Risky ripples allow bats and frogs to eavesdrop on a multisensory sexual display.

Animal displays are often perceived by intended and unintended receivers in more than one sensory system. In addition, cues that are an incidental consequence of signal production can also be perceived by different receivers, even when the receivers use different sensory systems to perceive them. Here we show that the vocal responses of male túngara frogs (Physalaemus pustulosus) increase twofold when call-induced water ripples are added to the acoustic component of a rival's call. Hunting bats (Trachops cirrhosus) can echolocate this signal by-product and prefer to attack model frogs when ripples are added to the acoustic component of the call. This study illustrates how the perception of a signal by-product by intended and unintended receivers through different sensory systems generates both costs and benefits for the signaler.

Rodlet cells in Murray cod, Maccullochella peelii peelii (Mitchell), affected with chronic ulcerative dermatopathy.

We have previously identified an unknown cell type in the gills of Murray cod affected with chronic ulcerative dermatopathy (CUD), a condition that causes severe erosion of epidermis surrounding cephalic and lateral line sensory canals. The condition arises in aquaculture facilities that utilize groundwater, with the cause of the condition suggested to be an unknown contaminant(s). Light and transmission electron microscopy were used to characterize and quantify the unknown cells in CUD-affected Murray cod. The cells were identified as rodlet cells and were characterized by their oval or round shape, basally located nucleus, thick fibrillar capsule surrounding the cell, and multiple rodlet sacs containing a central electron-dense core within the cell. Rodlet cells were present in the gills, kidney and intestine of non-CUD-affected and CUD-affected Murray cod; however, differences in the numbers were observed between the groups of fish. A significantly greater number of rodlet cells were observed in the gills and collecting ducts of CUD-affected fish. This is the first report of rodlet cells in Murray cod, and we suggest that the increased rodlet cell numbers in CUD-affected Murray cod may be in response to unknown water contaminant(s) present in the groundwater that give rise to CUD.

Biennial reproductive cycle in an extensive matrotrophic viviparous batoid: the sandyback stingaree Urolophus bucculentus from south-eastern Australia.

Urolophus bucculentus, the largest urolophid species found in southern Australia, exhibits a biennial reproductive cycle. Ovulation occurs during October to January followed by a 15-19 month period of gestation followed by parturition during April to May and a short rest period while the ovarian follicles continue to develop for subsequent ovulation. Male breeding condition peaks during April to June to coincide with the period of parturition. Urolophus bucculentus has the highest matrotrophic contribution reported for any urolophid species, with a mean wet mass gain from egg in utero (4 g) to full-term embryo in utero (250 g) of c. 6250% (maximum c. 7200%), and perhaps explains the biennial female reproductive cycle where 50% of females contribute to each year's recruitment. Litter size (one to five) increases with total length (L(T) ). Females reach a longer maximum L(T) (L(Tmax) ) than do males (885 v. 660 mm). The L(T) at maturity for males and females at 50% mature (L(T50) ) is c. 414 mm (63% of L(Tmax) ) for males and c. 502 mm (57% of L(Tmax) ) for females, length at maternity indicates that recruitment production occurs later in life at c. 632 mm L(T) (71% of L(Tmax) ).

LC-PUFA biosynthesis in rainbow trout is substrate limited: use of the whole body fatty acid balance method and different 18:3n-3/18:2n-6 ratios.

Five experimental diets with constant total C(18) PUFA and varying 18:3n-3/18:2n-6 ratios were fed to rainbow trout over an entire production cycle. The whole-body fatty acid balance method demonstrated a clear trend of progressively reduced fatty acid bioconversion activity along the n-3 and n-6 pathways, up to the production of 20:5n-3 and 20:4n-6, respectively. This suggests that the pathway exhibits a "funnel like" progression of activity rather than the existence of a single rate limiting step. The production of 22:5n-3 and 22:6n-3 was more active than that of 20:5n-3. However, despite this trend in reduced apparent in vivo net enzyme activity, the efficiency of the various bioconversion steps (measured as % of bioconverted substrate) confirmed an opposing trend. A 3.2-fold higher Δ-6 desaturase affinity towards 18:3n-3 over 18:2n-6 and an 8-fold greater Δ-5 desaturase affinity towards 20:4n-3 over 20:3n-6 were recorded. The main results of the study were that (1) rainbow trout are quite efficient at bioconverting 18:3n-3 to 22:6n-3, and (2) the LC-PUFA biosynthetic pathway is substrate limited. Fillet n-3 LC-PUFA concentrations increased with the increasing dietary supply of 18:3n-3. Despite an almost identical dietary supply of n-3 LC-PUFA, originating from the fish meal fraction of the diets, the fillets of trout fed the diet richest in 18:3n-3 were 2-fold higher in n-3 LC-PUFA than fish fed low 18:3n-3 diets. Nevertheless, fillets of trout fed a fish oil control diet contained more than double the amount of n-3 LC-PUFA compared to fish fed the diets richest in 18:3n-3.

