http://physiolgenomics.physiology.org Physiological Genomics RSS feed -- current issue 1531-2267 Apr 22 2008 12:00:00:000AM Physiological Genomics 1094-8341 http://physiolgenomics.physiology.org/icons/banner/title.gif http://physiolgenomics.physiology.org http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/139?rss=1 Genomic evidence reveals that gene expression in humans is precisely controlled in cellular, tissue-type, temporal, and condition-specific manners. Completely understanding the regulatory mechanisms of gene expression is therefore one of the most important issues in genomic medicine. Surprisingly, recent analyses of the human and animal genomes have demonstrated that the majority of RNA transcripts are relatively small, noncoding RNAs (sncRNAs), rather than large, protein coding message RNAs (mRNAs). Moreover, these sncRNAs may represent a novel important layer of regulation for gene expression. The most important breakthrough in this new area is the discovery of microRNAs (miRNAs). miRNAs comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target mRNAs. As a group, miRNAs may directly regulate ~30% of the genes in the human genome. In keeping with the nomenclature of RNomics, which is to study sncRNAs on the genomic scale, "microRNomics" is coined here to describe a novel subdiscipline of genomics that studies the identification, expression, biogenesis, structure, regulation of expression, targets, and biological functions of miRNAs on the genomic scale. A growing body of exciting evidence suggests that miRNAs are important regulators of cell differentiation, proliferation/growth, mobility, and apoptosis. These miRNAs therefore play important roles in development and physiology. Consequently, dysregulation of miRNA function may lead to human diseases such as cancer, cardiovascular disease, liver disease, immune dysfunction, and metabolic disorders. microRNomics may be a newly emerging approach for human disease biology.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00034.2008 American Physiological Society 2 33 147 2008-04-22 139 Review http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/148?rss=1 Retinoic acid (RA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activate distinct ligand-dependent transcription factors, and both cause cardiac malformation and heart failure in zebrafish embryos. We hypothesized that they cause this response by hyperactivating a common set of genes critical for heart development. To test this, we used microarrays to measure transcript changes in hearts isolated from zebrafish embryos 1, 2, 4, and 12 h after exposure to 1 µM RA. We used hierarchical clustering to compare the transcriptional responses produced in the embryonic heart by RA and TCDD. We could identify no early responses in common between the two agents. However, at 12 h both treatments produced a dramatic downregulation of a common cluster of cell cycle progression genes, which we term the cell cycle gene cluster. This was associated with a halt in heart growth. These results suggest that RA and TCDD ultimately trigger a common transcriptional response associated with heart failure, but not through the direct activation of a common set of genes. Among the genes rapidly induced by RA was Nr2F5, a member of the COUP-TF family of transcriptional repressors. We found that induction of Nr2F5 was both necessary and sufficient for the cardiotoxic response to RA.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00214.2007 American Physiological Society 2 33 158 2008-04-22 148 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/159?rss=1 Differentiation of hematopoietic stem and progenitor cells is an intricate process controlled in large part at the level of transcription. While some key megakaryocytic transcription factors have been identified, the complete network of megakaryocytic transcriptional control is poorly understood. Using global gene expression microarray analysis, Gene Ontology-based functional annotations, and a novel interlineage comparison with parallel, isogenic granulocytic cultures as a negative control, we closely examined the mRNA level of transcriptional regulators in megakaryocytes derived from human mobilized peripheral blood CD34⁺ hematopoietic cells. This approach identified 199 differentially expressed transcription factors or transcriptional regulators. We identified and detailed the transcriptional kinetics of most known megakaryocytic transcription factors including GATA1, FLI1, and MAFG. Furthermore, many genes with transcription