Class 3 Hox genes in insects and the origin of zen.

We have cloned, from a beetle and a locust, genes that are homologous to the class 3 Hox genes of vertebrates. Outside the homeobox they share sequence motifs with the Drosophila zerknüllt (zen) and z2 genes, and like zen, are expressed only in extraembryonic membranes. We conclude that the zen genes of Drosophila derive from a Hox class 3 sequence that formed part of the common ancestral Hox cluster, but that in insects this (Hox) gene has lost its role in patterning the anterio-posterior axis of the embryo, and acquired a new function. In the lineage leading to Drosophila, the zen genes have diverged particularly rapidly.

Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase.

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes. Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome. In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools. We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity. These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.

Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.

Identification of functional domains and evolution of Tc1-like transposable elements.

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.

In late 1994 and early 1995, Ebola (EBO) virus dramatically reemerged in Africa, causing human disease in the Ivory Coast and Zaire. Analysis of the entire glycoprotein genes of these viruses and those of other EBO virus subtypes has shown that the virion glycoprotein (130 kDa) is encoded in two reading frames, which are linked by transcriptional editing. This editing results in the addition of an extra nontemplated adenosine within a run of seven adenosines near the middle of the coding region. The primary gene product is a smaller (50-70 kDa), nonstructural, secreted glycoprotein, which is produced in large amounts and has an unknown function. Phylogenetic analysis indicates that EBO virus subtypes are genetically diverse and that the recent Ivory Coast isolate represents a new (fourth) subtype of EBO virus. In contrast, the EBO virus isolate from the 1995 outbreak in Kikwit, Zaire, is virtually identical to the virus that caused a similar epidemic in Yambuku, Zaire, almost 20 years earlier. This genetic stability may indicate that EBO viruses have coevolved with their natural reservoirs and do not change appreciably in the wild.

Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)

The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.

Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation.

ADPglucose pyrophosphorylase (glucose-1-phosphate adenylyltransferase; ADP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in alpha-glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC- strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides an efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity.

Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

Growth hormone secretagogues: characterization, efficacy, and minimal bioactive conformation.

Another class of growth hormone (GH) secretagogues has been discovered by altering the backbone structure of a flexible linear GH-releasing peptide (GHRP). In vitro and in vivo characterization confirms these GH secretagogues as the most potent and smallest (M(r) < 500) reported. Anabolic efficacy is demonstrated in rodents with intermittent delivery. A convergent model of the bioactive conformation of GHRPs is developed and is supported by the NMR structure of a highly potent cyclic analog of GHRP-2. The model and functional data provide a logical framework for the further design of low-molecular weight secretagogues and illustrate the utility of an interdisciplinary approach to elucidating potential bound-state conformations of flexible peptide ligands.

Stress and antidepressants differentially regulate neurotrophin 3 mRNA expression in the locus coeruleus.

The mechanisms by which stress and anti-depressants exert opposite effects on the course of clinical depression are not known. However, potential candidates might include neurotrophic factors that regulate the development, plasticity, and survival of neurons. To explore this hypothesis, we examined the effects of stress and antidepressants on neurotrophin expression in the locus coeruleus (LC), which modulates many of the behavioral and physiological responses to stress and has been implicated in mood disorders. Using in situ hybridization, we demonstrate that neurotrophin 3 (NT-3) is expressed in noradrenergic neurons of the LC. Recurrent, but not acute, immobilization stress increased NT-3 mRNA levels in the LC. In contrast, chronic treatment with antidepressants decreased NT-3 mRNA levels. The effect occurred in response to antidepressants that blocked norepinephrine uptake, whereas serotonin-specific reuptake inhibitors did not alter NT-3 levels. Electroconvulsive seizures also decreased NT-3 expression in the LC as well as the hippocampus. Ntrk3 (neurotrophic tyrosine kinase receptor type 3; formerly TrkC), the receptor for NT-3, is expressed in the LC, but its mRNA levels did not change with stress or antidepressant treatments. Because, NT-3 is known to be trophic for LC neurons, our results raise the possibility that some of the effects of stress and antidepressants on LC function and plasticity could be mediated through NT-3. Moreover, the coexpression of NT-3 and its receptor in the LC suggests the potential for autocrine mechanisms of action.

Orchestration of energy efficiency capabilities for a sustainable network management

The energy demand for operating Information and Communication Technology (ICT) systems has been growing, implying in high operational costs and consequent increase of carbon emissions. Both in datacenters and telecom infrastructures, the networks represent a significant amount of energy spending. Given that, there is an increased demand for energy eficiency solutions, and several capabilities to save energy have been proposed. However, it is very dificult to orchestrate such energy eficiency capabilities, i.e., coordinate or combine them in the same network, ensuring a conflict-free operation and choosing the best one for a given scenario, ensuring that a capability not suited to the current bandwidth utilization will not be applied and lead to congestion or packet loss. Also, there is no way in the literature to do this taking business directives into account. In this regard, a method able to orchestrate diferent energy eficiency capabilities is proposed considering the possible combinations and conflicts among them, as well as the best option for a given bandwidth utilization and network characteristics. In the proposed method, the business policies specified in a high-level interface are refined down to the network level in order to bring highlevel directives into the operation, and a Utility Function is used to combine energy eficiency and performance requirements. A Decision Tree able to determine what to do in each scenario is deployed in a Software Defined Network environment. The proposed method was validated with diferent experiments, testing the Utility Function, checking the extra savings when combining several capabilities, the decision tree interpolation and dynamicity aspects. The orchestration proved to be valid to solve the problem of finding the best combination for a given scenario, achieving additional savings due to the combination, besides ensuring a conflict-free operation.

