Impulsive choices in mice lacking imprinted Nesp55.

Genomic imprinting is the process whereby germline epigenetic events lead to parent-of-origin specific monallelic expression of a number of key mammalian genes. The imprinted gene Nesp is expressed from the maternal allele only and encodes for Nesp55 protein. In the brain, Nesp55 is found predominately in discrete areas of the hypothalamus and midbrain. Previously, we have shown that loss of Nesp55 gives rise to alterations in novelty-related behaviour. Here, we extend these findings and demonstrate, using the Nespm/+ mouse model, that loss of Nesp55 leads to impulsive choices as measured by a delayed-reinforcement task, whereby Nespm/+ mice were less willing to wait for a delayed, larger reward, preferring instead to choose an immediate, smaller reward. These effects were highly specific as performance in another component of impulsive behaviour, the ability to stop a response once started as assayed in the stop-signal reaction time task, was equivalent to controls. We also showed changes in the serotonin system, a key neurotransmitter pathway mediating impulsive behaviour. First, we demonstrated that Nesp55 is co-localized with serotonin and then went on to show that in midbrain regions there were reductions in mRNA expression of the serotonin-specific genes Tph2 and Slc6a4, but not the dopamine-specific gene Th in Nespm/+ mice; suggesting an altered serotonergic system could contribute, in part, to the changes in impulsive behaviour. These data provide a novel mode of action for genomic imprinting in the brain and may have implications for pathological conditions characterized by maladaptive response control.

Urinary biomarkers in the early diagnosis of acute kidney injury.

A change in the serum creatinine is not sensitive for an early diagnosis of acute kidney injury. We evaluated urinary levels of matrix metalloproteinase-9 (MMP-9), N-acetyl-beta-D-glucosaminidase (NAG), and kidney injury molecule-1 (KIM-1) as biomarkers for the detection of acute kidney injury. Urine samples were collected from 44 patients with various acute and chronic kidney diseases, and from 30 normal subjects in a cross-sectional study. A case-control study of children undergoing cardio-pulmonary bypass surgery included urine specimens from each of 20 patients without and with acute kidney injury. Injury was defined as a greater than 50% increase in the serum creatinine within the first 48 h after surgery. The biomarkers were normalized to the urinary creatinine concentration at 12, 24, and 36 h after surgery with the areas under the receiver-operating characteristic curve compared for performance. In the cross-sectional study, the area under the curve for MMP-9 was least sensitive followed by KIM-1 and NAG. Combining all three biomarkers achieved a perfect score diagnosing acute kidney injury. In the case-control study, KIM-1 was better than NAG at all time points, but combining both was no better than KIM-1 alone. Urinary MMP-9 was not a sensitive marker in the case-control study. Our results suggest that urinary biomarkers allow diagnosis of acute kidney injury earlier than a rise in serum creatinine.

Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors.

Small-molecule-regulated gene expression offers the promise of titrating the dose and duration of action of DNA-based therapies. To this end, we show that engineered zinc-finger protein transcription factors (ZFP TFs) can be coupled with a drug-inducible regulatory domain to permit small-molecule control of endogenous gene transcription. We constructed a drug-responsive ZFP TF via the fusion of a ZFP DNA-binding domain (DBD) targeting the human VEGF-A gene and an effector domain containing a truncated progesterone receptor ligand-binding domain linked to the NFkappaB p65 activation domain. Introduction of this engineered ZFP TF into human or murine cells allowed expression of the chromosomal VEGF-A gene to be induced upon addition of mifepristone, a synthetic steroid analog. Mifepristone-dependent VEGF-A induction was rapid, dose-dependent and reversible. Moreover, stable lines expressing the drug-responsive ZFP TF could be maintained in a state of continuous induction for at least 30 days without loss of viability. Potent VEGF-A induction was demonstrated using different engineered ZFP DBDs, thus this approach may represent a general solution to small-molecule regulation of targeted endogenous genes.

Echocardiographic characterization of fundamental mechanisms of abnormal diastolic filling in diabetic rats with a parameterized diastolic filling formalism.