Therapeutic potential of RhoA/Rho kinase inhibitors in pulmonary hypertension.

A burgeoning body of evidence suggests that RhoA/Rho kinase (ROCK) signalling plays an important role in the pathogenesis of various experimental models of pulmonary hypertension (PH), including chronic hypoxia-, monocrotaline-, bleomycin-, shunt- and vascular endothelial growth factor receptor inhibition plus chronic hypoxia-induced PH. ROCK has been incriminated in pathophysiologic events ranging from mediation of sustained abnormal vasoconstriction to promotion of vascular inflammation and remodelling. In addition, the 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, statins, which inhibit activation of RhoA by preventing post-translational isoprenylation of the protein and its translocation to the plasma membrane ameliorate PH in several different rat models, and may also be effective in PH patients. Also, phosphorylation of RhoA and prevention of its translocation to the plasma membrane are involved in the protective effect of the type 5-PDE inhibitor, sildenafil, against hypoxia- and bleomycin-induced PH. Collectively, these and other observations indicate that independent of the cause of PH, activation of the RhoA/ROCK pathway serves as a point of convergence of various signalling cascades in the pathogenesis of the disease. We propose that ROCK inhibitors and other drugs that inhibit this pathway might be useful in the treatment of various forms of PH.

The Cardiff paediatric laryngoscope blade: a comparison with the Miller size 1 and Macintosh size 2 laryngoscope blades.

The Cardiff paediatric laryngoscope blade is a single blade that has been designed for use in children from birth to adolescence. This open, randomised, crossover study compared the Cardiff blade with the straight, size 1, Miller laryngoscope blade in 39 infants under 1 years of age and the curved, size 2, Macintosh blade in 39 children aged 1-16 years. The same laryngoscopic view was obtained with the Cardiff and Miller blades in 26 patients; the view was better with the Cardiff blade in seven patients and better with the Miller blade in six (median (IQR [range]) grade of laryngoscopy 1 (1-2 [1-3]) vs. 1 (1-2 [1-3]), respectively; p = 0.405). The Cardiff blade was faster at gaining a view than the Miller blade (mean (SD) time 8.5 (2.9) s vs. 10.2 (3.5) s, respectively; 95% CI for difference -2.8 to -0.4; p = 0.009). The Cardiff and Macintosh blades produced the same view in 32 patients; the view was better with the Cardiff blade in seven patients (median (IQR [range]) grade of laryngoscopy 1 (1-1 [1-3]) vs. 1 (1-2 [1-3]), respectively; p = 0.008). There was no difference in time to gain these views: mean (SD) 8.7 (3.0) s vs. 9.3 (2.7) s, respectively (95% CI for difference -1.58 to 0.40; p = 0.237). The Cardiff paediatric laryngoscope blade compares favourably with these two established laryngoscope blades in children.

DNA repair in a chromatin environment.

DNA mutations and aberrations are a problem for all forms of life. Eukaryotes specifically have developed ways of identifying and repairing various DNA mutations in a complex and refractory chromatin environment. The chromatin structure is much more than a packaging unit for DNA; it is dynamic. Cells utilize and manipulate chromatin for gene regulation, genome organization and maintenance of genome integrity. Once a DNA aberration has occurred, the various DNA repair machineries interact with chromatin proteins, such as the histone variant H2A.X, and chromatin remodeling machines of the SWI/SNF family to gain access and repair the lesion in a timely manner. Recent studies have thus begun to address the roles of chromatin proteins in DNA repair as well as to dissect the functions of DNA repair machinery in vitro on more physiological, nucleosomal templates.

DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection.

We previously demonstrated that DNA-polylactic-polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actin-related mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (beta-galactosidase) transfection than SMC grown on native collagen (18.3+/-1.2 vs 1.0+/-0.1%, P<0.001). The denatured-collagen effect was completely blocked with anti-alpha(v)beta(3) integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by alpha(v)beta(3) integrins, showed a 33-fold increase in transfection compared to control (P<0.001); this effect was also blocked with anti-alpha(v)beta(3) antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actin-enriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.0+/-1.8 vs 3.02+/-0.9%, P<0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen-PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (P<0.001) in SMC cultures; the increased transfection was blocked by anti-alpha(v)beta(3) antibody. Pig coronary studies comparing denatured-collagen-PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.

