Gene expression and the concept of the phenotype

While the definition of the 'genotype' has undergone dramatic changes in the transition from classical to molecular genetics, the definition of the 'phenotype' has remained for a long time within the classical framework. In addition, while the notion of the genotype has received significant attention from philosophers of biology, the notion of the phenotype has not. Recent developments in the technology of measuring gene-expression levels have made it possible to conceive of phenotypic traits in terms of levels of gene expression. We demonstrate that not only has this become possible but it has also become an actual practice. This suggests a significant change in our conception of the phenotype: as in the case of the 'genotype', phenotypes can now be conceived in quantitative and measurable terms on a comprehensive molecular level. We discuss in what sense gene expression profiles can be regarded as phenotypic traits and whether these traits are better described as a novel concept of phenotype or as an extension of the classical concept. We argue for an extension of the classical concept and call for an examination of the type of extension involved.     

The effects of side-artifacts on the elastic modulus of trabecular bone

Determining accurate density-mechanical property relationships for trabecular bone is critical for correct characterization of this important structure-function relation. When testing any excised specimen of trabecular bone, an unavoidable experimental artifact originates from the sides of the specimen where peripheral trabeculae lose their vertical load-bearing capacity due to interruption of connectivity, a phenomenon denoted here as the 'side-artifact'. We sought in this study to quantify the magnitude of such side-artifact errors in modulus measurement and to do so as a function of the trabecular architecture and specimen size. Using parametric computational analysis of high-resolution micro-CT-based finite-element models of cores of elderly human vertebral trabecular bone, a specimen-specific correction factor for the side-artifact was quantified as the ratio of the side-artifact-free apparent modulus (Etrue) to the apparent modulus that would be measured in a typical experiment (Emeasured). We found that the width over which the peripheral trabeculae were mostly unloaded was between 0.19 and 0.58 mm. The side-artifact led to an underestimation error in Etrue of over 50% in some specimens, having a mean (+/-SD) of 27+/-11%. There was a trend for the correction factor to linearly increase as volume fraction decreased (p=0.001) and as mean trabecular separation increased (p<0.001). Further analysis indicated that the error increased substantially as specimen size decreased. Two methods used for correcting for the side-artifact were both successful in bringing Emeasured into statistical agreement with Etrue. These findings have important implications for the interpretation of almost all literature data on trabecular bone mechanical properties since they indicate that such properties need to be adjusted to eliminate the substantial effects of side-artifacts in order to provide more accurate estimates of in situ behavior.     

Ontogeny of gonadotropin-releasing hormone (GnRH) neurons in hybrid striped bass Morone sp.: whole-mount in situ hybridization analysis

The ontogeny of gonadotropin-releasing hormone (GnRH) mRNA-producing neurons was studied in developing hybrid striped bass (white bass Morone chrysops female × striped bass Morone saxatilis male), 1–55 days post-fertilization (dpf), by whole-mount in situ hybridization. Neurons that produce salmon (s) GnRH were first detected at 32 h post-fertilization in the olfactory placodes. These neurons migrated posteriorly during development and reached their final position at the olfactory bulbs-telencephalon boundary, possibly the terminal nerve ganglion (TNg) by 11 dpf. First signal of chicken (c) GnRH-II neurons appeared in the midbrain 2 dpf and remained there throughout development. A signal of seabream (sb) GnRH mRNA was first detected at 21 dpf in the preoptic area (POA) and as a bilateral continuum along the ventral telencephalon at 32–55 dpf. The expression of all three forms of GnRH increased throughout development. These results suggest that cGnRH-II neurons originate in the mid-brain, and that sGnRH neurons originate in the olfactory placodes and migrate caudally to the TNg. Neurons that express sbGnRH seem to originate at the preoptic area and then to migrate anteriorly along the ventral telencephalon. An olfactory placodal origin of these neurons, however, cannot be ruled out.     

Multiple roles of alpha-smooth muscle actin in mechanotransduction

Alpha-smooth muscle actin (SMA), an actin isoform that contributes to cell-generated mechanical tension, is normally restricted to cells of vascular smooth muscle, but SMA can also be expressed in certain non-muscle cells, most notably myofibroblasts. These cells are present in healing wounds, scars, and fibrocontractive lesions where they contribute to fibrosis. In myofibroblasts, cell-generated traction forces associated with SMA contribute to matrix remodeling, but exogenous mechanical forces can also increase SMA expression. Force-induced SMA utilizes a feed-forward amplification loop involving a priori SMA in focal adhesions, the binding of the p38 MAP kinase to SMA filaments, activation of Rho and binding of serum response factor to the CArG-B box of the SMA promoter. Thus, in addition to its importance as a structural protein in tissue remodeling and contraction, SMA may serve as a mechanotransducer, based on its ability to physically link mechanosensory elements and to enhance its own, force-induced expression.     