factor activity or transcription factor binding activity were identified in megakaryocytes that had not previously been associated with that lineage, including BTEB1, NR4A2, FOXO1A, MEF2C, HDAC5, VDR, and several genes associated with the tumor suppressor p53 (HIPK2, FHL2, and TADA3L). Protein expression and nuclear localization were confirmed in megakaryocytic cells for four of the novel candidate megakaryocytic transcription factors: FHL2, MXD1, E2F3, and RFX5. In light of the hypothesis that transcription factors expressed in a particular differentiation program are important contributors to such a program, these data substantially expand our understanding of transcriptional regulation in megakaryocytic differentiation of stem and progenitor cells.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00127.2007 American Physiological Society 2 33 169 2008-04-22 159 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/170?rss=1 Using serial analysis of gene expression, we collected quantitative transcriptome data in 11 regions of the adult wild-type mouse brain: the orbital, prelimbic, cingulate, motor, somatosensory, and entorhinal cortices, the caudate-putamen, the nucleus accumbens, the thalamus, the substantia nigra, and the ventral tegmental area. With >1.2 million cDNA tags sequenced, this database is a powerful resource to explore brain functions and disorders. As an illustration, we performed interregional comparisons and found 315 differential transcripts. Most of them are poorly characterized and 20% lack functional annotation. For 78 differential transcripts, we provide independent expression level measurements in mouse brain regions by real-time quantitative RT-PCR. We also show examples where we used in situ hybridization to achieve infrastructural resolution. For 30 transcripts, we next demonstrated that regional enrichment is conserved in the human brain. We then quantified the expression levels of region-enriched transcripts in the R6/2 mouse model of Huntington disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease and observed significant alterations in the striatum, cerebral cortex, thalamus and substantia nigra of R6/2 mice and in the striatum of MPTP-treated mice. These results show that the gene expression data provided here for the mouse brain can be used to explore pathophysiological models and disclose transcripts differentially expressed in human brain regions.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00125.2007 American Physiological Society 2 33 179 2008-04-22 170 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/180?rss=1 Excretion of uric acid, a compound of considerable medical importance, is largely determined by the balance between renal secretion and reabsorption. The latter process has been suggested to be principally mediated by urate transporter 1 (URAT1; slc22a12), but the role of various putative urate transporters has been much debated. We have characterized urate handling in mice null for RST, the murine ortholog of URAT1, as well as in those null for the related organic anion transporters Oat1 and Oat3. Expression of mRNA of other putative urate transporters (UAT, MRP2, MRP4, Oatv1) was unaffected in the knockouts, as were general indexes of renal function (glomerular filtration rate, fractional excretion of fluid and electrolytes). While mass spectrometric analyses of urine and plasma revealed significantly diminished renal reabsorption of urate in RST-null mice, the bulk of reabsorption, surprisingly, was preserved. Oat1- and Oat3-null mice manifested decreased secretion rather than reabsorption, indicating that these related transporters transport urate in the "opposite" direction to RST. Moreover, metabolomic analyses revealed significant alteration in the concentration of several molecules in the plasma and urine of RST knockouts, some of which may represent additional substrates of RST. The results suggest that RST, Oat1, and Oat3 each contribute to urate handling, but, at least in mice, the bulk of reabsorption is mediated by a transporter(s) that remains to be identified. We discuss the data in the context of recent human genetic studies that suggest that the magnitude of the contribution of URAT1 to urate reabsorption might vary with ethnic background.