Developmental regulation of skull morphology II: ontogenetic dynamics of covariance

Canalization may play a critical role in molding patterns of integration when variability is regulated by the balance between processes that generate and remove variation. Under these conditions, the interaction among those processes may produce a dynamic structure of integration even when the level of variability is constant. To determine whether the constancy of variance in skull shape throughout most of postnatal growth results from a balance between processes generating and removing variation, we compare covariance structures from age to age in two rodent species, cotton rats (Sigmodon fulviventer) and house mice (Mus musculus domesticus). We assess the overall similarity of covariance matrices by the matrix correlation, and compare the structures of covariance matrices using common subspace analysis, a method related to common principal components (PCs) analysis but suited to cases in which variation is so nearly spherical that PCs are ambiguous. We find significant differences from age to age in covariance structure and the more effectively canalized ones tend to be least stable in covariance structure. We find no evidence that canalization gradually and preferentially removes deviations arising early in development as we might expect if canalization results from compensatory differential growth. Our results suggest that (co)variation patterns are continually restructured by processes that equilibrate variance, and thus that canalization plays a critical role in molding patterns of integration.     

Protein abundance of urea transporters and aquaporin 2 change differently in nephrotic pair-fed vs. non-pair-fed rats

Salt and water retention is a hallmark of nephrotic syndrome (NS). In this study, we test for changes in the abundance of urea transporters, aquaporin 2 (AQP2), Na-K-2Cl cotransporter 2 (NKCC2), and Na-Cl cotransporter (NCC), in non-pair-fed and pair-fed nephrotic animals. Doxorubicin-injected male Sprague-Dawley rats (n = 10) were followed in metabolism cages. Urinary excretion of protein, sodium, and urea was measured periodically. Kidney inner medulla (IM), outer medulla, and cortex tissue samples were dissected and analyzed for mRNA and protein abundances. At 3 wk, all doxorubicin-treated rats developed features of NS, with a ninefold increase in urine protein excretion (from 144 ± 21 to 1,107 ± 165 mg/day; P < 0.001) and reduced urinary sodium excretion (from 0.17 to 0.12 meq/day; P < 0.001). Urine osmolalities were reduced in the nephrotic animals (1,057 ± 37, treatment vs. 1,754 ± 131, control). Unlike animals fed ad libitum, UT-A1 protein abundance was unchanged in nephrotic pair-fed rats. Glycosylated AQP2 was reduced in the IM base of both nephrotic groups. Abundances of NKCC2 and NCC were consistently reduced (71 ± 7 and 33 ± 13%, respectively) in both nephrotic pair-fed animals and animals fed ad libitum. In pair-fed nephrotic rats, we observed an increase in the cleaved form of membrane-bound γ-epithelial sodium channel (ENaC). However, α- and β-ENaC subunits were unaltered. NKCC2 and AQP2 mRNA levels were similar in treated vs. control rats. We conclude that dietary protein intake affects the response of medullary transport proteins to NS.     

Airway Epithelial Expression of TLR5 Is Downregulated in Healthy Smokers and Smokers with Chronic Obstructive Pulmonary Disease

The TLRs are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. On the basis of the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th to 12th order) epithelium from healthy nonsmokers (n = 60), healthy smokers (n = 73), and smokers with COPD (n = 36). Using the criteria of detection call of present (P call) ≥50%, 6 of 10 TLRs (TLRs 1-5 and 8) were expressed. Compared with nonsmokers, the most striking change was for TLR5, which was downregulated in healthy smokers (1.4-fold, p < 10(-10)) and smokers with COPD (1.6-fold, p < 10(-11)). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, was highly induced by flagellin in TLR5 high-expressing cells compared with TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced downregulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.     

N-Acyl and N-sulfonyloxazolidine-2,4-diones are pseudo-irreversible inhibitors of serine proteases

The synthesis, inhibitory activity and mode of action of oxazolidine-2,4-diones against porcine pancreatic elastase, here used as a model for human neutrophil elastase, are reported. The nature of N-substitution at the oxazolidine-2,4-dione scaffold has large effect on the inhibitory potency against elastase. N-Acyl and N-sulfonyloxazolidine-2,4-diones emerged as potent pseudo-irreversible inhibitors, displaying high second-order rate constants for PPE inactivation. The title compounds were also shown to be potent inhibitors of human neutrophil elastase (HNE) and proteinase-3, and weak inhibitors of human cathepsin G. The results herein presented show that the oxazolidine-2,4-diones represent a new promising class of serine protease inhibitors.     

