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We conducted maximum dose bioassays of insecticide for the control of diamondback moth (DBM), Plutella xylostella (Linnaeus), in cole crops, from 2016 to 2019 at several commercial locations in Georgia and Florida. The nominal maximum dose was defined as the highest labeled rate of an insecticide at the beginning of the survey in the equivalent of 935 liters/ha dilution. The results indicated low insecticide efficacy for high labeled rates of the following insecticides by common name (Insecticide Resistance Action Committee group number in parentheses). Our 4-yr survey identified very low levels of DBM larval control (<47%) by lambda-cyhalothrin (3), methoxyfenozide (18), pyriproxyfen (7C), novaluron (15), bifenthrin (3), chlorantraniliprole (28), indoxacarb (22A), and methomyl (1A). The best products for DBM control (>74%) listed in decreasing average levels of efficacy were naled (1B), cyclaniliprole (28), tolfenpyrad (21A), emamectin benzoate (6), and cyantraniliprole (28). Intermediate levels of control (61-71%) were obtained with Bacillus thuringiensis subspecies aizawai (11A), Bacillus thuringiensis, subsp. kurstaki, strain ABTS-351 (11A), and spinetoram (5). This rapid bioassay provided the grower with a ranking of insecticide efficacy for the control the DBM population for that farm site. These data allowed growers to make an informed decision on control quickly and plan for resistance management rotations for DBM that season., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate β-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.

Delivery of small interfering RNAs (siRNAs) into primary T cells is quite challenging because they are non-proliferating cells and are difficult to transfect with non-viral approaches. Because sonoporation is independent of the proliferation status of cells and siRNA acts in the cell cytoplasm, we investigated whether sonoporation could be used to deliver siRNA into mouse and human T cells. Cells mixed with Definity microbubbles and siRNA were sonicated with a non-focused transducer of center frequency 2.20 MHz producing ultrasound at a 10% duty cycle, pulse repetition frequency of 2.20 kHz and spatial average temporal average ultrasound intensity of 1.29 W/cm 2 for 5 s and then examined for siRNA fluorescence by flow cytometry analysis. These sonoporation conditions resulted in high-efficiency transfection of siRNA in mouse and human T cells. Further, the efficacy of siRNA delivery by sonoporation was illustrated by the successful visualization of decreased methylation-controlled J protein expression in mouse and human CD8 T cells via Western blot analysis. The results provide the first evidence that sonoporation is a novel approach to delivery of siRNA into fresh isolated mouse and human T cells in vitro, and might be used for in vivo studies in the future., (Copyright © 2019. Published by Elsevier Inc.)

Objective: Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression., Methods: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy., Results: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid β-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment., Conclusion: GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The development and use of tailored interventions in overcoming barriers to optimum health in long-term care facility residents is of the highest importance. One successful approach to improving health outcomes is the use of a checklist by health care providers. Despite the evidence of the success of such checklists in nursing and medicine, there is little evidence on their use in improving dental outcomes. This study investigated whether an intervention comprising the daily use of a checklist for oral care by nursing staff supplemented by random inspections by a charge nurse resulted in lower dental plaque scores in patient participants at a long-term care facility (n=19) as compared with in those at another long-term care facility that did not receive the intervention (n=13). All participants received a dental cleaning at baseline. At a follow-up examination at 6 to 8 weeks post-baseline, significant differences were observed in the plaque scores between the participants at each location, with the median plaque scores in those undergoing the intervention being less than half of those in the patients that did not (20.8% vs. 52.8%, p<0.001). After adjusting for age in a linear regression model, this difference remained significant. The use of a daily checklist for oral care supplemented by random inspections by a charge nurse was associated with lower plaque scores (p<0.001). These results warrant further research, including prospective studies aimed at establishing how use of both clinical supervision and a checklist for oral health may influence plaque scores in geriatric patients in long-term care facilities over time.