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00207.2007 American Physiological Society 2 33 192 2008-04-22 180 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/193?rss=1 Ovarian follicles develop in groups yet individual follicles follow different growth trajectories. This growth and development are regulated by endocrine and locally produced growth factors that use a myriad of receptors and signal transduction pathways to exert their effects on theca and granulosa cells. We hypothesize that differential growth may be due to differences in hormonal responsiveness that is partially mediated by differences in expression of genes involved in signal transduction. We used the bovine dominant follicle model, microarrays, quantitative real-time PCR and RNA interference to examine this. We identified 83 genes coding for signal transduction molecules and validated a subset of them associated with different stages of the follicle wave. We suggest important roles for CAM kinase-1 and EphA4 in theca cells and BCAR1 in granulosa cells for the development of dominant follicles and for betaglycan and FIBP in granulosa cells of regressing subordinate follicles. Inhibition of genes for betaglycan and FIBP in granulosa cells in vitro suggests that they inhibit estradiol production in regressing subordinate follicles.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00274.2007 American Physiological Society 2 33 204 2008-04-22 193 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/205?rss=1 Polymorphisms that have been proven to influence gene functions are called functional polymorphisms. It is significant to know the distribution of functional polymorphisms in the rat, widely used in animal models for human diseases. In this study, we assessed 16 functional polymorphisms consisting of 3 coat color and 13 disease-associated genes in 136 rat strains, as a part of the genetic profiling program of the National Bio Resource Project for the Rat (NBRP-Rat). Polymorphisms of Cdkn1a, Fcgr3, Grp10, Lss, and Fdft1, which were proven to function in prostate tumorigenesis, glomerulonephritis, hyperphagia, and cholesterol biosynthesis, were shared among various inbred strains. These findings indicated that most rat strains harbored the disease-associated alleles and suggested that many unidentified functional polymorphisms might exist in inbred rat strains. The functional polymorphisms shared in inbred strains were also observed within outbred stocks available commercially. Therefore, this implies that experimental plans based on either rat inbred strains or outbred stocks need to be carefully designed with a full understanding of the genetic characteristics of the animals. To select the most suitable strains for experiments, the NBRP-Rat will periodically improve and update the genetic profiles of rat strains.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00222.2007 American Physiological Society 2 33 211 2008-04-22 205 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/212?rss=1 The metabolic syndrome (involving obesity, hypertension, dyslipidemia, insulin resistance, and a proinflammatory/prethrombotic state) is a major risk factor for cardiovascular disease. Its incidence continues to rise, in part because of the epidemic increase in obesity. The Lyon hypertensive (LH) rat is a model for hypertension and several other features of the metabolic syndrome, having high body weight, plasma cholesterol, and triglycerides, increased insulin-to-glucose ratio, and salt-sensitive hypertension. Previous genetic studies in LH/Mav rats and a normotensive control (LN/Mav) identified quantitative trait loci (QTLs) on rat chromosome (RNO)17 for multiple features of the metabolic syndrome. To further evaluate the role of RNO17 in the LH rat, we generated a consomic strain (LH-17^BN) by substituting LH RNO17 with that of the sequenced Brown Norway (BN/NHsdMcwi) rat. Male LH and BN rats and LH-17^BN rats were characterized for blood pressure and metabolic and morphological parameters. Similar to the protective effect of LN alleles, the LH-17^BN rat also showed decreased body weight, triglycerides, and blood pressure; however, there was no significant difference in cholesterol or insulin-to-glucose ratio. Therefore, the substitution of the LH chromosome 17 is sufficient to recapitulate some, but not all, of the traits previously mapped to this chromosome. This could be due to the lack of a susceptible LH genome background or due to the introgression of chromosome 17 from another strain. Regardless, this study provides a single-chromosome genetic model for further dissection of blood pressure and morphological and metabolic traits on this chromosome.