N-CoR-HDAC corepressor complexes: roles in transcriptional regulation by nuclear hormone receptors.

Many nuclear hormone receptors (NHRs) actively repress the expression of their primary response genes through the recruitment of transcriptional corepressor complexes to regulated promoters. N-CoR and the highly related SMRT were originally isolated and characterized by their ability to interact exclusivelywith the unliganded forms of NHRs and confer transcriptional repression. Recently, both the N-CoR and SMRT corepressors have been found to exist in vivo in multiple, distinct macromolecular complexes. While these corepressor complexes differ in overall composition, a general theme is that they contain histone deacetylase enzymatic activity. Several of these complexes contain additional transcriptional corepressor proteins with functional ties to chromatin structure. Together, these data suggest that modulation of chromatin structure plays a central role in N-CoR mediated transcriptional repression from unliganded NHRs.

A case of fatal tumor embolus following trauma in a patient with undiagnosed Wilms' tumor.

Involvement of the inferior vena cava with tumor thrombus has been reported in 5 to 10% of patients with Wilms' tumor. Preoperative imaging usually alerts the surgeon to the extent of the intravascular extension. We present a case report of trauma to a previously undiagnosed Wilms' tumor that resulted in a fatal intraoperative pulmonary tumor embolus.

Prx1 controls vascular smooth muscle cell proliferation and tenascin-C expression and is upregulated with Prx2 in pulmonary vascular disease.

Prx1 and Prx2 are homeobox transcription factors expressed during vasculogenesis. To begin to elucidate how Prx1 and Prx2 are regulated and function in the adult vasculature, in situ hybridization studies were performed. Prx1 and Prx2 mRNAs were not detected in normal adult rat pulmonary arteries; however, both genes were induced with vascular disease, colocalizing to sites of tenascin-C (TN-C) expression. Because catabolism of the extracellular matrix (ECM) is a critical step in the development of vascular disease, we investigated whether changes in vascular smooth muscle cell (SMC)-ECM interactions regulate Prx1 and Prx2. A10 SMCs cultured on native type I collagen showed low levels of Prx1 and Prx2 mRNA expression, whereas cells cultured on denatured collagen showed higher levels of expression of both genes. At a functional level, transfection of SMCs with a Prx1 expression plasmid significantly increased their growth. Because TN-C also promotes SMC growth and its expression is also upregulated by denatured collagen, we tested and thereafter showed that Prx1 expression significantly enhances TN-C gene promoter activity 20-fold. Similar experiments conducted with truncated Prx1 proteins showed that the N-terminal portion and the homeodomain of Prx1 were necessary to induce the bulk of TN-C promoter activity. These findings support the hypothesis that Prx genes are regulated by changes in SMC adhesion and play key morphoregulatory roles during the development and progression of pulmonary vascular disease in adults.

Matrix metalloproteinase-2 is associated with tenascin-C in calcific aortic stenosis.

We previously showed that the expression of tenascin (TN-C), an extracellular matrix glycoprotein found in developing bone and atherosclerotic plaque, and matrix metalloproteinase-2 (MMP-2) are coordinated and interdependent in cultured vascular smooth muscle cells. In this study, we hypothesized that TN-C and MMP-2 are mechanistically involved in the pathobiology of calcific aortic stenosis. Human calcific aortic stenosis cusps demonstrated immunohistochemically prominent deposition of TN-C, MMP-2, and alkaline phosphatase activity, as well as MMP-2 gelatinolytic activity. Although far lesser amounts of TN-C were noted in several of the grossly non-calcified valve cusps, MMP-2 and AP were never detected. Further, when aortic valve interstitial cells (both sheep and human) were cultivated on collagen supplemented with TN-C, both MMP-2 mRNA expression and MMP-2 gelatinolytic activity (both pro and active forms), were up-regulated compared to control. These observations support the view that accumulation of first TN-C and then MMP-2 are associated with progression of calcification. The residual presence of these proteins in severe calcifications is indicative of their involvement in the pathogenesis.

Multiple N-CoR complexes contain distinct histone deacetylases.