Abnormalities of diastolic function (DF) precede systolic dysfunction in diabetic cardiomyopathy. Transmitral Doppler flow analysis is the primary method for noninvasively assessing DF. We used model-based Doppler E-wave analysis to evaluate diastolic function differences between normal and diabetic rat hearts. Control rats and those with diabetes underwent echocardiography with analysis by traditional Doppler indexes and by the parameterized diastolic filling (PDF) formalism, generating 3 parameters, x0, c, and k, that uniquely characterize each E-wave. Significant intergroup differences in the E/A ratios (P <.01), isovolumic relaxation times (P <.01), and the modeling parameter c (P <.05) were found. There were no significant differences in shortening fraction, deceleration time, myocardial collagen content, or the parameters x0 and k between diabetic and control rats. These results indicate that differences in diastolic function may be noninvasively quantified and that diabetic hearts may exhibit defects in uncoupling of the contractile apparatus without concomitant increases in chamber stiffness.

High-frequency ultrasound for quantitative characterization of myocardial edema.

Myocardial edema has been associated with impaired ventricular compliance and diastolic filling. To determine the sensitivity of high-frequency (40 MHz) ultrasound to myocardial edema, we employed a model in which myocardial edema was induced by immersion of tissue in isotonic saline. The effect of freezing tissue on edema formation was also evaluated. Rat hearts were arrested at end-diastole and insonified fresh within 15 min of excision (n = 5) or following being frozen for 24 h and thawed (n = 4). Measurements of attenuation, backscatter, tissue thickness and speed of sound were performed at baseline and hourly for 4 h, and compared with direct measurements of myocardial edema. Fresh tissue demonstrated a greater propensity for the development of edema than frozen tissue. Integrated backscatter increased in both tissues, whereas the magnitude and slope of attenuation decreased as edema evolved. We conclude that high-frequency ultrasound sensitively detects myocardial edema, and we propose that the extension of these methods to clinical frequencies may prove useful for monitoring and treatment of cardiac edematous disease states.

Transplant coronary artery disease in pediatric heart transplant recipients.

BACKGROUND: Transplant coronary artery disease (TxCAD) contributes to a large percentage of late morbidity and mortality among adult heart transplant recipients. Intracoronary ultrasound (ICUS) is a sensitive tool in the diagnosis of TxCAD in adult patients and has allowed analysis of factors contributing to disease development. Experience with ICUS in pediatrics, however, has been limited. By using ICUS we sought to determine the overall prevalence of TxCAD in pediatrics and to characterize factors associated with its development in this population. METHODS: Eighty-six studies were performed in 51 pediatric patients a median of 3.4 years after heart transplantation. Evaluation included angiography and ICUS in 83 and angiography alone in 3 studies. Donor and recipient characteristics were obtained. The ICUS images were analyzed for intimal thickening and compared with coronary angiograms. The presence of any intimal thickening on ICUS was considered TxCAD. An intimal index and point of maximal intimal thickening (MIT) were measured. Vessel disease was graded 0 to 4 based on these results. Four patients had evidence of vasculopathy by angiography, whereas 32 patients (63%) had evidence of intimal proliferation by ICUS. Grade 2 or greater disease was present in 19 (37%) patients. A positive correlation was found when comparing time from transplant with intimal index and MIT (p < 0.001). No other factors were found to predict the development of disease. The overall prevalence of disease was 74% in patients studied at least 5 years after transplant. Intracoronary ultrasound can be performed safely in pediatric patients. Transplant coronary artery disease is common in infants and children after heart transplantation, although its prevalence appears to be less than in adult recipients at similar time intervals. We found no factor other than time from transplant was associated with development of disease.

Reconstitution of virus-mediated expression of interferon alpha genes in human fibroblast cells by ectopic interferon regulatory factor-7.