Congenital hyperreninemic hypoaldosteronism in Israel: sequence analysis of CYP11B2 gene

BACKGROUND/AIMS: Isolated aldosterone biosynthesis defect causing congenital hyperreninemic hypoaldosteronism with otherwise normal adrenal function usually results from aldosterone synthase deficiency. Patients present with manifestations of mineralocorticoid deficiency during the first weeks of life. The largest numbers of cases have been described in Iranian Jews, who carried concomitantly two homozygous missense mutations (R181W and V386A). In a few cases with presumed aldosterone synthase deficiency no mutations in CYP11B2 gene have been identified. We describe a molecular and endocrine evaluation of seven cases of congenital hyperreninemic hypoaldosteronism in Israel. PATIENTS/METHODS: Two of the six Jewish patients are of Iranian origin. The parents of five other patients originated from Yemen, Syria and Morocco. One patient is a Muslim-Arab. CYP11B2's exons, exon-intron boundaries and promoter region were sequenced by multiple PCR amplifications. Gene size determination was performed either by long-range PCR or by Southern blot analysis. RESULTS: Only two patients (Iranian Jews) carried a known homozygous R181W, V386A mutations, other two were compound heterozygotes for either the R181W or V386A and one additional novel amino acid substitution (A319V or D335G), and one patient was found to be a carrier of the two novel variations (A319V and D335G). We could not find a molecular defect in 2 patients: one was a carrier of the D335G mutation and the other had no detectable molecular change in the coding and promoter regions. CONCLUSION: The genetic and molecular basis of congenital hyperreninemic hypoaldosteronism is more heterogeneous than previously described. The significance of amino acid substitutions identified in this study remains to be determined.     

LDL induces Saos2 osteoblasts death via Akt pathways responsive to a neutral sphingomyelinase inhibitor

Atherosclerosis is epidemiologically associated with postmenopausal osteoporosis (OP) presumably by common etiologic factors, reflecting a state of co-morbidity in aging. Osteoblasts make a significant facet of this co-morbidity state. Since oxidized low-density lipoprotein (oxLDL) is a major factor in generation of vascular wall pathology, we examined the ability of native LDL (nLDL) and oxLDL to induce Saos2 osteoblasts growth arrest. OxLDL induced Saos2 cell death with morphological features of apoptosis that was inhibited mainly by caspase-9 and partially by caspase-3 but not by caspase-8 inhibitors. nLDL, like oxLDL, has induced cell death, where 60% (P = 0.00033) and 30% (P = 0.075, ns) of the cell death, respectively, could be inhibited by scyphostatin (a neutral sphingomyelinase [nSMase] inhibitor). Upon similar condition, nLDL inhibited the phosphorylation of Akt and two of its downstream targets, fork head receptor (FKHR) and glycogen synthase kinase-3 (GSK3). This is a pathway that stimulates cell survival and proliferation. nLDL has also induced an increase in the proapoptotic Bcl-Xs and it has diminished the potential antiapoptotic Src kinase activity. At the 4 h time-point, upon a substantial decrease in nLDL-induced Akt phosphorylation, scyphostatin has inhibited the reduction in FKHR and GSK3 phosphorylation but inexplicably not that of Akt. Scyphostatin has also corrected the reduction in Src kinase activity. Taken together, the results indicate that nLDL has induced apoptosis in Saos2 osteoblasts by inactivation of the pathway downstream to Akt using nSMase, and by involvement of Src kinase. Inferring that caspase-9 was the main executioner (rather than caspase-8 and-3) in Saos2 cell death, indicates that the nSMase-induced release of ceramide, directly activated the intrinsic mitochondrial apoptotic pathway. With regard to the Akt inactivation by nLDL, Saos2 osteoblasts responded in an opposite fashion to the response reported by others, in macrophages.     

Characterizing and controlling the inherent dynamics of cyclophilin-A

With the recent advances in NMR relaxation techniques, protein motions on functionally important timescales can be studied at atomic resolution. Here, we have used NMR-based relaxation experiments at several temperatures and both 600 and 900 MHz to characterize the inherent dynamics of the enzyme cyclophilin-A (CypA). We have discovered multiple chemical exchange processes within the enzyme that form a “dynamic continuum” that spans 20–30 Å comprising active site residues and residues proximal to the active site. By combining mutagenesis with these NMR relaxation techniques, a simple method of counting the dynamically sampled conformations has been developed. Surprisingly, a combination of point mutations has allowed for the specific regulation of many of the exchange processes that occur within CypA, suggesting that the dynamics of an enzyme may be engineered.     

Prefermentation of liquid dairy manure to support biological nutrient removal

A continuously operated, intermittently fed reactor (fermenter) system with a 2-d solids retention time was proposed for supporting biological nutrient removal from liquid dairy manure. The first objective of this study was to select a material with high fermentation potential to be used as the fermenter feed. Primary sludge, liquid separated dairy manure, and flushed dairy manure were investigated for their fermentation potential. Liquid separated dairy manure had the highest fermentation potential, 0.73 mg volatile fatty acid as chemical oxygen demand/mg of initial volatile suspended solids (VSS). The second objective was to investigate the performance of a pilot-scale fermenter operated under an average organic loading rate (OLR) of 3 kg-VSS/m³/d. The reactor utilized 18% of the manure fermentation potential. Performance comparison of the pilot-scale fermenter and a lab-scale fermenter with an average OLR of 7 kg-VSS/m³/d highlighted the need to increase the OLR of the pilot-scale fermenter so that it can exploit a higher fraction of the manure fermentation potential. A continuously operated, intermittently fed fermenter with 2-d SRT can utilize the majority of the manure fermentation potential and support a downstream BNR reactor provided that it receives a sufficiently high OLR.