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00262.2007 American Physiological Society 2 33 217 2008-04-22 212 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/218?rss=1 To further define genes that are differentially expressed during cysteine deprivation and to evaluate the roles of amino acid deprivation vs. oxidative stress in the response to cysteine deprivation, we assessed gene expression in human hepatoma cells cultured in complete or cysteine-deficient medium. Overall, C3A cells responded to cysteine deprivation by activation of the eukaryotic initiation factor (eIF)2 kinase-mediated integrated stress response to inhibit global protein synthesis; increased expression of genes containing amino acid response elements (ASNS, ATF3, CEBPB, SLC7A11, and TRIB3); increased expression of genes for amino acid transporters (SLC7A11, SLC1A4, and SLC3A2), aminoacyl-tRNA synthetases (CARS), and, to a limited extent, amino acid metabolism (ASNS and CTH); increased expression of genes that act to suppress growth (STC2, FOXO3A, GADD45A, LNK, and INHBE); and increased expression of several enzymes that favor glutathione synthesis and maintenance of protein thiol groups (GCLC, GCLM, SLC7A11, and TXNRD1). Although GCLC, GCLM, SLC7A11, HMOX, and TXNRD1 were upregulated, most genes known to be upregulated via oxidative stress were not affected by cysteine deprivation. Because most genes known to be upregulated in response to eIF2 phosphorylation and activating transcription factor 4 (ATF4) synthesis were differentially expressed in response to cysteine deprivation, it is likely that many responses to cysteine deprivation are mediated, at least in part, by the general control nondepressible 2 (GCN2)/ATF4-dependent integrated stress response. This conclusion was supported by the observation of similar differential expression of a subset of genes in response to leucine deprivation. A consequence of sulfur amino acid restriction appears to be the upregulation of the cellular capacity to cope with oxidative and chemical stresses via the integrated stress response.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00263.2007 American Physiological Society 2 33 229 2008-04-22 218 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/230?rss=1 Congenital heart defects (CHD) are common in Down syndrome (DS, trisomy 21). Recently, cardiac sympathetic-parasympathetic imbalance has also been documented in DS adults free of any CHD. The KCNJ6 gene located on human chromosome 21 encodes for the Kir3.2/GIRK2 protein subunits of G protein-regulated K⁺ (K_G) channels and could contribute to this altered cardiac regulation. To elucidate the role of its overexpression, we used homozygous transgenic (Tg^+/+) mice carrying copies of human KCNJ6. These mice showed human Kir3.2 mRNA expression in the heart and a 2.5-fold increased translation in the atria. Phenotypic alterations were assessed by recording electrocardiogram of urethane anesthetized mice. Chronotropic responses to direct (carbachol) and indirect (methoxamine) muscarinic stimulation were enhanced in Tg^+/+ mice with respect to wild-type (WT) mice. Alternating periods of slow and fast rhythm induced by CCPA (2-chloro-N-cyclopentyl-adenosine) were amplified in Tg^+/+ mice, resulting in a reduced negative chronotropic effect. These drugs reduced the atrial P wave amplitude and area. P wave variations induced by methoxamine and CCPA were respectively increased and reduced in the Tg^+/+ mice, while PR interval and ventricular wave showed no difference between Tg^+/+ and WT. These results indicate that Tg^+/+ mice incorporating the human KCNJ6 exhibit altered Kir3.2 expression and responses to drugs that would activate K_G channels. Moreover, these altered expression and responses are limited to sino-atrial node and atria that normally express large amounts of K_G channels. These data suggest that KCNJ6 could play an important role in altered cardiac regulation in DS patients.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00143.2007 American Physiological Society 2 33 239 2008-04-22 230 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/240?rss=1 Alzheimer's Disease (AD) is the most widespread form of dementia during the later stages of life. If improved therapeutics are not developed, the prevalence of AD will drastically increase in the coming years as the world's population ages. By identifying differences in neuronal gene expression profiles between healthy elderly persons and individuals diagnosed with AD, we may be able to better understand the molecular mechanisms that drive AD pathogenesis, including the formation of amyloid plaques and neurofibrillary tangles. In