Type I interferons constitute an important part of the innate immune response against viral infection. Unlike the expression of interferon (IFN) B gene, the expression of IFNA genes is restricted to the lymphoid cells. Both IFN regulatory factor 3 and 7 (IRF-3 and IRF-7) were suggested to play positive roles in these genes expression. However, their role in the differential expression of individual subtypes of human IFNA genes is unknown. Using various IFNA reporter constructs in transient transfection assay we found that overexpression of IRF-3 in virus infected 2FTGH cells selectively activated IFNA1 VRE, whereas IRF-7 was able to activate IFNA1, A2, and A4. The binding of recombinant IRF-7 and IRF-3 to these VREs correlated with their transcriptional activation. Nuclear proteins from infected and uninfected IRF-7 expressing 2FTGH cells formed multiple DNA-protein complexes with IFNA1 VRE, in which two unique DNA-protein complexes containing IRF-7 were detected. In 2FTGH cells, virus stimulated expression of IFNB gene but none of the IFNA genes. Reconstitution of IRF-7 synthesis in these cells resulted, upon virus infection, in the activation of seven endogenous IFNA genes in which IFNA1 predominated. These studies suggest that IRF-7 is a critical determinant for the induction of IFNA genes in infected cells.

High-frequency ultrasound detection of the temporal evolution of protein cross linking in myocardial tissue.

The progressive increase in stiffening of the myocardium associated with the aging process and abetted by comorbid conditions such as diabetes may be linked to an excessive number of collagen cross links within the myocardial extra-cellular matrix. To determine whether ultrasound can delineate changes in the physical properties of heart tissue undergoing cross linking, the authors employed a model in which increased cross linking was induced by treating rat myocardial tissue with specific chemical fixatives. Rat hearts (n=5 each group) were arrested at end-diastole, insonified (30 to 50 MHz) fresh within a few minutes of excision in a phosphate buffered solution, placed in a fixative (10% formalin or 2.5% glutaraldehyde) and insonified at 30-minute intervals thereafter for 24 hours. Ultrasonic attenuation increased in tissues cross linked with formalin (maximal change: 27.2+/-3.4 dB/cm) and glutaraldehyde (maximal change: 40.2+/-5.6 dB/cm) over a 24-hour period. The frequency dependence of the attenuation coefficient increased as a function of the extent of collagen cross links in formalin (maximal change: 0.8+/-0.3 dB/cm-MHz) and glutaraldehyde (maximal change: 0.9+/-0.6 dB/cm-MHz). This study represents the first time that the precise time course of myocardial protein cross linking in situ has been characterized by using real time monitoring, and the physiologic effect has been delineated on microscopic material properties.

Long-term therapy for pulmonary hypertension in children.

This review recounts recent advances in the understanding and treatment of the processes that cause pulmonary hypertension in infancy and childhood. New discoveries have begun to unveil connections between the basic physiological mechanisms responsible for the regulation of pulmonary vascular tone and the abnormal responses of the pulmonary vasculature in a variety of disease conditions. These discoveries raise hope for new therapeutic interventions that may improve the high mortality and morbidity of both children and adults with pulmonary vascular disease. In the meantime, treatment efforts continue to be focused on the relief of pulmonary vasoconstriction with inhaled nitric oxide and intravenous prostacyclin in the short term and oral calcium channel blockers as the mainstay of long-term therapy. Lung transplantation often remains as the only viable option for continued survival when the pulmonary vascular disease is progressive.

Patterns and potential value of cardiac troponin I elevations after pediatric cardiac operations.

BACKGROUND: Perioperative myocardial injury is a major determinant of postoperative cardiac dysfunction for congenital heart disease, but its assessment during this period is difficult. The objective of this study was to determine the suitability of using postoperative serum concentrations of cardiac troponin I (cTnI) for this purpose. METHODS: Cardiac troponin I levels were measured serially in the serum of patients undergoing uncomplicated repairs of atrial septal defect (n = 23), ventricular septal defect (n = 16) or tetralogy of Fallot (n = 16). The concentrations were correlated with intraoperative parameters (cardiopulmonary bypass time, aortic cross-clamp time, and cardiac bypass temperature), and postoperative parameters (magnitude of inotropic support, duration of intubation, and postoperative intensive care and hospital stay). RESULTS: Postoperative absolute cTnI levels were lesion specific, with a pattern of increase and decrease similar for each lesion. For the total cohort, significant correlations between postoperative cTnI levels at all times (r = 0.43 to 0.83, p < 0.05) until 72 hours were noted for all parameters, except for cardiac bypass temperature. When evaluated as individual procedure groups, no significant relationships were noted in the atrial septal defect group, whereas postoperative cTnI levels were more strongly correlated with all intraoperative and postoperative parameters in the ventricular septal defect group than in the tetralogy of Fallot group. CONCLUSIONS: This study suggests that cTnI values immediately after operation reflect the extent of myocardial damage from both incisional injury and intraoperative factors. Cardiac tropinin I levels in the first hours after operation for congenital heart disease are a potentially useful prognostic indicator for difficulty of recovery.

