A Framework for Navigating Institutional Review Board (IRB) Oversight in the Complicated Zone of Research.

The treatment therapies and technologies currently emerging from the rapidly evolving health care industry must undergo full examination in a clinical setting if they are to be marketed to the public. All elements of clinical studies involving human subjects must undergo thorough IRB review before study activities can commence. Regulations regarding IRB oversight apply to all clinical studies-including retrospective examinations of private medical data and identifiable biological samples. It is not uncommon for researchers to be unsure whether, or on what level, IRB review and oversight are required for a particular project. Yet, if human subjects or their private medical data are utilized in a study, peer-reviewed journals will require relevant IRB approval information be provided as a requirement for publication. This article examines IRB processes and review types, offers insight into the IRB decision-making process, and emphasizes the importance of engaging an IRB consultant early in the clinical study design process.

Relationship between glycogen accumulation and the laforin dual specificity phosphatase.

Laforin, encoded by the EPM2A gene, is a dual specificity protein phosphatase that has a functional glycogen-binding domain. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons and other tissues. We examined the level of laforin protein in several mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin as judged by Western analysis. Mice completely lacking muscle glycogen or with 10% normal muscle glycogen had reduced laforin. Mice defective in the GAA gene encoding lysosomal alpha-glucosidase (acid maltase) overaccumulate glycogen in the lysosome but did not have elevated laforin. We propose, therefore, that laforin senses cytosolic glycogen accumulation which in turn determines the level of laforin protein.

Gene expression profiling of mice with genetically modified muscle glycogen content.

Glycogen, a branched polymer of glucose, forms an energy re-serve in numerous organisms. In mammals, the two largest glyco-gen stores are in skeletal muscle and liver, which express tissue-specific glycogen synthase isoforms. MGSKO mice, in which mGys1 (mouse glycogen synthase) is disrupted, are devoid of muscle glycogen [Pederson, Chen, Schroeder, Shou, DePaoli-Roach and Roach (2004) Mol. Cell. Biol. 24, 7179-7187]. The GSL30 mouse line hyper-accumulates glycogen in muscle [Manchester, Skurat, Roach, Hauschka and Lawrence (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 10707-10711]. We performed a microarray analysis of mRNA from the anterior tibialis, medial gastrocnemius and liver of MGSKO mice, and from the gastroc-nemius of GSL30 mice. In MGSKO mice, transcripts of 79 genes varied in their expression in the same direction in both the anterior tibialis and gastrocnemius. These included several genes encoding proteins proximally involved in glycogen metabolism. The Ppp1r1a [protein phosphatase 1 regulatory (inhibitor) sub-unit 1A] gene underwent the greatest amount of downregulation. In muscle, the downregulation of Pfkfb1 and Pfkfb3, encoding isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphospha-tase, is consistent with decreased glycolysis. Pathways for branched-chain amino acid, and ketone body utilization appear to be downregulated, as is the capacity to form the gluconeogenic precursors alanine, lactate and glutamine. Expression changes among several members of the Wnt signalling pathway were identified, suggesting an as yet unexplained role in glycogen meta-bolism. In liver, the upregulation of Pfkfb1 and Pfkfb3 expression is consistent with increased glycolysis, perhaps as an adaptation to altered muscle metabolism. By comparing changes in muscle expression between MGSKO and GSL30 mice, we found a subset of 44 genes, the expression of which varied as a function of muscle glycogen content. These genes are candidates for regulation by glycogen levels. Particularly interesting is the observation that 11 of these genes encode cardiac or slow-twitch isoforms of muscle contractile proteins, and are upregulated in muscle that has a greater oxidative capacity in MGSKO mice.

Glucose metabolism in mice lacking muscle glycogen synthase.

Glycogen is an important component of whole-body glucose metabolism. MGSKO mice lack skeletal muscle glycogen due to disruption of the GYS1 gene, which encodes muscle glycogen synthase. MGSKO mice were 5-10% smaller than wild-type littermates with less body fat. They have more oxidative muscle fibers and, based on the activation state of AMP-activated protein kinase, more capacity to oxidize fatty acids. Blood glucose in fed and fasted MGSKO mice was comparable to wild-type littermates. Serum insulin was lower in fed but not in fasted MGSKO animals. In a glucose tolerance test, MGSKO mice disposed of glucose more effectively than wild-type animals and had a more sustained elevation of serum insulin. This result was not explained by increased conversion to serum lactate or by enhanced storage of glucose in the liver. However, glucose infusion rate in a euglycemic-hyperinsulinemic clamp was normal in MGSKO mice despite diminished muscle glucose uptake. During the clamp, MGSKO animals accumulated significantly higher levels of liver glycogen as compared with wild-type littermates. Although disruption of the GYS1 gene negatively affects muscle glucose uptake, overall glucose tolerance is actually improved, possibly because of a role for GYS1 in tissues other than muscle.

Serine/threonine/tyrosine phosphorylation of the LHX3 LIM-homeodomain transcription factor.

LHX3 is a LIM homeodomain transcription factor with essential roles in pituitary and motor neuron development in mammals. Patients with mutations in the LHX3 gene have combined pituitary hormone deficiency and other symptoms. In this study, we show that the LHX3 protein can be modified post-translationally by phosphorylation. LHX3 can serve as a substrate for protein kinase C and casein kinase II. Overexpression of these kinases reduces the transcriptional capacity of LHX3 to activate target genes. Following exposure of LHX3 to cellular kinases, mass spectrometry was used to map the phosphorylation of five amino acid residues within the human LHX3a isoform. Two phosphorylated residues (threonine 63 and serine 71) lie within the first LIM domain of the protein. Three other modified amino acids (tyrosine 227, serine 234, and serine 238) are located in the carboxyl terminus. Targeted replacement of these amino acids with non-modifiable residues significantly reduced the ability of LHX3 to activate both synthetic and pituitary hormone reporter genes. However, the amino acid replacements did not significantly affect the capability of LHX3 to interact with the NLI, PIT1, and MRG1 partner proteins, or its ability to bind to a high affinity DNA site. In conclusion, we have identified unique amino acids within LHX3 that are important for its transcriptional activity and are phosphorylated.

Regulation of the follicle-stimulating hormone beta gene by the LHX3 LIM-homeodomain transcription factor.

FSH is a critical hormone regulator of gonadal function that is secreted from the pituitary gonadotrope cell. Human patients and animal models with mutations in the LHX3 LIM-homeodomain transcription factor gene exhibit complex endocrine diseases, including reproductive disorders with loss of FSH. We demonstrate that in both heterologous and pituitary gonadotrope cells, specific LHX3 isoforms activate the FSH beta-subunit promoter, but not the proximal LHbeta promoter. The related LHX4 mammalian transcription factor can also induce FSHbeta promoter transcription, but the homologous Drosophila protein LIM3 cannot. The actions of LHX3 are specifically blocked by a dominant negative LHX3 protein containing a Kruppel-associated box domain. Six LHX3-binding sites were characterized within the FSHbeta promoter, including three within a proximal region that also mediates gene regulation by other transcription factors and activin. Mutations of the proximal binding sites demonstrate their importance for LHX3 induction of the FSHbeta promoter and basal promoter activity in gonadotrope cells. Using quantitative methods, we show that the responses of the FSHbeta promoter to activin do not require induction of the LHX3 gene. By comparative genomics using the human FSHbeta promoter, we demonstrate structural and functional conservation of promoter induction by LHX3. We conclude that the LHX3 LIM homeodomain transcription factor is involved in activation of the FSH beta-subunit gene in the pituitary gonadotrope cell.