Catalase as a source of both X- and V-factor for Haemophilus influenzae

Haemophilus influenzae requires two growth factors, designated factor X (porphyrin) and factor V (NAD). Mammalian catalases contain both bound heme and NADPH. This study shows that catalase can supply both factors X and V to H. influenzae in vitro, thus representing a potential in vivo source of these essential growth factors.     

Identification of modulating residues defining the catalytic cleft of insulin-regulated aminopeptidase.

Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the 'entrance' of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys1-vasopressin (2-9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine1-angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH2-terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.     

Structure of the glucanase inhibitor protein (GIP) family from phytophthora species suggests coevolution with plant endo-beta-1,3-glucanases

During invasion of their plant hosts, species of the oomycete genus Phytophthora secrete glucanase inhibitor proteins (GIPs) into the plant apoplast, which bind and inhibit the activity of plant extracellular endo-beta-1,3-glucanases (EGases). GIPs show structural homology to the chymotrypsin class of serine proteases (SP) but lack proteolytic activity due to the absence of an intact catalytic triad and, thus, belong to a broader class of proteins called serine protease homologs (SPH). To study the evolutionary relationship between GIPs and functional SP, database searches were used to identify 48 GIP homologs in the P. sojae, P. ramorum, and P. infestans genomes, composing GIPs, SPH, and potentially functional SP. Analyses of P. infestans-inoculated tomato leaves showed that P. infestans GIPs and tomato EGases are present in the apoplast and form stable complexes in planta. Studies of the temporal expression of a four-membered GIP family from P. infestans (PiGIP1 to PiGIP4) further revealed that the genes show distinctly different patterns during an infection timecourse. Codon evolution analyses of GIP homologs identified several positively selected peptide sites and structural modeling revealed them to be in close proximity to rapidly evolving EGase residues, suggesting that the interaction between GIPs and EGases has the hallmarks of a coevolving molecular arms race.     

Mice with the testicular feminization mutation demonstrate a role for androgen receptors in the regulation of anxiety-related behaviors and the hypothalamic-pituitary-adrenal axis

Testosterone (T) appears to play a role in anxiety and sensorimotor gating in rodents, but whether T acts through the androgen receptor (AR) to influence these behaviors is less clear. We compared adult genetic male mice with the testicular feminization mutation (Tfm), which lack functional ARs, to wild type male littermates (wt males) on an assay of sensorimotor gating (prepulse inhibition of the acoustic startle response; PPI) and several tests thought to reflect anxiety: open field exposure, novel object exposure, elevated plus maze (EPM), and light/dark (LD) box. PPI was similar between groups, but indices of anxiety in the novel object and LD box tests were increased in Tfm males with no significant differences found in the open field or EPM. Since Tfm male mice have decreased circulating T, the same tests were conducted in mice that were gonadectomized (wt males) or sham-operated (Tfm males) as adults and supplemented with T or nothing (B). While T treatment reduced indices of anxiety in the novel object and LD box tests in wt males, it was ineffective in Tfm males. Increased indices of anxiety in Tfm males appear to be related to hyper-activation of the hypothalamic–pituitary–adrenal axis since levels of the stress hormone corticosterone were elevated in Tfm males compared to wt males at baseline and at several time points after exposure to a novel object. These findings demonstrate that ARs influence anxiety and stress responses in mice.     

Cystic echinococcosis in a wild population of the brush-tailed rock-wallaby (Petrogale penicillata), a threatened macropodid

Infection of small macropodids with the larval stage of Echinococcus granulosus can cause fatalities as well as significant pulmonary impairment and other adverse sequelae. The brush-tailed rock-wallaby (Petrogale penicillata) is a small macropodid listed as vulnerable on the IUCN's Red List of Threatened Species. This study used radiographic techniques to determine the prevalence and severity of pulmonary hydatid infection and growth rates of hydatid cysts in a wild population of this macropodid. The overall prevalence was 15.3% (9/59 animals) with 20.0% (8/40 animals) of adults infected. During the study period, the death of at least 1 infected animal was directly attributed to pulmonary hydatidosis. Rapid cyst growth occurred in some animals (up to 43% increase in cyst volume in 3 months). Cyst volume reduced lung capacity by up to 17%. Secondary pulmonary changes were uncommon but, in 1 animal, resulted in reduction in lung capacity by approximately 50%. Infection was associated with a higher blood urea concentration, but no significant differences in other blood variables were detected. These results indicate that hydatid infection may be a significant risk to threatened populations of small macropodids and should be addressed in conservation management plans for these animals.     

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.     

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.