Mixed-model reanalysis of primate data suggests tissue and species biases in oligonucleotide-based gene expression profiles

An emerging issue in evolutionary genetics is whether it is possible to use gene expression profiling to identify genes that are associated with morphological, physiological, or behavioral divergence between species and whether these genes have undergone positive selection. Some of these questions were addressed in a recent study (Enard et al. 2002) of the difference in gene expression among human, chimp, and orangutan, which suggested an accelerated rate of divergence in gene expression in the human brain relative to liver. Reanalysis of the Affymetrix data set using analysis of variance methods to quantify the contributions of individuals and species to variation in expression of 12,600 genes indicates that as much as one-quarter of the genome shows divergent expression between primate species at the 5% level. The magnitude of fold change ranges from 1.2-fold up to 8-fold. Similar conclusions apply to reanalysis of Enard et al. 2002 parallel murine data set. However, biases inherent to short oligonucleotide microarray technology may account for some of the tissue and species effects. At high significance levels, more differences were observed in the liver than in the brain in each of the pairwise species comparisons, so it is not clear that expression divergence is accelerated in the human brain. Further, there is an apparent bias toward upregulation of gene expression in the brain in both primates and mice, whereas genes are equally likely to be up- or downregulated in the liver when these species diverge. A small subset of genes that are candidates for adaptive divergence may be identified on the basis of a high ratio of interspecific to intraspecific divergence.     

Glycerol-3-phosphate acquisition in spirochetes: distribution and biological activity of glycerophosphodiester phosphodiesterase (GlpQ) among Borrelia species

Relapsing-fever spirochetes achieve high cell densities (>10(8)/ml) in their host's blood, while Lyme disease spirochetes do not (<10(5)/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood. Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B. hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids. Sequence analysis of B. hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B. burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.     

Structure of homo- and hetero-oligomeric meprin metalloproteases. Dimers,tetramers, and high molecular mass multimers

Meprin A and B, metalloproteases consisting of evolutionarily related alpha and/or beta subunits, are membrane-bound and secreted enzymes expressed by kidney and intestinal epithelial cells, leukocytes, and cancer cells. Previous work established that the multidomain meprin subunits (each approximately 80 kDa) form disulfide-bridged homo- and heterodimers, and differ in substrate and peptide bond specificities. The work herein clearly demonstrates that meprin dimers differ markedly in their ability to oligomerize. Electrophoresis, light scattering, size exclusion chromatography, and electron microscopy were used to characterize quaternary structures of recombinant rat meprins. Meprin B, consisting of meprin beta subunits only, was dimeric under a wide range of conditions. By contrast, meprin alpha homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from approximately 1.0 to 6.0 MDa. The size of the meprin alpha homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin alphabeta heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin beta, which has a transmembrane domain in vivo, restricts the oligomerization potential of meprin molecules and localizes meprins to the plasma membrane. By contrast, the propensity of secreted meprin alpha homodimers to self-associate concentrates proteolytic potential into high molecular mass multimers and thus allows for autocompartmentalization. The work indicates that different mechanisms exist to localize and concentrate the proteolytic activity of membrane-bound and secreted meprin metalloproteinases.     

Genome and Hormones: Gender Differences in Physiology: Selected Contribution: Cerebrovascular NOS and cyclooxygenase are unaffected by estrogen in mice lacking estrogen receptor-alpha

Estrogen alters reactivity of cerebral arteries by modifyingproduction of endothelium-dependent vasodilators. Estrogen receptors (ER) are thought to be involved, but the responsible ER subtype isunknown. ER- knockout (ERKO) mice were used to test whether estrogen acts via ER-. Mice were ovariectomized, with or without estrogen replacement, and cerebral blood vessels were isolated 1 molater. Estrogen increased levels of endothelial nitric oxide synthaseand cyclooxygenase-1 in vessels from wild-type mice but was ineffectivein ERKO mice. Endothelium-denuded middle cerebral artery segmentsfrom all animals constricted when pressurized. In denuded arteries fromERKO but not wild-type mice, estrogen treatment enhancedconstriction. In endothelium-intact, pressurized arteries fromwild-type estrogen-treated mice, diameters were larger compared witharteries from untreated wild-type mice. In addition, contractileresponses to indomethacin were greater in arteries from wild-typeestrogen-treated mice compared with arteries from untreated wild-typemice. In contrast, estrogen treatment of ERKO mice had no effect ondiameter or indomethacin responses of endothelium-intact arteries. ThusER- regulation of endothelial nitric oxide synthase andcyclooxygenase-1 pathways appears to contribute to effects of estrogenon cerebral artery reactivity.

     

alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation

Smooth muscle exhibitsmechanosensitivity independent of neural input, suggesting thatmechanosensitive pathways reside within smooth muscle cells. The nativeL-type calcium current recorded from human intestinal smooth muscle ismodulated by stretch. To define mechanosensitive mechanisms involved inthe regulation of smooth muscle calcium entry, we cloned the_1C L-type calcium channel subunit (Ca_V1.2)from human intestinal smooth muscle and expressed the channel in aheterologous system. This channel subunit retained mechanosensitivitywhen expressed alone or coexpressed with a ₂ calciumchannel subunit in HEK-293 or Chinese hamster ovary cells. Theheterologously expressed human cardiac _1C splice formalso demonstrated mechanosensitivity. Inhibition of kinase signalingdid not affect mechanosensitivity of the native channel. Truncation of the _1C COOH terminus, which containsan inhibitory domain and a proline-rich domain thought to mediatemechanosensitive signaling from integrins, did not disruptmechanosensitivity of the expressed channel. These data demonstratemechanical regulation of calcium entry through molecularly identifiedL-type calcium channels in mammalian cells and suggest that themechanosensitivity resides within the pore forming_1C-subunit.

     

Mildly acidic pH activates the extracellular molecular chaperone clusterin

Many features of the chaperone action of clusterin are similar to those of the intracellular small heat shock proteins (sHSPs) that, like clusterin, exist in solution as heterogeneous aggregates. Increased temperature induces dissociation of some sHSP aggregates and an enhanced chaperone action, suggesting that a dissociated form is the active chaperone species. We recently reported that clusterin aggregates dissociate at mildly acidic pH. To further explore the similarities between clusterin and the sHSPs, we tested the effects of temperature and pH on the structure of clusterin and its chaperone action. Our results demonstrate that increased temperature does not induce dissociation of clusterin aggregates, or other major structural changes, and has little effect on its chaperone action. However, we show that the chaperone action of clusterin is enhanced at mildly acidic pH. Clusterin is the first chaperone shown to be activated by reduced pH. This unique mode of activation appears to result from an increase in regions of solvent-exposed hydrophobicity, which is independent of any major changes in secondary or tertiary structure. We propose a model in which low pH-induced dissociation of clusterin aggregates increases the abundance of the heterodimeric chaperone-active species, which has greater hydrophobicity exposed to solution.     

A novel synthesis of 2-arylpyrrolo[1,2-a]pyrimid-7-ones and their structure-activity relationships as potent GnRH receptor antagonists

In the process of developing GnRH receptor antagonists, a novel base-catalyzed cyclization of compounds 5a-b was discovered, which led to the formation of the 2-aryl pyrrolo[1,2-a]pyrimid-7-one core structures 6a-b. These intermediates were further modified at positions 1, 2, 4 and 6 to afford a series of potent GnRH antagonists with low nanomolar K(i) values.