Silencing of miR-370 in Human Cholangiocarcinoma by Allelic Loss and Interleukin-6 Induced Maternal to Paternal Epigenotype Switch

Cholangiocarcinoma (CCA) is a highly lethal malignant tumor arising from the biliary tract epithelium. Interleukin-6 (IL-6) is a major mediator of inflammation and contributor to carcinogenesis within the biliary tree. Previous studies suggested that enforced IL-6 contributes to cholangiocarcinogenesis through hypermethylation of several genes implicated in CCA. However, the precise mechanisms of IL-6 effects in CCA remain unclear. We now demonstrate that microRNA (miR)-370 is underexpressed in a large cohort of human CCA vs. normal liver tissues. In addition, we show that IL-6 induces a time-dependent silencing of miR-370. In addition, demethylation of CCA cells results in upregulation of miR-370. Furthermore, we demonstrate that miR-370 is imprinted, and that the Intergenic Differentially Methylated Region (IG-DMR) responsible for imprinting regulation of this genomic locus is hypermethylated in response to IL-6 treatment. In addition, the IG-DMR is hypermethylated in human CCA specimens compared to normal matched controls, in the same location as the IL-6 induced hypermethylation. Finally, miR-370 was found to regulate WNT10B in luciferase as well as western blotting experiments. Our data indicate that the paternal allele of miR-370 is normally silenced through genomic imprinting and that the overexpression of IL-6 in CCA effectively suppresses the expression of miR-370 from the maternal allele, lending support to the theory that miR-370 silencing in human CCA follows a classic two-hit mechanism.     

Four loci explain 83% of size variation in the horse

Horse body size varies greatly due to intense selection within each breed. American Miniatures are less than one meter tall at the withers while Shires and Percherons can exceed two meters. The genetic basis for this variation is not known. We hypothesize that the breed population structure of the horse should simplify efforts to identify genes controlling size. In support of this, here we show with genome-wide association scans (GWAS) that genetic variation at just four loci can explain the great majority of horse size variation. Unlike humans, which are naturally reproducing and possess many genetic variants with weak effects on size, we show that horses, like other domestic mammals, carry just a small number of size loci with alleles of large effect. Furthermore, three of our horse size loci contain the LCORL, HMGA2 and ZFAT genes that have previously been found to control human height. The LCORL/NCAPG locus is also implicated in cattle growth and HMGA2 is associated with dog size. Extreme size diversification is a hallmark of domestication. Our results in the horse, complemented by the prior work in cattle and dog, serve to pinpoint those very few genes that have played major roles in the rapid evolution of size during domestication.     

Novel role of NADPH oxidase in ischemic myocardium: a study with Nox2 knockout mice

Several potential sources of reactive oxygen species (ROS) in cells exist. One source is NADPH oxidase, which is especially important for superoxide radical production. Nox2 is a primary regulatory subunit of NADPH oxidase. In the present study, we examined the role of ROS and NADPH oxidase in ischemic preconditioning (IP)-mediated cardioprotection by using Nox2(-/-) mice. Both wild-type (WT) and Nox2(-/-) mice were subjected to either 30?min of ischemia followed by 2?h of reperfusion (IR) or IP prior to 30?min ischemia and 2?h of reperfusion. Reduction in left ventricular developed pressure (60.1 versus 63?mmHg), dp/dt (max) (893 versus 1,027?mmHg/s), and aortic flow (0.9 versus 1.8?ml/min) was observed in Nox2(-/-)IPIR compared to WTIPIR along with increased infarct size (33% versus 22%) and apoptosis after 120?min of reperfusion. Differentially regulated genes were demonstrated by comparing gene expression in WTIPIR versus Nox2(-/-) IPIR hearts. Selected differentially regulated genes such as β-catenin, SRPK3, ERDR1, ACIN1, Syntaxin-8, and STC1 were validated by real-time PCR. Taken together, this is the first report identifying important, differentially expressed genes during ischemic preconditioning in Nox2(-/-) mice by using microarray analysis.     

