Genetic variation in the kakerori (Pomarea dimidiata), an endangered endemic bird successfully recovering in the Cook Islands

The Cook Islands endemic kakerori (Pomarea dimidiata) underwent a severe population decline following the introduction of ship rats (Rattus rattus) in the late 1800s. By 1989, the sole population on Rarotonga consisted of 29 known birds. Subsequent intensive management efforts enabled this population to recover to around 250?C300 birds in recent years. This study, using microsatellite and mitochondrial DNA markers, assesses the level of genetic diversity and the genetic structure of the contemporary kakerori population on Rarotonga. No mitochondrial control region and cytochrome b haplotype diversity was found in the 11 samples examined at each locus. In 81 samples genotyped at 7 polymorphic microsatellite loci, an average of 4 alleles per locus were found, with an average observed heterozygosity of 0.65. No subpopulation division was found in this population. There was no evidence of inbreeding, but genetic bottleneck tests showed that the population had indeed experienced a significant genetic bottleneck. Recovery of the kakerori was successful in the past two decades despite low genetic diversity in terms of allelic diversity. Our data suggested that low allelic diversity did not hamper population expansion and the continued survival of this species, however, longer-term effects are still possible.     

Leaf photosynthesis and carbohydrates of CO₂-enriched maize and grain sorghum exposed to a short period of soil water deficit during vegetative development

Among C₄ species, sorghum is known to be more drought tolerant than maize. The objective was to evaluate differences in leaf gas exchanges, carbohydrates, and two enzyme activities of these nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) C₄ subtype monocots in response to water deficit and CO₂ concentration ([CO₂]). Maize and sorghum were grown in pots in sunlit environmental-controlled chambers. Treatments included well watered (WW) and water stressed (WS) (water withheld at 26 days) and daytime [CO₂] of 360 (ambient) and 720 (elevated) μmol mol⁻¹. Midday gas exchange rates, concentrations of nonstructural carbohydrates, and activities of sucrose-phosphate synthase (SPS) and adenosine 5′-diphosphoglucose pyrophosphorylase (ADGP) were determined for fully expanded leaf sections. There was no difference in leaf CO₂ exchange rates (CER) between ambient and elevated [CO₂] control plants for both maize and sorghum. After withholding water, leaf CER declined to zero after 8 days in maize and 10 days for sorghum. Sorghum had lower stomatal conductance and transpiration rates than maize, which resulted in a longer period of CER under drought. Nonstructural carbohydrates of both control maize and sorghum were hardly affected by elevated [CO₂]. Under drought, however, increases in soluble sugars and decreases in starch were generally observed for maize and sorghum at both [CO₂] levels. For stressed maize and sorghum, decreases in starch occurred earlier and were greater at ambient [CO₂] than at elevated [CO₂]. For maize, drought did not meaningfully affect SPS activity. However, a decline in SPS activity was observed for drought-stressed sorghum under both [CO₂] treatments. There was an increase in ADGP activity in maize under drought for both [CO₂] treatments. Such a response in ADGP to drought, however, did not occur for sorghum. The generally more rapid response of maize than sorghum to drought might be related to the more rapid growth of leaf area of maize.     

Hypothesis/review: The structural basis of sweetness perception of sweet-tasting plant proteins can be deduced from sequence analysis

Human perception of sweetness, behind the felt pleasure, is thought to play a role as an indicator of energy density of foods. For humans, only a small number of plant proteins taste sweet. As non-caloric sweeteners, these plant proteins have attracted attention as candidates for the control of obesity, oral health and diabetic management. Significant advances have been made in the characterization of the sweet-tasting plant proteins, as well as their binding interactions with the appropriate receptors. The elucidation of sweet-taste receptor gene sequences represents an important step towards the understanding of sweet taste perception. However, many questions on the molecular basis of sweet-taste elicitation by plant proteins remain unanswered. In particular, why homologues of these proteins do not elicit similar responses? This question is discussed in this report, on the basis of available sequences and structures of sweet-tasting proteins, as well as of sweetness-sensing receptors. A simple procedure based on sequence comparisons between sweet-tasting protein and its homologous counterparts was proposed to identify critical residues for sweetness elicitation. The open question on the physiological function of sweet-tasting plant proteins is also considered. In particular, this review leads us to suggest that sweet-tasting proteins may interact with taste receptor in a serendipity manner.     

Immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3

Passive administration of porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing antibodies (NAbs) can effectively protect pigs against PRRSV infection. However, after PRRSV infection, pigs typically develop a weak and deferred NAb response. One major reason for such a meager NAb response is the phenomenon of glycan shielding involving GP5, a major glycoprotein carrying one major neutralizing epitope. We describe here a type II PRRSV field isolate (PRRSV-01) that is highly susceptible to neutralization and induces an atypically rapid, robust NAb response in vivo. Sequence analysis shows that PRRSV-01 lacks two N-glycosylation sites, normally present in wild-type (wt) PRRSV strains, in two of its envelope glycoproteins, one in GP3 (position 131) and the other in GP5 (position 51). To determine the influence of these missing N-glycosylation sites on the distinct neutralization phenotype of PRRSV-01, a chimeric virus (FL01) was generated by replacing the structural genes of type II PRRSV strain FL12 cDNA infectious clone with those from PRRSV-01. N-glycosylation sites were reintroduced into GP3 and GP5 of FL01, separately or in combination, by site-directed mutagenesis. Reintroduction of the N-glycosylation site in either GP3 or GP5 allowed recovery of in vivo and in vitro glycan shielding capacity, with an additive effect when these sites were reintroduced into both glycoproteins simultaneously. Although the loss of these glycosylation sites has seemingly occurred naturally (presumably by passage through cell cultures), PRRSV-01 virus quickly regains these glycosylation sites through replication in vivo, suggesting that a strong selective pressure is exerted at these sites. Collectively, our data demonstrate the involvement of an N-glycan moiety located in GP3 in glycan shield interference.     

A new species of Rhinolophus (Chiroptera: Rhinolophidae) from China

A new species of the genus Rhinolophus is described from Yunnan Province, southwestern China. The new taxon belongs to the Rhinolophus "philippinensis-group" and is distinguished by differences in the nose-leaf structures, craniodental characteristics, and bacular features.     

New insight into the Nox4 subcellular localization in HEK293 cells: first monoclonal antibodies against Nox4

Nox4, a member of Nox family of NADPH oxidase expressed in nonphagocytic cells, is a major source of reactive oxygen species in many cell types. But understanding of the role of Nox4 in the production of ROS and of regulation mechanism of oxidase activity is largely unknown. This study reports for the first time the generation and characterization of 5 mAbs against a recombinant Nox4 protein (AA: 206-578). Among 5 novel mAbs, 3 mAbs (8E9, 5F9, 6B11) specifically recognized Nox4 protein in HEK293 transfected cells or human kidney cortex by western blot analysis; mAb 8E9 reacted with intact tet-induced T-REx™ Nox4 cells in FACS studies. The other 2 mAbs 10B4 and 7C9 were shown to have a very weak reactivity after purification. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4, while mAbs 6B11 and 5F9 are directed to its cytosolic tail. Contrary to mAb 6B11, mAb 5F9 failed to detect Nox4 at the plasma membrane. Cell-free oxidase assays demonstrated a moderate but significant inhibition of constitutive Nox4 activity by mAbs 5F9 and 6B11. In conclusion, 5 mAbs raised against Nox4 were generated for the first time. 3 of them will provide powerful tools for a structure/function relationship of Nox4 and for physiopathological investigations in humans.     