Attentional processes associated with victimization history and posttraumatic symptomatology in women exposed to intimate partner violence

Exposure to intimate partner violence (IPV) puts women at risk for severe and chronic physical and mental health consequences, including elevations in IPV-related psychopathology and increased risk for future victimization. Previous research has examined attention as one of the key information processing mechanisms associated with elevated psychopathology and risk for victimization; however, the nature of attentional processing in response to IPV-related information in women exposed to IPV is poorly understood. Therefore, the current study aimed to further understanding of associations between attentional processing, IPV exposure, and related distress using measures of eye movement and subjective interpretations of IPV-related information. A sample of women exposed to IPV (n = 57) viewed sets of negative, positive, and neutral relationship images for 15 s each while having their eye movements monitored and later provided subjective ratings and interpretations of levels of risk and safety in those images. We examined associations of outcome measures with proximal victimization experiences and IPV-related psychopathology (i.e., depression, posttraumatic stress disorder (PTSD), anxiety, and dissociation). Results indicated a bias to attend to negative relationship images relative to positive and neutral images, though this attention bias fluctuated over time and varied as a function of symptomatology such that depression corresponded with increases in attention to negative images over time and PTSD corresponded with decreases in attention to negative images. The general attention bias for negative images appeared to be explained by rumination on and/or difficulty disengaging from negative images, which was related to general elevations in psychopathology as well as exposure to revictimization by different perpetrators. Subjective interpretations and perception of danger cues were related to victimization history and level and type of IPV-related distress. We replicated these procedures with a sample of undergraduate students without IPV histories or related symptomatology (n = 33) and found that the overall attention bias for negative images was not replicated, despite general similarities in patterns of attention over time. Results therefore indicated associations between attentional processing and IPV exposure and related symptomatology. Implications for models of IPV-related psychopathology and attentional processing as well as directions for future study and interventions are discussed.

The Clinical Utility of the Adult Attachment Projective Picture System: A Clinician's Perspective

The Adult Attachment Projective Picture System (AAP) is the first performance- based measure of adult attachment to be developed. The purpose of the measure is to provide a clinical understanding of an adult client's attachment status and associated coping mechanisms. The AAP is a relatively new measure that has yet to be examined from a utility perspective. In the current study, seven psychologists completed a structured survey in order to identify their perspectives of the AAP and its utility as a clinical instrument. A phenomenological qualitative analysis of the data was conducted to derive themes about the AAP and its clinical utility. Analyses aimed to answer the following: What clinical considerations do clinician's focus on when deciding to use this measure? What are common factors among clinician's who do use the measure as well as those who do not? What aspects of the measure are user-friendly and what aspects are difficult? General themes that emerged include (a) the clinical information provided by the AAP is viewed by those who use it as unique and beneficial; (b) time commitment and cost for the clinician are common considerations when clinician's are deciding whether or not to use the AAP or when pursuing training; (c) the AAP provides an increased understanding of one's relational capacities and defenses; and (d) the coding system and transcription process are difficult aspects of the AAP and influence how and/or when it is used. In addition to these themes, multiple respondents discussed potential changes for the AAP that would increase their future use of the instrument. Finally, the implications of these results are discussed.

Trauma Treatment in Forensic Settings: Clinicians' Perspectives, Barriers to Effective Treatment, and Suggestions for Clinical Practice

Between 30% and 90% of the prison population is estimated to have survived traumatic experiences such as sexual, emotional, and physical abuse prior to incarceration (Anonymous, 1999; Fondacaro, Holt, & Powell, 1999; Messina & Grella, 2006; Pollard & Baker, 2000; Veysey, De Cou, & Prescott, 1998). Similarly, information from the Bureau of Justice Statistics (as reported in Warren, 2001) estimated that more than half of the women in state prisons have experienced past physical and sexual abuse. Thus, given the astonishing number of inmates who appear to be victims of some kind of trauma, it seems likely that those who work with these inmates (e.g., prison staff, guards, and treatment providers) will in some way encounter challenges related to the inmates' trauma history. These difficulties may appear in any number of forms including inmates' behavioral outbursts, increased emotionality, sensitivity to triggering situations, and chronic physical or mental health needs (Veysey, et al., 1998). It is also likely that these individuals with trauma histories would benefit greatly from treatment while incarcerated. This treatment could be utilized to minimize symptoms of posttraumatic stress, decrease behavioral problems, and help the inmate function more effectively in society when released from incarceration (Kokorowski & Freng, 2001; Tucker, Cosio, Meshreki, 2003). Few studies have explored the types of trauma treatment that are effective with inmate populations or made specific suggestions for clinicians working in forensic settings (Kokorowski & Freng, 2001). Essentially, there appears to be a large gap in terms of the need for trauma treatment for inmates and the lack of literature addressing what to do about it. However, clinicians across the country seem to be quietly attempting to fulfill this need for trauma treatment with incarcerated populations. They are providing this greatly needed treatment every day. in the face of enormous challenges and often without recognition or the opportunity to share their valuable work with the larger community.