Distinct Structural Changes in the GABAA Receptor Elicited by Pentobarbital and GABA

The barbiturate pentobarbital binds to γ-aminobutyric acid type A (GABA_A) receptors, and this interaction plays an important role in the anesthetic action of this drug. Depending on its concentration, pentobarbital can potentiate (∼10-100 μM), activate (∼100-800 μM), or block (∼1-10 mM) the channel, but the mechanisms underlying these three distinct actions are poorly understood. To investigate the drug-induced structural rearrangements in the GABA_A receptor, we labeled cysteine mutant receptors expressed in Xenopus oocytes with the sulfhydryl-reactive, environmentally sensitive fluorescent probe tetramethylrhodamine-6-maleimide (TMRM). We then used combined voltage clamp and fluorometry to monitor pentobarbital-induced channel activity and local protein movements simultaneously in real time. High concentrations of pentobarbital induced a decrease in TMRM fluorescence (F_TMRM) of labels tethered to two residues in the extracellular domain (α₁L127C and β₂L125C) that have been shown previously to produce an increase in F_TMRM in response to GABA. Label at β₂K274C in the extracellular end of the M2 transmembrane helix reported a small but significant F_TMRM increase during application of low modulating pentobarbital concentrations, and it showed a much greater F_TMRM increase at higher concentrations. In contrast, GABA decreased F_TMRM at this site. These results indicate that GABA and pentobarbital induce different structural rearrangements in the receptor, and thus activate the receptor by different mechanisms. Labels at α₁L127C and β₂K274C change their fluorescence by substantial amounts during channel blockade by pentobarbital. In contrast, picrotoxin blockade produces no change in F_TMRM at these sites, and the pattern of F_TMRM signals elicited by the antagonist SR95531 differs from that produced by other antagonists. Thus, with either channel block by antagonists or activation by agonists, the structural changes in the GABA_A receptor protein differ during transitions that are functionally equivalent.     

Absence of tissue inhibitor of metalloproteinases 3 disrupts alveologenesis in the mouse

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs) throughout lung development. We examined lungs from TIMP3 null mice and found significant air space enlargement compared with wild type (WT) animals during a time course spanning early alveologenesis (post‐partum days 1, 5, 9 and 14). Trichrome staining revealed a similar pattern of collagen distribution in the walls of nascent alveoli; however, the alveolar walls of TIMP3 mutant mice appeared to be thinner than controls. Assessment of MMP2 and MMP9 activities by gelatin zymography demonstrated a significant elevation in the active form of MMP2 at post‐partum days 1 and 5. Treatment of null pregnant dams with a broad spectrum synthetic metalloproteinase inhibitor, GM6001, on embryonic day 16.5 enhanced the formation of primitive alveoli during the saccular stage of lung development as evidenced by a partial, but significant, rescue of alveolar size in post‐partum day 1 animals. We propose that increased MMP activity in the absence of TIMP3 enhances ECM proteolysis, upsetting proper formation of primitive alveolar septa during the saccular stage of alveologenesis. Therefore, TIMP3 indirectly regulates alveolar formation in the mouse. To our knowledge, ours is the first study to demonstrate that in utero manipulation of the TIMP/MMP proteolytic axis, to specifically inhibit proteolysis, significantly affects lung development.     

LEAF SHAPE EVOLUTION IN THE SOUTH AFRICAN GENUS PELARGONIUM L' HÉR. (GERANIACEAE)

Leaf shapes reflect complex assemblages of shape-determining elements, yet evolutionary studies tend to treat leaf shape as a single attribute, for example cordate or linear. As with all complex structures, individual elements of a leaf could theoretically evolve independently and at different rates to the extent permitted by genetic and functional limitations. We examined relative evolutionary lability of shape-determining elements in the highly diverse South African plant genus Pelargonium (Geraniaceae). We used SIMMAP to calculate Bayesian posterior probabilities for ancestral states of leaf-shape characters for major nodes across multiple phylogenetic trees. Trees were derived from a Bayesian analysis of DNA sequence data from four partitions. We found that shape elements differed in rates of character-state transformations across the tree. Leaf base, apex, and overall outline had low rates. Transformations in venation occurred at slightly higher rates and were associated with shifts in venation among major clades. Leaf margin type and overall leaf size showed intermediate rates, whereas high rates were observed in the extent of lamina lobing and functional leaf size. The results indicate that suites of elements characteristic of the recently evolved xerophytic lineage, for example pinnate venation, dissected lamina, and entire margins, were acquired piecemeal over nested levels of the phylogeny.     

An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA

Rad54 protein is a key member of the RAD52 epistasis group required for homologous recombination in eukaryotes. Rad54 is a duplex DNA translocase that remodels both DNA and protein–DNA complexes, and functions at multiple steps in the recombination process. Here we use biochemical criteria to demonstrate the existence of this important protein in a prokaryotic organism. The Sulfolobus solfataricus Rad54 (SsoRad54) protein is a double-strand DNA-dependent ATPase that can alter the topology of duplex DNA. Like its eukaryotic homolog, it interacts directly with the S. solfataricus Rad51 homologue, SsoRadA, to stimulate DNA strand exchange. Confirmation of this protein as an authentic Rad54 homolog establishes an essential phylogenetic bridge for identifying Rad54 homologs in the archaeal and bacterial domains.     