shRNA targeting SFRP2 promotes the apoptosis of hypertrophic scar fibroblast

Hypertrophic scars result from a dysregulated process in wound healing. Although the basic mechanism is unclear, increased proliferation and decreased cell apoptosis are noticed in the development of hypertrophic scar. In previous study, we found that secreted frizzled-related protein 2 (SFRP2), which was associated with cell proliferation, apoptosis, and differentiation, was dramatically upregulated in hypertrophic scar (HS) tissue. In this study short hairpin RNA (shRNA) targeting SFRP2 was employed to characterize SFRP2 function in hypertrophic scar-derived fibroblasts (HSFb). Cell proliferation was assessed by MTT, dynamic growth curves, and BRDU assays. Meanwhile, Cell apoptosis was detected using fluorescence-activated cell sorting (FACS). Caspase-3 activity was assayed by spectrophotometry. Fibroblast populated collagen lattice (FPCL) model was employed to evaluate the contractility of HSFb. Further, real-time PCR and western blot were used to measure the mRNA and protein expressions of α-SMA in HSFb. In addition, mRNA levels of type I and III procollagen were assayed by quantitative real-time PCR. The results revealed that shRNA targeting SFRP2 significantly promoted the apoptosis of HSFb, while it had no effect on the cell proliferation. Decreased synthesis of a-smooth muscle actin (α-SMA) in HSFb and reduced contraction of fibroblasts in the FPCL model were observed. Quantitative RT-PCR suggested that the mRNAs of type I and III procollagen were significantly downregulated. In conclusion, as a novel anti-apoptosis gene, SPRP2 was present in hypertrophic scars. Importantly, shRNA targeting SFRP2 may provide a new approach to preventing the formation of HS.     

Stereoselective pharmacokinetics of stable isotope (+/−)‐[13C]‐pantoprazole: Implications for a rapid screening phenotype test of CYP2C19 activity

Aims: We have previously shown that the (±)‐[¹³C]‐pantoprazole breath test is a promising noninvasive probe of CYP2C19 activity. As part of that trial, plasma, breath test indices and CYP2C19 (*2, *3, and *17) genotype were collected. Here, we examined whether [¹³C]‐pantoprazole exhibits enantioselective pharmacokinetics and whether this enantioselectivity is correlated with indices of breath test. Methods: Plasma (−)‐ and (+)‐[¹³C]‐pantoprazole that were measured using a chiral HPLC were compared between CYP2C19 genotypes and correlated with breath test indices. Results: The AUC_(0‐∞) of (+)‐[¹³C]‐pantoprazole in PM (*2/*2, n = 4) was 10.1‐ and 5.6‐fold higher that EM (*1/*1or *17, n = 10) and IM (*1/*2or *3, n = 10) of CYP2C19, respectively (P < 0.001). The AUC_(0‐∞) of (−)‐[¹³C]‐pantoprazole only significantly differed between PMs and EMs (1.98‐fold; P = 0.05). The AUC_(0‐∞) ratio of (+)‐/(−)‐[¹³C]‐pantoprazole was 3.45, 0.77, and 0.67 in PM, IM, and EM genotypes, respectively. Breath test index, delta over baseline show significant correlation with AUC_(0‐∞) of (+)‐[¹³C]‐pantoprazole (Pearson's r = 0.62; P < 0.001). Conclusions: [¹³C]‐pantoprazole exhibits enantioselective elimination. (+)‐[¹³C]‐pantoprazole is more dependent on CYP2C19 metabolic status and may serve as a more attractive probe of CYP2C19 activity than (−)‐[¹³C]‐pantoprazole or the racemic mixture. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells

Mitochondrial toxicity has been a serious concern, not only in preclinical drug development but also in clinical trials. In mitochondria, there are several distinct metabolic processes including fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), and each process contains discrete but often intimately linked steps. Interruption in any one of those steps can cause mitochondrial dysfunction. Detection of inhibition to OXPHOS can be complicated in vivo because intermediate endogenous metabolites can be recycled in situ or circulated systemically for metabolism in other organs or tissues. Commonly used assays for evaluating mitochondrial function are often applied to ex vivo or in vitro samples; they include various enzymatic or protein assays, as well as functional assays such as measurement of oxygen consumption rate, membrane potential, or acidification rates. Metabolomics provides quantitative profiles of overall metabolic changes that can aid in the unraveling of explicit biochemical details of mitochondrial inhibition while providing a holistic view and heuristic understanding of cellular bioenergetics. In this paper, we showed the application of quantitative NMR metabolomics to in vitro myotube cells treated with mitochondrial toxicants, rotenone and antimycin A. The close coupling of the TCA cycle to the electron transfer chain (ETC) in OXPHOS enables specific diagnoses of inhibition to ETC complexes by discrete biochemical changes in the TCA cycle.     

Discovery of novel hepatoselective HMG-CoA reductase inhibitors for treating hypercholesterolemia: a bench-to-bedside case study on tissue selective drug distribution

The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.