Leeches (Annelida: Hirudinea) possess powerful salivary anticoagulants and, accordingly, are frequently employed in modern, authoritative medicine. Members of the almost exclusively marine family Piscicolidae account for 20% of leech species diversity, and they feed on host groups (e.g., sharks) not encountered by their freshwater and terrestrial counterparts. Moreover, some species of Ozobranchidae feed on endangered marine turtles and have been implicated as potential vectors for the tumor-associated turtle herpesvirus. In spite of their ecological importance and unique host associations, there is a distinct paucity of data regarding the salivary transcriptomes of either of these families. Using next-generation sequencing, we profiled transcribed, putative anticoagulants and other salivary bioactive compounds that have previously been linked to blood feeding from 7 piscicolid species (3 elasmobranch feeders; 4 non-cartilaginous fish feeders) and 1 ozobranchid species (2 samples). In total, 149 putative anticoagulants and bioactive loci were discovered in varying constellations throughout the different samples. The putative anticoagulants showed a broad spectrum of described antagonistic pathways, such as inhibition of factor Xa and platelet aggregation, which likely have similar bioactive roles in marine fish and turtles. A transcript with homology to ohanin, originally isolated from king cobras, was found in Cystobranchus vividus but is otherwise unknown from leeches. Estimation of selection pressures for the putative anticoagulants recovered evidence for both positive and purifying selection along several isolated branches in the gene trees, and positive selection was also estimated for a few select codons in a variety of marine species. Similarly, phylogenetic analyses of the amino acid sequences for several anticoagulants indicated divergent evolution.

Aims: To evaluate the effects of hippotherapy on physical capacities of children with cerebral palsy., Methods: Thirteen children (4-12 years old) with cerebral palsy classified in Gross Motor Function Classification System Level I or II were included in this prospective quasi-experimental ABA design study. Participants received 10 weeks of hippotherapy (30 min per week). Gross motor function and proficiency were measured with the Bruininks-Oseretski Motor Proficiency short form [BOT2-SF]) and the Gross Motor Function Measure-88 [GMFM-88] (Dimension D and E) twice before the program (T1 and T1'), immediately after (T2), and 10 weeks following the end of the program (T3)., Results: Mean scores for dimensions D and E of the GMFM-88 Dimension scores (p = .005) and three out of the eight items of the BOT2-SF (fine motor precision (p = .013), balance (p = .025), and strength (p = .012) improved between baseline and immediately after intervention; mean scores immediately following and 10 weeks following intervention did not differ., Conclusions: Hippotherapy provided by a trained therapist who applies an intense and graded session for 10 weeks can improve body functions and performance of gross motor and fine motor activities in children with cerebral palsy.

Increased moment-to-moment predictability, or inertia, of negative affect has been identified as an important dynamic marker of psychological maladjustment, and increased vulnerability to depression in particular. However, little is known about the processes underlying emotional inertia. The current article examines how the emotional context, and people's responses to it, are related to emotional inertia. We investigated how individual differences in the inertia of negative affect (NA) are related to individual differences in exposure, reactivity, and recovery from emotional events, in daily life (assessed using experience sampling) as well as in the lab (assessed using an emotional film-clip task), among 200 participants commencing their first year of tertiary education. This dual-method approach allowed us to assess affective responding on different timescales, and in response to standardized as well as idiographic emotional stimuli. Our most consistent finding, across both methods, was that heightened NA inertia is related to decreased NA recovery following negative stimuli, suggesting that higher levels of inertia may be mostly driven by impairments in affect repair following negative events., ((c) 2015 APA, all rights reserved).)

That emotions change in response to emotion-eliciting events is a natural part of human life. However, it is equally important for emotions to return to baseline once the emotion-eliciting events have passed. This suggests that the ability to emotionally react to and recover from emotion-eliciting events is critical for healthy psychological functioning. But why do individuals differ in their emotion reactivity and recovery? The present work postulates that the ability to update emotional information in working memory (WM) may explain individual differences in emotion reactivity and recovery. Two studies are presented, which examined whether updating ability was related to emotion reactivity and recovery. In Study 1, we assessed participants' self-reported affect as they viewed negative and positive films. Our results revealed that better updating ability was related to greater emotion reactivity and facilitated (i.e., quicker) recovery from watching negative films. In Study 2, participants recalled a recent angering event, and were then instructed to either ruminate about or reappraise the event. Results revealed that updating ability was again related to greater emotion reactivity and facilitated (i.e., successful) emotion recovery in response to the angering event, and that this was unrelated to the emotion regulation strategy used. These findings identify the ability to update emotional information in WM as a possible mechanism in emotion responding.