Cardioprotection by HO-4038, a novel verapamil derivative, targeted against ischemia and reperfusion-mediated acute myocardial infarction

Many cardiac interventional procedures, such as coronary angioplasty, stenting, and thrombolysis, attempt to reintroduce blood flow (reperfusion) to an ischemic region of myocardium. However, the reperfusion is accompanied by a complex cascade of cellular and molecular events resulting in oxidative damage, termed myocardial ischemia-reperfusion (I/R) injury. In this study, we evaluated the ability of HO-4038, an N-hydroxypiperidine derivative of verapamil, on the modulation of myocardial tissue oxygenation (Po(2)), I/R injury, and key signaling molecules involved in cardioprotection in an in vivo rat model of acute myocardial infarction (MI). MI was created in rats by ligating the left anterior descending coronary artery (LAD) for 30 min followed by 24 h of reperfusion. Verapamil or HO-4038 was infused through the jugular vein 10 min before the induction of ischemia. Myocardial Po(2) and the free-radical scavenging ability of HO-4038 were measured using electron paramagnetic resonance spectroscopy. HO-4038 showed a significantly better scavenging ability of reactive oxygen radicals compared with verapamil. The cardiac contractile functions in the I/R hearts were significantly higher recovery in HO-4038 compared with the verapamil group. A significant decrease in the plasma levels of creatine kinase and lactate dehydrogenase was observed in the HO-4038 group compared with the verapamil or untreated I/R groups. The left ventricular infarct size was significantly less in the HO-4038 (23 +/- 2%) compared with the untreated I/R (36 +/- 4%) group. HO-4038 significantly attenuated the hyperoxygenation (36 +/- 1 mmHg) during reperfusion compared with the untreated I/R group (44 +/- 2 mmHg). The HO-4038-treated group also markedly attenuated superoxide production, increased nitric oxide generation, and enhanced Akt and Bcl-2 levels in the reperfused myocardium. Overall, the results demonstrated that HO-4038 significantly protected hearts against I/R-induced cardiac dysfunction and damage through the combined beneficial actions of calcium-channel blocking, antioxidant, and prosurvival signaling activities.     

Drought stress increases the production of 5-hydroxynorvaline in two C4 grasses

Plants produce various compounds in response to water deficit. Here, the presence and identification of a drought-inducible non-protein amino acid in the leaves of two C₄ grasses is first reported. The soluble amino acids extracted from the leaves of three different species were measured by high-performance liquid chromatography of derivatives formed with o-phthaldialdehyde and β-mercaptoethanol. One amino acid that increased in amount with drought stress had a retention time not corresponding to any common amino acid. Its identity was determined by metabolite profiling, using ¹H NMR and GC-MS. This unusual amino acid was present in the dehydrated leaves of Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel, but was absent from Paspalum dilatatum Poir. Its identity as 2-amino-5-hydroxypentanoic acid (5-hydroxynorvaline, 5-HNV) was confirmed by synthesis and co-chromatography of synthetic and naturally occurring compounds. The amount of 5-HNV in leaves of the more drought tolerant C₄ grasses, C. dactylon and Z. japonica, increased with increasing water deficit; therefore, any benefits from this unusual non-protein amino acid for drought resistance should be further explored.     

The genetic basis of phenotypic convergence in beach mice: similar pigment patterns but different genes

Convergent evolution is a widespread phenomenon seen in diverseorganisms inhabiting similar selective environments. However,it is unclear if similar phenotypes are produced by the sameor different genes and mutations. Here we analyze the molecularmechanisms underlying convergent pigment pattern among subspeciesof the beach mouse (Peromyscus polionotus) inhabiting the Gulfand Atlantic coasts of Florida. In these two geographic regions,separated by more than 300 km, "beach mice" have lighter coloredcoats than do their mainland counterparts, produced by naturalselection for camouflage against the pale coastal sand dunes.We measured color pattern in eight beach mouse subspecies andshowed that three of the Gulf Coast subspecies are more phenotypicallysimilar to an Atlantic coast subspecies than to their Gulf Coastneighbors. However, light-colored beach mice do not form a monophyleticgroup. Previous results implicated a single derived amino acidchange in the melanocortin-1 receptor (Mc1r) as a major contributorto pigment pattern in the Gulf Coast beach mice; despite phenotypicsimilarities, the derived Mc1r allele was not found in the Atlanticcoast beach mouse populations. Here we show that Atlantic coastbeach mice have high levels of Mc1r polymorphism but they lackunique alleles. Functional assays revealed that single aminoacid mutations segregating in Atlantic coast beach mice do notcause any change in Mc1r activity compared with the activityof Mc1r from dark-colored mice. These joint results show thatconvergent pigment patterns in recently diverged beach mousesubspecies—whose developmental constraints are presumablysimilar—have evolved through a diversity of genetic mechanisms.