this study, we expression profiled histopathologically normal cortical neurons collected with laser capture microdissection (LCM) from six anatomically and functionally discrete postmortem brain regions in 34 AD-afflicted individuals, using Affymetrix Human Genome U133 Plus 2.0 microarrays. These regions include the entorhinal cortex, hippocampus, middle temporal gyrus, posterior cingulate cortex, superior frontal gyrus, and primary visual cortex. This study is predicated on previous parallel research on the postmortem brains of the same six regions in 14 healthy elderly individuals, for which LCM neurons were similarly processed for expression analysis. We identified significant regional differential expression in AD brains compared with control brains including expression changes of genes previously implicated in AD pathogenesis, particularly with regard to tangle and plaque formation. Pinpointing the expression of factors that may play a role in AD pathogenesis provides a foundation for future identification of new targets for improved AD therapeutics. We provide this carefully phenotyped, laser capture microdissected intraindividual brain region expression data set to the community as a public resource.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00242.2007 American Physiological Society 2 33 256 2008-04-22 240 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/257?rss=1 The cardiac troponin C (cTnC) mutation, L29Q, has been found in a patient with familial hypertrophic cardiomyopathy. We previously showed that L29, together with neighboring residues, Asp2, Val28, and Gly30, plays an important role in determining the Ca²⁺ affinity of site II, the regulatory site of mammalian cardiac troponin C (McTnC). Here we report on the Ca²⁺ binding characteristics of L29Q McTnC and D2N/V28I/L29Q/G30D McTnC (NIQD) utilizing the Phe²⁷ -> Trp (F27W) substitution, allowing one to monitor Ca²⁺ binding and release. We also studied the effect of these mutants on Ca²⁺ activation of force generation in single mouse cardiac myocytes using cTnC replacement, together with sarcomere length (SL) dependence. The Ca²⁺-binding affinity of site II of L29Q McTnC^F27W and NIQD McTnC^F27W was ~1.3- and ~1.9-fold higher, respectively, than that of McTnC^F27W. The Ca²⁺ disassociation rate from site II of L29Q McTnC^F27W and NIQD McTnC^F27W was not significantly different than that of control (McTnC^F27W). However, the rate of Ca²⁺ binding to site II was higher in L29Q McTnC^F27W and NIQD McTnC^F27W relative to control (~1.5-fold and ~2.0-fold respectively). The Ca²⁺ sensitivity of force generation was significantly higher in myocytes reconstituted with L29Q McTnC (~1.4-fold) and NIQD McTnC (~2-fold) compared with those reconstituted with McTnC. Interestingly, the change in Ca²⁺ sensitivity of force generation in response to an SL change (1.9, 2.1, and 2.3 µm) was significantly reduced in myocytes containing L29Q McTnC or NIQD McTnC. These results demonstrate that the L29Q mutation enhances the Ca²⁺-binding characteristics of cTnC and that when incorporated into cardiac myocytes, this mutant alters myocyte contractility.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00154.2007 American Physiological Society 2 33 266 2008-04-22 257 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/267?rss=1 Idiopathic dilated cardiomyopathy (IDCM) constitutes a large portion of patients with heart failure of unknown etiology. Up to 50% of all transplant recipients carry this clinical diagnosis. Female-specific gene expression in IDCM has not been explored. We report sex-related differences in the gene expression profile of ventricular myocardium from patients undergoing cardiac transplantation. We produced and sequenced subtractive cDNA libraries, using human left ventricular myocardium obtained from male transplant recipients with IDCM and nonfailing human heart donors. With the resulting sequence data, we generated a custom human heart failure microarray for IDCM containing 1,145 cardiac-specific oligonucleotide probes. This array was used to characterize RNA samples from female IDCM transplant recipients. We identified a female gene expression pattern that consists of 37 upregulated genes and 18 downregulated genes associated with IDCM. Upon functional analysis of the gene expression pattern, deregulated genes unique to female IDCM were those that are involved in energy metabolism and regulation of transcription and translation. For male patients we found deregulation of genes related to muscular contraction. These data suggest that 1) the gene expression pattern we have detected for IDCM may be specific for this disease and 2) there is a sex-specific profile to IDCM. Our observations further suggest for the first time ever novel targets for treatment of IDCM in women and men.