Appraisals and core affect are both considered central to the experience of emotion. In this study we examine the bidirectional relationships between these two components of emotional experience by examining how core affect changes following how people appraise events and how appraisals in turn change following how they feel in daily life. In an experience sampling study, participants recorded their core affect and appraisals of ongoing events; data were analyzed using cross-lagged multilevel modeling. Valence-appraisal relationships were found to be characterized by congruency: the same appraisals that were associated with a change in pleasure-displeasure (motivational congruency, other-agency, coping potential, and future expectancy), changed themselves as a function of pleasure-displeasure. In turn, mainly secondary appraisals of who is responsible and how one is able to cope with events were associated with changes in arousal, which itself is followed by changes in the future appraised relevance of events. These results integrate core affect and appraisal approaches to emotion by demonstrating the dynamic interplay of how appraisals are followed by changes in core affect which in turn change our basis for judging future events.

Well-controlled laboratory column experiments were conducted to understand the influence of Pseudomonas aeruginosa (P. aeruginosa) biofilms on the transport of selected engineered nanoparticles (ENPs) in granular porous media representative of groundwater aquifers or riverbank filtration settings. To understand the importance of particle size on retention in the biofilm-coated granular (quartz sand) matrix, column experiments were carried out using nanosized (20 nm) and micrometer-sized (1 μm) sulfate-functionalized polystyrene latex particles (designated as 20 nSL and 1 mSL, respectively). Additional experiments conducted with nanosized (20 nm) carboxyl-modified latex particles (20nCL) and carboxyl-modified CdSe/ZnS quantum dots (QDs) provide information on the influence of particle surface chemistry on retention. Biofilm grown on the surface of the sand was characterized by total biomass quantification, confocal laser scanning microscopy (CLSM), and electrokinetic analysis. All four particles exhibit increased retention in the biofilm-coated packed bed: e.g., the attachment efficiency (α) of the 1 mSL particle increases from 0.40 to 1.7, whereas α for the 20 nSL particle increases from 0.04 to 0.10 in the biofilm-coated system. Particle surface chemistry can also influence the affinity of the ENPs for the biofilm coating as revealed by the greater attachment of the 20 nSL particle onto the biofilm-coated sand (α = 0.10) than its carboxylated counterpart (α = 0.04). Column experiments conducted using sand coated with growth medium (LB) or extracellular polymeric substances (EPS) extracted from P. aeruginosa biofilms further reveal that particle surface chemistry influences the interaction between the different ENPs and these coated sand surfaces. Namely, coating of sand surfaces with LB medium or bacterial EPS does not affect the transport of the sulfonated nanoparticle, but the LB coating leads to decreased retention of the carboxylated latex nanoparticle. Furthermore, our results show that EPS coatings are not necessarily good surrogates for biofilm-coated sand. Electrokinetic characterization of the clean and coated sand surfaces also reveals that the extent of particle retention is not controlled by electrical double layer interactions. Future studies should thus be aimed at improving our understanding of the fundamental mechanisms (both colloidal and noncolloidal) governing nanoparticle transport and fate in biofilm-laden granular aquatic environments.

The purpose of this case report is to describe the impact of an 11-week hippotherapy program on the gross motor functions of two children (respectively 28 and 37 months old) diagnosed with Down syndrome. Hippotherapy is a strategy that uses the horse's motion to stimulate and enhance muscle contraction and postural control. The children were assessed by the Gross Motor Function Measure (GMFM) and accelerometry. The results indicate that both children improved on many dimensions of the GMFM. Power spectral analysis of the acceleration signals showed improvement in postural control of either the head or trunk, because the children adopted two different adaptative strategies to perturbation induced by the moving horse.