N-CoR (nuclear receptor corepressor) is a corepressor for multiple transcription factors including unliganded thyroid hormone receptors (TRs). In vitro, N-CoR can interact with the Sin3 corepressor, which in turn binds to the histone deacetylase Rpd3 (HDAC1), predicting the existence of a corepressor complex containing N-CoR, Sin3, and histone deacetylase. However, previous biochemical studies of endogenous Sin3 complexes have failed to find an N-CoR association. Xenopus laevis eggs and oocytes contain all of the necessary components for transcriptional repression by unliganded TRs. In this study, we report the biochemical fractionation of three novel macromolecular complexes containing N-CoR, two of which possess histone deacetylase activity, from Xenopus egg extract. One complex contains Sin3, Rpd3, and RbAp48; the second complex contains a Sin3-independent histone deacetylase; and the third complex lacks histone deacetylase activity. This study describes the first biochemical isolation of endogenous N-CoR-containing HDAC complexes and illustrates that N-CoR associates with distinct histone deacetylases that are both dependent and independent of Sin3. Immunoprecipitation studies show that N-CoR binds to unliganded TR expressed in the frog oocyte, confirming that N-CoR complexes are involved in repression by unliganded TR. These results suggest that N-CoR targets transcriptional repression of specific promoters through at least two distinct histone deacetylase pathways.

Programming the transcriptional state of replicating methylated dna.

CpG methylation is maintained in daughter chromatids by the action of DNA methyltransferase at the replication fork. An opportunity exists for transcription factors at replication forks to bind their cognate sequences and thereby prevent remethylation by DNA methyltransferase. To test this hypothesis, we injected a linearized, methylated, and partially single-stranded reporter plasmid into the nuclei of Xenopus oocytes and followed changes in the transcriptional activity after DNA replication. We find that dependent on Gal4-VP16, the action of DNA methyltransferase, and replication-coupled chromatin assembly DNA replication provides a window of time in which regulatory factors can activate or repress gene activity. Demethylation in the promoter region near the GAL4 binding sites of the newly synthesized DNA did not occur even though the Gal4 binding sites were occupied and transcription was activated. We conclude that "passive" demethylation at the replication fork is not simply dependent on the presence of DNA binding transcriptional activators.

Purification of the MeCP2/histone deacetylase complex from Xenopus laevis.

DNA methylation has long been associated with stable transcriptional silencing and a repressive chromatin structure (reviewed in refs. 1,2). Differential methylation is associated with imprinting, carcinogenesis, silencing of repetitive DNA, and allows for differentiating cells to efficiently shut off unnecessary genes. In vertebrates, where 60-90% of genomic CpG dinucleotides are methylated, methylation-dependent repression is vital for proper embryonic development (3). Microinjection experiments using methylated DNA templates implicate chromatin structure as an underlying mechanism of methylation-dependent silencing (4,5). Methyl-specific transcriptional repression requires chromatin assembly, and can be partially relieved by the histone deacetylase inhibitor Trichostatin A. In addition, several proteins have been identified that specifically bind to methylated DNA (6-8). Two of these methyl-DNA binding proteins, MeCP1 and MeCP2, have been shown to mediate transcriptional repression (6,7). MeCP1 is a relatively uncharacterized complex that requires at least 12 symmetrical methyl-CpGs for DNA binding (6). MeCP2 is a single polypeptide containing a methyl-binding domain capable of binding a single methyl-CpG, and a transcriptional repression domain (9). Recently MeCP2 was shown to interact with the Sin3 corepressor and histone deacetylase (10,11). Changes in the acetylation state of the core histone tails correlates with changes in transcription (reviewed in refs. 12,13), and several transcriptional repression complexes containing histone deacetylases have recently been described (10,14,15).

Tenascin-C in development and disease: gene regulation and cell function.

Tenascin-C (TN-C) is a modular and multifunctional extracellular matrix (ECM) glycoprotein that is exquisitely regulated during embryonic development and in adult tissue remodeling. TN-C gene transcription is controlled by intracellular signals that are generated by multiple soluble factors, integrins and mechanical forces. These external cues are interpreted by particular DNA control elements that interact with different classes of transcription factors to activate or repress TN-C expression in a cell type- and differentiation-dependent fashion. Among the transcriptional regulators of the TN-C gene that have been identified, the homeobox family of proteins has emerged as a major player. Downstream from TN-C, intracellular signals that are relayed via specific cell surface receptors often impart contrary cellular functions, even within the same cell type. A key to understanding this behavior may lie in the dual ability of TN-C-enriched extracellular matrices to generate intracellular signals, and to define unique cellular morphologies that modulate these signal transduction pathways. Thus, despite the contention that TN-C null mice appear to develop and act normally, TN-C biology continues to provide a wealth of information regarding the complex nature of the ECM in development and disease.