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00265.2007 American Physiological Society 2 33 277 2008-04-22 267 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/278?rss=1 Pulmonary hypertension (PH) and cancer pathology share growth factor- and MAPK stress-mediated signaling pathways resulting in endothelial and smooth muscle cell dysfunction and angioproliferative vasculopathy. In this study, we assessed sorafenib, an antineoplastic agent and inhibitor of multiple kinases important in angiogenesis [VEGF receptor (VEGFR)-1–3, PDGF receptor (PDGFR)-β, Raf-1 kinase] as a potential PH therapy. Two PH rat models were used: a conventional hypoxia-induced PH model and an augmented PH model combining dual VEGFR-1 and -2 inhibition (SU-5416, single 20 mg/kg injection) with hypoxia. In addition to normoxia-exposed control animals, four groups were maintained at 10% inspired O₂ fraction for 3.5 wk (hypoxia/vehicle, hypoxia/SU-5416, hypoxia/sorafenib, and hypoxia/SU-5416/sorafenib). Compared with normoxic control animals, rats exposed to hypoxia/SU-5416 developed hemodynamic and histological evidence of severe PH while rats exposed to hypoxia alone displayed only mild elevations in hemodynamic values (pulmonary vascular and right ventricular pressures). Sorafenib treatment (daily gavage, 2.5 mg/kg) prevented hemodynamic changes and demonstrated dramatic attenuation of PH-associated vascular remodeling. Compared with normoxic control rats, expression profiling (Affymetrix platform) of lung RNA obtained from hypoxia [false discovery rate (FDR) 6.5%]- and hypoxia/SU-5416 (FDR 1.6%)-challenged rats yielded 1,019 and 465 differentially regulated genes (fold change >1.4), respectively. A novel molecular signature consisting of 38 differentially expressed genes between hypoxia/SU-5416 and hypoxia/SU-5416/sorafenib (FDR 6.7%) was validated by either real-time RT-PCR or immunoblotting. Finally, immunoblotting studies confirmed the upregulation of the MAPK cascade in both PH models, which was abolished by sorafenib. In summary, sorafenib represents a novel potential treatment for severe PH with the MAPK cascade a potential canonical target.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00169.2007 American Physiological Society 2 33 291 2008-04-22 278 Research Articles http://physiolgenomics.physiology.org/cgi/content/abstract/33/2/292?rss=1 The ability to monitor transcription factor (TF) activation in the central nervous system (CNS) has the potential to provide novel information regarding the molecular mechanisms underlying a wide range of neurobiological processes. However, traditional biochemical assays limit the mapping of TF activity to select time points. In vivo bioluminescence imaging (BLI) has emerged as an attractive technology for visualizing internal molecular events in the same animal over time. Here, we evaluated the utility of BLI, in combination with virally mediated delivery of reporter constructs to cardiovascular nuclei, for monitoring of TF activity in these discrete brain regions. Following viral gene transfer of NF-B-driven luciferase reporter to the subfornical organ (SFO), BLI enabled daily measurements of baseline TF activity in the same animal for 1 mo. Importantly, systemic endotoxin, a stimulator of NF-B activity, induced dramatic and dose-dependent increases in NF-B-dependent bioluminescence in the SFO up to 30 days after gene transfer. Cotreatment with a dominant-negative IB mutant significantly prevented endotoxin-dependent NF-B activation, confirming the specificity of the bioluminescence signal. NF-B-dependent luminescence signals were also stable and inducible 1 mo following delivery of luciferase reporter construct to the paraventricular nucleus or rostral ventrolateral medulla. Lastly, using targeted adenoviral delivery of an AP-1 responsive luciferase reporter, we showed similar baseline and endotoxin-induced AP-1 activity in these same brain regions as with NF-B reporters. These results demonstrate that BLI, in combination with virally mediated gene transfer, is a powerful method for longitudinal monitoring and quantification of TF activity in targeted CNS nuclei in vivo.

]]> 2008-04-22 info:doi/10.1152/physiolgenomics.00296.2007 American Physiological Society 2 33 299 2008-04-22 292 Toolbox