In psittacine birds, the antemortem diagnosis of aspergillosis is usually based on the clinical signalment combined with the results of diagnostic tests such as radiography, routine hematologic and biochemical analysis, and biopsy. For several years, plasma protein electrophoresis has been used as an ancillary diagnostic technique in forming a diagnosis and treatment plan in avian species. More recently, a commercially available assay to measure galactomannan, an Aspergillus species antigen, has been described for clinical use in humans, cattle, horses, dogs, and gyr falcons. This report describes several confirmed cases of aspergillosis, with accompanying clinical data, including plasma protein electrophoresis and galactomannan assay results, in addition to results of traditional evaluations by hematology, radiography, and biopsy. In clinical cases in psittacine birds, the galactomannan assay appears useful for detecting circulating Aspergillus antibody.

Previous work in our laboratory described the in vitro killing of Borrelia burgdorferi when co-cultured with saliva from adult Amblyomma americanum. Borreliacidal activity was not evident using Ixodes scapularis saliva. Mixing trypsin with saliva eliminated the borreliacidal activity of A. americanum saliva, while incorporating a trypsin inhibitor restored all borreliacidal activity, indicating this factor was of protein or peptide origin. One-dimensional PAGE indicated at least 7 major protein differences between I. scapularis and A. americanum saliva. To determine the borreliacidal factor, A. americanum saliva was fractionated by gel filtration and subsequent killing of B. burgdorferi was associated with a single fraction. Two-dimensional gel analysis indicated protein and/or peptide(s) in borreliacidal fractions running between 38 and 64 kDa. Finally, admixing saliva with the phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine completely eliminated the ability of A. americanum saliva to kill B. burgdorferi. These studies indicate the borreliacidal activity found in A. americanum saliva is likely due to phospholipase A2 enzymatic activity.

In humans, two glucocorticoid receptor (GR) splice variants exist: GRalpha and GRbeta, which are identical between amino acids 1-727 and then diverge. Whereas GRalpha (the canonical GR) acts as a ligand-activated transcription factor, GRbeta does not bind traditional glucocorticoid agonists, lacks GRalpha's transactivational activity, and acts as a dominant-negative inhibitor of GRalpha. It has been suggested that this receptor isoform is involved in the induction of glucocorticoid resistance in asthma patients. Unfortunately, a GR beta-isoform has been detected in only humans, and therefore, an animal model for studies on this isoform is lacking. In the present study, we demonstrate that in zebrafish a GR isoform exists that diverges from the canonical zebrafish GR at the same position as human GRbeta from human GRalpha. The zebrafish GR beta-isoform acts as a dominant-negative inhibitor in reporter assays, and the extent of inhibition and the effective GRalpha/GRbeta ratio is similar to studies performed with the human GR isoforms. In addition, the subcellular localization of zebrafish GRbeta is similar to its human equivalent. Finally, expression levels of GRalpha and GRbeta were determined in adult zebrafish tissues and at several developmental stages. Both receptor isoforms were detected throughout the body, and GRbeta mRNA levels were relatively low compared with GRalpha mRNA levels, as in humans. Thus, for the first time, a GR beta-isoform has been identified in a nonhuman animal species, shedding new light on the relevance of this GR splice variant and providing a versatile animal model for studies on the GR system.

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules found in lipoprotein particles. This particular lipase also has a role in the binding of lipoprotein particles to different cell-surface receptors. LPL has been identified in the brain but has no specific function yet. This study aimed at elucidating the role of LPL in the brain in response to injury. Mice were subjected to hippocampal deafferentation using the entorhinal cortex lesion and mRNA and protein expression were assessed over a time-course of degeneration/reinnervation. Hippocampal LPL levels peaked at 2 days post-lesion (DPL) both at the mRNA and protein levels. No change was observed for receptors of the LDL-receptor family or RAP at DPL 2 in the hippocampus but the glia-specific syndecan-4 was found to be significantly upregulated at DPL 2. These results suggest that LPL is involved in the recycling of cholesterol and lipids released from degenerating terminals after a lesion through a syndecan-4-dependent pathway.