A regulatory domain within the virus-response element of the interferon alpha 1 gene acts as a transcriptional repressor sequence and determinant of cell-specific gene expression.

Type-I interferons are encoded by a multigene family, the major members of which are at least 13 IFN A subtypes and a single IFN B gene. IFNs A and B are induced in response to similar stimuli, such as virus infection and double-stranded RNA, but in different cell types: the induction of IFN A is almost exclusively restricted to cells of lymphoid origin, while IFN B has been found to be induced in a variety of cell types including fibroblasts. The virus-responsive enhancer element in the promoter region of IFN A family members is largely responsible for the differential expression of individual subtypes in responsive cells. In this paper we describe experiments which address the issue of the differential expression of IFN A and IFN B in different cell types. We show that IFN-beta is induced in a variety of cells of different origin, while not all of these are able to secrete IFN-alpha. By transfection of reporter gene constructs comprising the virus-responsive enhancer from the IFN A1 and IFN B genes, we show that this differential response is mediated at the level of transcription via these control elements. More detailed analysis of the function of these regions identifies specific sequences within the IFN A1 virus response element that has an inhibitory effect on expression in cells that are normally inducible, and is also implicated in the overall suppression of IFN A induction in non-inducible cells.

Relative transcriptional inducibility of the human interferon-alpha subtypes conferred by the virus-responsive enhancer sequence.

This paper addresses the role of transcriptional regulation in the determination of the levels of expression of different interferon-alpha subtypes secreted from Namalwa cells following infection with Sendai virus. Using RT-PCR to determine the relative abundance of mRNA species coding for the various subtypes, we found a general correlation with corresponding protein levels, indicative of a role for transcriptional control in the determination of levels of individual subtypes. We have used reporter gene constructs to compare the inducibility of the virus-response elements from the IFNA1, A2, A4, and A14 subtype genes cloned upstream of a secreted alkaline phosphatase gene. The inducibility of these reporter gene constructs broadly correlated with the relative mRNA abundances in both transiently and stably transfected Namalwa cells. During work with stable cell lines, we found that G418, the drug used for the selection of transfected cells, inhibited the induction of interferon by both Sendai virus and double-stranded RNA. This inhibition was reversible when G418 was removed from the medium 24 h before the addition of virus.

Transactivation by the herpes simplex virus virion protein Vmw65 and viral permissivity in a neuronal cell line with reduced levels of the cellular transcription factor Oct-1.

Transactivation of the herpes simplex virus (HSV) immediate-early (IE) genes is dependent upon the formation of a complex between the viral protein Vmw65 and the cellular transactivation factor Oct-1. Differentiation of the proliferating ND7 neuronal cell to a nondividing phenotype results in a large fall in the amount of Oct-1 to a level characteristic of nondividing neuronal cells but does not dramatically affect the level of IE gene expression following infection or the ability of Vmw65 to transactivate the IE promoter in transfection experiments. This suggests that the low levels of Oct-1 in nonproliferating neuronal cells do not play a key role in the failure of IE gene expression following initial infection of these cells, which is an essential step in the establishment of latent infections with HSV.

The octamer binding site in the HPV16 regulatory region produces opposite effects on gene expression in cervical and non-cervical cells.

The upstream regulatory region (URR) of the tumorigenic human papillomaviruses HPV 16 and 18 contains an octamer binding site which is located adjacent to a binding site for the ubiquitous transcription factor NFI. The octamer site binds both the constitutively expressed transcription factor Oct-1 and a novel cervical octamer binding protein. In contrast the URR of the non-tumorigenic viruses HPV6 and HPV11 lacks the octamer binding site although the adjacent NFI site is conserved. Inactivation of the octamer binding site results in a higher level of gene expression in cells which contain only Oct-1 and a lower level in cells containing the cervical octamer binding protein indicating that that whilst Oct-1 binding reduces promoter activity, the cervical protein increases it. In agreement with this, over-expression of Oct-1 reduces the level of gene activity directed by this region of the HPV 16/18 URR and inhibits its activation by NFI whilst having no effect on the corresponding region of the HPV 6/11 URR. The significance of these effects is discussed in terms of the cervical-specific activity of the HPV16/18 URR and its role in HPV-mediated transformation.

