1 microM) or the non-selective alpha-adrenoceptor antagonist, phentolamine (1 microM). 3. Adenosine (100 microM) reduced the amplitude of e.j.ps, both in the presence and in the absence of phentolamine (1 microM). This inhibitory effect of adenosine is most likely caused by a reduction in transmitter release as there was no detectable change in spontaneous ej.p. amplitudes. 4. In the presence of phentolamine, application of the adenosine uptake inhibitor, S-(p-nitrobenzyl)-6-thioinosine (0.1 microM), had no effect on ej.p. amplitudes. 5. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (100 microM), significantly increased the amplitudes of all e.j.ps evoked during trains of 20 stimuli at 1 Hz, both in the presence and in the absence of phentolamine (1 microM). 6. These results suggest that endogenous adenosine modulates neurotransmitter release by an action at prejunctional A1 adenosine receptors only when alpha2-adrenoceptors are blocked.

Novel Saccharomyces cerevisiae gene, MRK1, encoding a putative protein kinase with similarity to mammalian glycogen synthase kinase-3 and Drosophila Zeste-White3/Shaggy.

A Saccharomyces cerevisiae gene was identified that would encode a protein similar to the mammalian protein kinase glycogen synthase kinase-3 (GSK-3) and the Drosophila Zeste-White3/Shaggy gene product. The open reading frame predicts a 375 amino acid polypeptide with a putative protein kinase domain that displays 70% and 39% identity, respectively, to two known yeast proteins, Mds1p and Mck1p. The new gene, designated MRK1 (Mds1p Related Kinase), is located on chromosome IV. Disruption of MRK1 was not lethal and did not elicit any alteration in glycogen accumulation. In addition, an mck1 mds1 mrk1 triple disruptant was viable.

Interactions between cAMP-dependent and SNF1 protein kinases in the control of glycogen accumulation in Saccharomyces cerevisiae.

The synthesis of glycogen in Saccharomyces cerevisiae is stimulated by nutrient limitation and requires both glycogen synthase and the glycogen branching enzyme. Of the two glycogen synthase genes present in yeast, GSY2 appears to be more important for the accumulation of glycogen upon entry into stationary phase. In cells grown on glucose, GSY2 mRNA levels increased approximately 10-fold during the transition from logarithmic to stationary phase. Growth of cells in glycerol, however, resulted in constitutive expression of GSY2 mRNA and the corresponding protein, GS-2, suggestive of glucose repression of GSY2. Mutants defective in the SNF1 gene, which encodes a protein kinase important in glucose repression mechanisms, are known not to accumulate glycogen. A modest 2-4-fold decrease in total GS-2 level was observed, and upon entry into stationary phase, the enzyme was blocked in the inactive, phosphorylated state in snf1 strains. The GS-2 protein is thought to be regulated by covalent phosphorylation of three COOH-terminal sites (Hardy, T.A., and Roach, P.J. (1993) J. Biol. Chem. 268, 23799-23805), removal of which results in constitutively active glycogen synthase that bypasses phosphorylation controls. Expression of COOH-terminally truncated GS-2 in snf1 cells restored glycogen accumulation, and so we propose that the SNF1 kinase controls the phosphorylation state of GS-2. Cyclic AMP pathways also exert control over glycogen accumulation. In bcy1 cells, which have constitutively active cyclic AMP-dependent protein kinase, greatly reduced levels of both GS-2 message and protein were observed. With wild type GSY2 placed under control of the ADH1 promoter, bcy1 cells did not accumulate glycogen despite increased GS-2. Overexpression of truncated GS-2, however, resulted in definite though reduced glycogen accumulation; the glycogen synthesized was structurally distinct from wild type with properties characteristic of less branched polysaccharide. We conclude that the cAMP pathway controls both the expression and the phosphorylation state of GS-2. Furthermore, other factor(s) necessary for glycogen biosynthesis, such as the branching enzyme GLC3, must also be under negative control by the cAMP pathway. The results demonstrate interactive controls of GS-2 by the cAMP-dependent and SNF1 protein kinases.

MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.

Control of yeast glycogen synthase-2 by COOH-terminal phosphorylation.