Natural variations in maternal care in the rat influence the development of neuronal systems that regulate endocrine and behavioral responses to stress. Thus, as adults, rats that received higher levels of maternal licking/grooming (LG) in infancy are less 'fearful' in response to novelty, compared with adult offspring of Low LG mothers. The present study examined the influence of maternal care on behavioral and neuronal responses to a more specific, localizable form of threat using an electrified probe in the shock-probe burying test. Even under these conditions, adult offspring of High LG mothers displayed lower levels of fear reactivity (i.e. less shock-induced freezing and probe burying) throughout the test than did offspring of Low LG mothers. These differences in fearfulness were associated with differential patterns of cFos immunoreactivity (cFos-IR), 120 min following test exposure. Relative to control rats exposed to a non-electrified probe, cFos-IR was increased in the offspring of High LG mothers exposed to an electrified probe in the dentate gyrus, ventral subiculum, lateral and medial septum, nucleus accumbens and the dorsal periaqueductal gray. Shock-exposed offspring of Low LG dams displayed a very different pattern of neuronal activation characterized by both increases (area CA1 of the ventral hippocampus and the inferior colliculus) and decreases (paraventricular nucleus of the hypothalamus and the ventrolateral periaqueductal gray) in cFos-IR compared with the no-shock controls. Together these results suggest that maternal care serves to 'program' neuronal circuits that modulate fear-related responding in the rat resulting in qualitatively different neuronal responses to stress.

Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 microM(-)(1) s(-)(1)) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows ( approximately 50 s(-)(1)), and the stabilized final complexes are resistant to autoreduction (E degrees = +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s(-)(1)), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s(-)(1)). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.

Lipoproteins are macromolecular complexes composed of lipids and proteins. The role of these complexes is to provide cells of the organism with lipids to be used as a source of energy, building blocks for biomembrane synthesis, and lipophilic molecules (e.g., steroid hormones and vitamin E) for other physiological purposes, such as cell signaling and antioxidative mechanisms. Lipoproteins also promote the cellular efflux of cholesterol for its disposal into bile. Thus, lipoproteins play an important role in the maintenance of lipid homeostasis throughout the organism. Accordingly, lipoprotein particles have been found circulating in blood, lymph, and interstitial fluid. Despite the existence of the blood-brain barrier, lipoprotein particles have been shown to be also present in the cerebrospinal fluid (CSF). Although a portion of their protein components may filter through the barrier from the vascular compartment, experimental evidence indicates that these particles originate from the nervous tissue. The other protein components include apolipoproteins E, J, and D, and these have been shown to be synthesized by cells within the central nervous system (CNS). Furthermore, it was shown that lipoprotein particles can be isolated from the conditioned medium of astrocytic cultures. The differences in size, structure, and composition of in vitro assembled particles compared with those isolated from the CSF suggest that the particles are modified following their secretion in vivo. This is supported by observations that lipoprotein-modifying enzymes and transfer proteins are also present within CNS tissue and CSF. The fate of CSF lipoproteins is unclear but is probably related to the turnover and clearance of lipids from the CNS or, alternatively, the particles may be recaptured and recycled back into the CNS tissue. The presence of several cell surface receptors for apoE-containing lipoproteins on ependymal cells, as well as on neurons and glial cells, supports this notion and suggests that the isolated brain possesses its own system to maintain local lipid homeostasis. This is further exemplified by the salvage and recycling of lipids shown to occur following a lesion in order to allow surviving neurons to sprout and reestablish lost synapses. Not much is currently known about lipoprotein metabolism in neurodegenerative diseases, but lipid alterations have been repeatedly reported in Alzheimer brains in which neuronal loss and deafferentation are major features. Although the mechanism underlying the link between the epsilon4 allele of the apolipoprotein E gene and Alzheimer's disease is presently unclear, it may well be postulated that it is related to disturbances in brain lipoprotein metabolism.