Regulation of expression of the neuronal POU protein Oct-2 by nerve growth factor.

POU proteins are a class of homeobox-containing transcription factors that regulate tissue-specific gene expression and influence cell differentiation and function. We have investigated the possible role of such factors in mediating the actions of nerve growth factor (NGF) on sensory neurons. NGF has been found to have differential effects on the levels of three POU protein transcription factors that are expressed in adult rat sensory neurons. A sensory neuron octamer-binding protein with the properties of the transcription factor Oct-2 is up-regulated 3-4-fold by NGF, as measured by mobility shift assays using nuclear extracts from adult rat dorsal root ganglion neurons grown in the presence or absence of NGF. Quantitation of Oct-2 mRNA by polymerase chain reaction amplification of RNA from such cells shows a parallel increase in Oct-2 mRNA levels. In contrast, the levels of mRNA encoding the ubiquitous POU protein Oct-1 or the neuron-specific POU protein Brn-3, also present in sensory neurons, are unaffected by NGF. These observations suggest a role for Oct-2 in mediating transcriptional effects induced by NGF. In particular, as Oct-2 is known to inhibit herpes simplex virus immediate-early gene expression in neuronal cells, these findings provide a mechanism for the known action of NGF in the maintenance of latent herpes virus infections in sensory neurons.

Elevation of cyclic AMP levels in cell lines derived from latently infectable sensory neurons increases their permissivity for herpes virus infection by activating the viral immediate-early 1 gene promoter.

Immortalized cell lines derived from sensory neurons are relatively non-permissive for lytic infection with herpes simplex virus (HSV) and fail to transcribe the viral immediate-early genes following infection. Treatment of these cells with agents which raise the intra-cellular level of cyclic AMP results in increased activity of the IE1 gene which contains a cyclic AMP response element within its promoter and produces a consequent increase in permissivity for HSV infection. The significance of these effects for the regulation of HSV infection of neuronal cells are discussed in the light of the finding that cyclic AMP treatment can reactivate latent HSV infections.

Interferon-alpha treatment of Daudi cells down-regulates the octamer binding transcription/DNA replication factors Oct-1 and Oct-2.

Treatment of Daudi cells with alpha-interferon (alpha-IFN) results in a considerable decrease in the levels of the octamer-binding DNA replication/transcription factors Oct-1 and Oct-2 and specifically inhibits gene expression by octamer-containing promoters. The inhibitory effect on octamer-binding proteins also occurs after culturing cells with phorbol 12-myristate 13-acetate but it does not occur following alpha-IFN treatment of an alpha-IFN-resistant variant of the Daudi cell line or of HeLa cells. We discuss the potential role of the decreased levels of octamer-binding proteins in the inhibition of cell proliferation.

The octamer-binding protein Oct-2 represses HSV immediate-early genes in cell lines derived from latently infectable sensory neurons.

Transcription of herpes simplex virus (HSV) immediate-early (IE) genes does not occur in sensory neurons latently infected with the virus or following infection of neuronal cell lines. In neuronal cell lines this inability results from the weak activity of the viral IE promoters, which is caused by a neuron-specific repressor factor that binds specifically to the TAATGARAT motif in these promoters and to related octamer elements. Cells expressing this repressor contain an additional octamer-binding protein that is absent from permissive cells. We identify this factor as the lymphocyte- and neuron-specific octamer-binding protein Oct-2 and show that Oct-2 mRNA is also present in dorsal root ganglion neurons, the natural site of HSV latency in vivo. Moreover, artificially elevated expression of Oct-2 can repress the IE promoter. The potential role of Oct-2 in the initiation and maintenance of in vivo latent infection with HSV is discussed.