The budding yeast Saccharomyces cerevisiae expresses two isoforms of glycogen synthase, of which glycogen synthase-2 (GS-2) appears to be the most important determinant of glycogen accumulation (Farkas, I., Hardy, T. A., Goebl, M. G., and Roach, P. J. (1991) J. Biol. Chem. 266, 15602-15607). Partial proteolysis of purified yeast glycogen synthase activated the enzyme, mimicking the effects of dephosphorylation. The cleavage was localized to the COOH terminus of the molecule and trypsin treatment released 32P from enzyme labeled in vivo with 32P or in vitro by cyclic AMP-dependent protein kinase. Similarly, when cells were labeled with 32P, no radioactivity was incorporated into a mutant form of GS-2 truncated at residue 643 while the wild type enzyme was phosphorylated at both Ser and Thr residues. The 9 Ser and Thr residues COOH-terminal to position 643 were mutated individually to Ala, and the GS-2 mutants were expressed from a low copy plasmid in yeast that lacked functional chromosomal copies of the two glycogen synthase genes. Mutations at Ser-650, Ser-654, and Thr-667 resulted in significant activation of yeast glycogen synthase and elevation in the level of accumulated glycogen as compared with wild type. Likewise, expression of the truncated GS-2 resulted in hyperactive enzyme and the overaccumulation of glycogen. None of the other Ser or Thr mutations substantially affected glycogen synthase activity and glycogen storage. We conclude that Ser-650, Ser-654, and Thr-667 are regulatory phosphorylation sites in vivo. However, in vitro, cyclic AMP-dependent protein kinase modified Ser residue(s) COOH-terminal to position 659, and so the identity of the physiological GS-2 kinases is unclear. Yeast strains bearing glc7 and gac1 mutations are defective in genes encoding type 1 protein phosphatase components and are impaired in their ability to accumulate glycogen. Expression of the truncated GS-2 in these strains restored glycogen accumulation, as did the presence of GS-2 mutated at Ser-650, Ser-654, or Thr-667. These data are consistent with the hypothesis that type 1 phosphatase regulates GS-2 by controlling its phosphorylation state.

Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled.

In previous work, we identified a Saccharomyces cerevisiae glycogen synthase gene, GSY1, which codes for an 85-kDa polypeptide present in purified yeast glycogen synthase (Farkas, I., Hardy, T.A., DePaoli-Roach, A.A., and Roach, P.J. (1990) J. Biol. Chem. 265, 20879-20886). We have now cloned another gene, GSY2, which encodes a second S. cerevisiae glycogen synthase. The GSY2 sequence predicts a protein of 704 residues, molecular weight 79,963, with 80% identity to the protein encoded by GSY1. Amino acid sequences obtained from a second polypeptide of 77 kDa present in yeast glycogen synthase preparations matched those predicted by GSY2. GSY1 resides on chromosome VI, and GSY2 is located on chromosome XII. Disruption of the GSY1 gene produced a strain retaining about 85% of wild type glycogen synthase activity at stationary phase, while disruption of the GSY2 gene yielded a strain with only about 10% of wild type enzyme activity. The level of glycogen synthase activity in yeast cells disrupted for GSY1 increased in stationary phase, whereas the activity remained at a constant low level in cells disrupted for GSY2. Disruption of both genes resulted in a viable haploid that totally lacked glycogen synthase activity and was defective in glycogen deposition. In conclusion, yeast expresses two forms of glycogen synthase with activity levels that behave differently in the growth cycle. The GSY2 gene product appears to be the predominant glycogen synthase with activity linked to nutrient depletion.

Glycogen metabolism and signal transduction in mammals and yeast.

Mammalian glycogen synthase, with its complex multisite phosphorylation mechanisms, continues to provide interesting and novel examples of the regulation of protein function. The mammalian enzyme is phosphorylated in a hierarchal manner such that modification of certain sites requires the prior phosphorylation of other sites. Yeast contains two glycogen synthases that have extensive similarities to their mammalian counterpart but the greatest divergence in amino acid sequence is seen precisely in the regions likely to be involved in covalent control. We hope that examination of the control of the yeast glycogen synthase will be as informative as study of the mammalian enzymes, whether by revealing important parallels with the mammalian system or by uncovering major differences in mechanism.

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene.

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.

Human DNA (cytosine-5)methyltransferase selectively methylates duplex DNA containing mispairs.

The presence of the C.C mispair in a defined duplex oligodeoxynucleotide enhanced its capacity to serve as a substrate for highly purified human DNA methyltransferase. Analysis of tritiated reaction products showed that the C.C mispair acted as a "methylation acceptor" in that it was itself rapidly methylated. The m5C.G base pair also enhanced the capacity of the oligodeoxynucleotide to serve as a substrate for the enzyme. However, this complementary base pair was found to act as a "methylation director". That is, the presence of the m5C in one strand induced the enzyme to rapidly methylate at the cytosine residue on the opposite strand in an adjacent C.G base pair.

The influence of the dT.dG mispair on the activity of the human DNA(cytosine-5)methyltransferase.

Synthetic oligodeoxynucleotides containing a dT.dG mispair at a centrally located d(pCG) dimer are methylated at a moderate rate by highly purified human DNA(cytosine-5)methyltransferase (E.C. 2.1.1.37). The presence of a mispaired dT in one strand induced the enzyme to preferentially methylate the opposite strand.