Background: Nitrophorins are nitric oxide (NO) transport proteins from the saliva of blood-feeding insects, which act as vasodilators and anti-platelet agents. Rhodnius prolixus, an insect that carries the trypanosome that causes Chagas' disease, releases four NO-loaded nitrophorins during blood feeding, whereupon the ligand is released into the bloodstream or surrounding tissue of the host. Histamine, a signaling molecule released by the host upon tissue damage, is tightly bound by the nitrophorins; this may facilitate the release of NO and reduce inflammation in the host., Results: Recombinant nitrophorin 4 (NP4) was expressed in Escherichia coli, reconstituted with heme, and found to bind NO and histamine in a manner similar to that of the natural protein. The crystal structure of NP4 revealed a lipocalin-like eight-stranded beta barrel, with heme inserted into one end of the barrel. His59 ligates the proximal site on the heme, a solvent molecule (NH3) ligates the distal site, and three additional solvent molecules occupy the distal pocket. Buried in the protein interior are Glu55 and three solvent molecules. A detailed comparison with other lipocalins suggests that NP4 is closely related to the biliverdin-binding proteins from insects., Conclusions: The nitrophorins have a unique hemoprotein structure and are completely unlike the globins, the only other hemoproteins designed to transport dissolved gases. Compared with the recently described structure of NP1, the NP4 structure is considerably higher resolution, confirms the unusual placement of ionizable groups in the protein interior, and clarifies the solvent arrangement in the distal pocket. It also provides a striking example of structural homology where sequence homology is minimal.

The nitrophorins are heme-based proteins from the salivary glands of the blood-sucking insect Rhodnius prolixus that deliver nitric oxide gas (NO) to the victim while feeding, resulting in vasodilation and inhibition of platelet aggregation. The nitrophorins also bind tightly to histamine, which is released by the host to induce wound healing. Here we present three crystal structures of nitrophorin 1 (NP1): bound to cyanide, which binds in a manner similar to NO (2.3 A resolution); bound to histamine (2.0 A resolution); and bound to what appears to be NH3 from the crystallization solution (2.0 A resolution). The NP1 structures reveal heme to be sandwiched between strands of a lipocalin-like beta-barrel, and in an arrangement unlike any other gas-transport protein discovered to date. The heme is six-coordinate with a histidine (His 59) on the proximal side, and ligand in a spacious pocket on the distal side. The structures confirm that NO and histamine compete for the same binding pocket and become buried on binding. The dissociation constant for histamine binding was found to be 19 nM, approximately 100-fold lower than that for NO.

It has been reported that corticotropin-releasing factor (CRF) may regulate its own biosynthesis in the paraventricular nucleus of the hypothalamus (PVH). Whether this CRF autoregulation is mediated by local circuitry or from extra-PVH CRF neuronal fibers terminating on CRF perikarya within the PVH is unknown. In the present study, we sought to determine the origin(s) of this CRF innervation using retrograde transport of wheat germ-conjugated-gold particles (WGA-apoHRP-Au) combined with immunohistochemistry for CRF. The rats also received colchicine (100 microg, icv) 5-7 days after tracer injection and were perfused 24 h later. Results of retrograde labeling with pressure injections of WGA-apoHRP-Au centered to PVH and subsequent immunohistochemical staining for CRF demonstrated numerous retrogradely labeled CRF neurons in the perifornical hypothalamic nucleus (PeF), the dorsolateral hypothalamic area (DA) (medial and lateral portions) and the dorsomedial nucleus of the hypothalamus (DMH). Smaller groups of CRF-ir neurons that were retrogradely labeled were found in the bed nuclei of the stria terminalis (BnST), the Barrington's nucleus (Bar) and the dorsal raphé (DR). These CRFergic pathways to the PVH may represent an anatomical substrate underlying the function of the stress-integrative PVH neurons in the autonomic, behavioral and neuroendocrine regulation during the stress response, including CRF autoregulation.