Assembly with the Cul4A-DDB1DCAF1 ubiquitin ligase protects HIV-1 Vpr from proteasomal degradation

Many viruses subvert the host ubiquitin-proteasome system to optimize their life cycle. We recently documented such a mechanism for the human immunodeficiency virus type 1 Vpr protein, which promotes cell cycle arrest by recruiting the DCAF1 adaptor of the Cul4A-DDB1 ubiquitin ligase, a finding now confirmed by several groups. Here we examined the impact of Cul4A-DDB1(DCAF1) on Vpr stability. We show that the Vpr(Q65R) mutant, which is defective in DCAF1 binding, undergoes proteasome-mediated degradation at a higher rate than wild-type Vpr. DCAF1 overexpression stabilizes wild-type Vpr and leads to its cytoplasmic accumulation, whereas it has no effect on the Vpr(Q65R) mutant. Conversely, small interfering RNA-mediated silencing of DCAF1 decreases the steady state amount of the viral protein. Stabilization by DCAF1, which is conserved by Vpr species from human immunodeficiency virus type 2 and the SIVmac strain, results in increased G(2) arrest and requires the presence of DDB1, indicating that it occurs through assembly of Vpr with a functional Cul4A-DDB1(DCAF1) complex. Furthermore, in human immunodeficiency virus type 1-infected cells, the Vpr protein, issued from the incoming viral particle, is destabilized under DCAF1 or DDB1 silencing. Together with our previous findings, our data suggest that Cul4A-DDB1(DCAF1) acts at a dual level by providing Vpr with the equipment for the degradation of specific host proteins and by counter-acting its proteasome targeting by another cellular E3 ubiquitin ligase. This protection mechanism may represent an efficient way to optimize the activity of Vpr molecules that are delivered by the incoming virus before neosynthesis takes place. Targeting the Vpr-DCAF1 interaction might therefore present therapeutic interest.     

Bromelain induces cardioprotection against ischemia-reperfusion injury through Akt/FOXO pathway in rat myocardium

Bromelain (Br), a proteolytic enzyme extracted from the stem of the pineapple, is known to possess anti-inflammatory activity and has been shown to reduce blood viscosity, prevent the aggregation of blood platelets, and improve ischemia-reperfusion (I/R) injury in a skeletal muscle model. We investigated the capacity of Br to limit myocardial injury in a global I/R model. Adult male Sprague-Dawley rats were divided into two groups: control (PBS) and Br at 10 mg/kg in PBS administered via intraperitoneal injection (twice/day) for 15 consecutive days. On day 16, the hearts were excised and subjected to 30 min of global ischemia followed by 2 h of reperfusion. Br treatment showed higher left ventricular functional recovery throughout reperfusion compared with the controls [maximum rate of rise in intraventricular pressure (dP/dt max), 2,225 vs. 1,578 mmHg/s at 2 h reperfusion]. Aortic flow was also found to be increased in Br treatment when compared with that in untreated rats (11 vs. 1 ml). Furthermore, Br treatment reduced both the infarct size (34% vs. 43%) and the degree of apoptosis (28% vs. 37%) compared with the control animals. Western blot analysis showed an increased phosphorylation of both Akt and FOXO3A in the treatment group compared with the control. These results demonstrated for the first time that Br triggers an Akt-dependent survival pathway in the heart, revealing a novel mechanism of cardioprotective action and a potential therapeutic target against I/R injury.     

Cardioprotection with palm oil tocotrienols: comparision of different isomers

A recent study from our laboratory indicated the cardioprotective ability of the tocotrienol-rich fraction (TRF) from red palm oil. The present study compared cardioprotective abilities of different isomers of tocotrienol against TRF as recently tocotrienol has been found to function as a potent neuroprotective agent against stroke. Rats were randomly assigned to one of the following groups: animals were given, by gavage, either 0.35%, 1%, or 3.5% TRF for two different periods of time (2 or 4 wk) or 0.03, 0.3, and 3 mg/kg body wt of one of the isomers of tocotrienol (alpha, gamma, or delta) for 4 wk; control animals were given, by gavage, vehicle only. After 2 or 4 wk, rats were killed, and their hearts were then subjected to 30 min of global ischemia followed by 2 h of reperfusion. Dose-response and time-response experiments revealed that the optimal concentration for TRF was 3.5% TRF and 0.3 mg/kg body wt of tocotrienol given for 4 wk. TRF as well as all the isomers of tocotrienol used in our study provided cardioprotection, as evidenced by their ability to improve postischemic ventricular function and reduce myocardial infarct size. The gamma-isoform of tocotrienol was the most cardioprotective of all the isomers followed by the alpha- and delta-isoforms. The molecular mechanisms of cardioprotection afforded by tocotrienol isoforms were probed by evaluating their respective abilities to stabilize the proteasome, allowing it to maintain a balance between prodeath and prosurvival signals. Our results demonstrated that tocotrienol isoforms reduced c-Src but increased the phosphorylation of Akt, thus generating a survival signal.