Objectives: Because constructional ability is a crucial perceptual-motor skill that relates to daily functioning, it should be accurately assessed in clients with neurological dysfunction. This study examined three versions of the Puzzle Reproduction task (a constructional ability task) of the Loewenstein Occupational Therapy Cognitive Assessment (LOTCA) in order to determine whether a reduced-detail version of the task would be easier (i.e., require less time to complete) than the original version and whether a subplacement version would be more difficult to perform (i.e., require more time to complete) than the original version. In addition, the study examined whether older adult subjects would perform more slowly than younger adult subjects., Methods: Seventy-two right-handed adults with no disabilities were divided into two age groups: 18 to 30 years old (n = 36) and 58 to 70 years old (n = 36). Each subject was tested on one of three versions of the LOTCA Puzzle Reproduction task (i.e., original subplacement, simplified)., Results: For the older subjects, the simplified version of the task required significantly less time than the original version, although there was not a significant time difference between the original and subplacement versions. For the younger subjects, the subplacement versions. required significantly more time than the original version, but there was no significant time difference between the original and simplified versions. Results also indicated that older subjects took significantly longer to perform all three versions of the task than did the younger subjects., Conclusion: The findings support the use of the simplified version of the LOTCA Puzzle Reproduction task with older adults or with persons with major cognitive-perceptual difficulties. Further studies of the level of difficulty of the subplacement version are needed to examine whether this version is more sensitive to constructional deficits in a sample of person with neurological impairments because even mild constructional deficits have been shown to relate to disabilities in daily functioning.

A nitric oxide transport protein (nitrophorin I) from the salivary glands of the blood-sucking bug Rhodnius prolixus has been expressed as an insoluble form in Escherichia coli, reconstituted with heme, and characterized with respect to NO binding kinetics and equilibria. NO binding and absorption spectra for recombinant nitrophorin I were indistinguishable from those of the insect-derived protein. The degree of NO binding, the rate of NO release, and the Soret absorption maxima for nitrophorin I were all pH dependent. The NO dissociation constant rose 9-fold over the pH range 5.0-8.3, from 0.19 x 10(-6) to 1.71 x 10(-6). The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, from 1.2 x 10(-3) to 3.0 s(-1). Thus, the NO association rate must also be pH dependent and reduced at pH 5.0 by approximately 280-fold. These factors are consistent with nitrophorin function: NO storage in the apparent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible nature of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure determination by X-ray crystallography. The most promising crystal form contains two protein molecules in the asymmetric unit and diffracts beyond 2.0 A resolution.

The relationship between stress and hypertension has long been a topic of debate. Until recently, no animal (nor physical) model existed which incorporated these two parameters together, hence accurate assessments of their interactions were postulations, at best. The borderline hypertensive rat (BHR) is an animal model in which arterial hypertension will ensue only when the rat is subjected to a chronic stressor. Therefore, this animal model offers an opportunity to study the physiological consequences of a given stressor on the entire organism during the course of the development of hypertension. The cardiovascular and humoral effects of chronic exposure to a physiological stressor on the BHR, at rest and during exposure to an acute and novel stress, have been examined and are presented in this paper. The involvement of the central nervous system in the regulation of this stress induced hypertensive state has been hypothesized to play a major role in the regulation of the stress and hypertensive relationship. Of particular importance are the endogenous opioids and their influence on the maintenance of homeostasis during periods of physiological stress. The localization of the endogenous opioid enkephalin in the paraventricular nucleus of the hypothalamus (PVN), an important brain region in the regulation of the stress response further substantiates the involvement of the opioid system during stress induced hypertension. Immunohistochemical data on the localization of ENKergic fibers in the PVN and other brain regions are similarly discussed in this paper.

In this paper, Donald Champagne reviews the salivary vasodilators, points to effects of similar compounds that may be shared by the insect substances, and discusses the potential significance of these effects with regard to parasite transmission.

The bloodsucking bug Rhodnius prolixus has a salivary vasodilator, previously characterized as a nitrovasodilator, with salivary smooth muscle-relaxing and antiplatelet activity. Rhodnius salivary glands are bright red owing to the abundance of heme proteins. Electron paramagnetic resonance and optical spectroscopic experiments indicated that the salivary vasodilator is a nitrosylheme protein with an Fe(III) heme that binds nitric oxide (NO) reversibly. Dilution of the protein in neutral pH promoted NO release. This protein thus appears to be the NO carrier that helps R. prolixus to feed on blood.