Measuring thigmotaxis in larval zebrafish.

One of the most commonly used behavioral endpoints measured in preclinical studies using rodent models is thigmotaxis (or "wall-hugging"). Thigmotaxis is a well-validated index of anxiety in animals and humans. While assays measuring thigmotaxis in adult zebrafish have been developed, a thigmotaxis assay has not yet been validated in larval zebrafish. Here we present a novel assay for measurement of thigmotaxis in zebrafish larvae that is triggered by a sudden change in illumination (i.e. sudden light-to-darkness transition) and performed in a standard 24-well plate. We show that zebrafish larvae as young as 5 days post fertilization respond to this challenge by engaging in thigmotaxis. Thigmotaxis was significantly attenuated by anxiolytic (diazepam) and significantly enhanced by anxiogenic (caffeine) drugs, thus representing the first validated thigmotaxis assay for larval zebrafish. We also show that exposure to sudden darkness per se may represent an anxiogenic situation for larval zebrafish since less contrasting light-to-darkness transitions (achieved by lowering darkness degrees) significantly decreased thigmotaxis levels in a manner similar to what was achieved with diazepam. These findings suggest that stimuli such as exposure to sudden darkness could be used proficiently to trigger the expression of anxiety-like behaviors in laboratory settings. In sum, this is a versatile protocol allowing testing of both anxiolytic and anxiogenic drugs in a cost-effective manner (only 10 min). This assay is also amenable to medium to high-throughput capacity while constituting a valuable tool for stress and central nervous system research as well as for preclinical drug screening and discovery.

Black fly salivary gland extract inhibits proliferation and induces apoptosis in murine splenocytes.

Black flies are known to be vectors of pathogens including Onchocerca volvulus, which causes human onchocerciasis, and Vesicular Stomatitis Virus. Their salivary secretion has been shown to contain a complex cocktail of anti-haemostatic factors and immunomodulatory activities, which may contribute to efficient transmission of the pathogens. Black fly salivary gland extract (SGE) inhibits mitogen-stimulated mouse splenocyte proliferation, including proliferation of both CD4(+) and CD8(+) T cells. The factor responsible for the inhibition was determined to be a protein (or protein complex) of a size larger than 50 kDa. Moreover, exposure to SGE results in activation of caspase 3 and characteristic morphological changes in CD4(+) and CD8(+) T cells, suggesting that induction of apoptosis could, at least in part, be responsible for this inhibition.

Effect of apolipoprotein E deficiency on reactive sprouting in the dentate gyrus of the hippocampus following entorhinal cortex lesion: role of the astroglial response.

This study investigated the effect of apolipoporotein E (apoE) deficiency on hippocampal reactive sprouting responses of the septohippocampal cholinergic (SHC) and commissural/associational fibers (C/A) following an electrolytic lesion of the entorhinal cortex (ECL), using apoE knockout (apoEKO) and age-matched control wild-type mice. Based on recent evidence suggesting that apoE plays a role in the modulation of glial inflammation, we also tested the hypothesis that the pattern of the astroglial response to ECL might be related to the defective reinnervation previously reported in apoEKO mice. Consistent with our hypothesis, we report a differential pattern of astroglial response that concurred with impairments in the sprouting of the SHC and corresponding synaptic replacement in apoEKO mice at 14 and 30 days post-lesion (DPL), a time range covering the onset of axonal/terminal sprouting to synaptogenesis. We also report a limited sprouting of the C/A fiber system in apoEKO relative to control mice at 30 DPL, a period of active dendritic remodeling. The results of the present study confirm and extend previous findings that apoEKO mice display impaired regenerative capacity in response to ECL and argue that in addition to the effect of apoE on lipid trafficking, apoE may also influence the astroglial response to damage, and that both of these effects account for the defective reinnervation observed in apoEKO mice.

Saliva of the Yellow Fever mosquito, Aedes aegypti, modulates murine lymphocyte function.

Saliva of many vector arthropods contains factors that inhibit haemostatic responses in their vertebrate hosts. Less is known about the effect of vector saliva on host immune responses. We investigated the effect of Aedes aegypti salivary gland extracts on antigen-stimulated responses of transgenic OVA-TCR DO11 mouse splenocytes in vitro. T-cell proliferation was inhibited in a dose-dependent manner, with greater than 50% inhibition at 0.3 salivary gland pair (SGP) equivalents/mL. LPS-stimulated B-cell proliferation was also inhibited. Secretion of the Th1 cytokines IL-2 and IFN-gamma was reduced by 50% or more with 0.45-0.6 SGP/mL, as was secretion of the pro-inflammatory cytokines GM-CSF and TNF-alpha, and the Th2 cytokine IL-5. The Th2 cytokines IL-4 and IL-10 were similarly reduced with 0.6-2 SGP/mL. Inhibition of lymphocyte function involved modulation of viable T-cells at low salivary gland extract (SGE) concentrations, and decreased viability at higher concentrations. Dendritic cells were not killed by salivary gland extracts at concentrations as high as 25 salivary gland pairs/mL, but secretion of IL-12 was inhibited by 87% following exposure to 0.6 SGP/mL. Activity is present in saliva and extracts of female but not male salivary glands, and it is depleted from salivary glands of blood-fed mosquitoes. The activity is denatured by boiling and by digestion with the protease papain, indicating a protein; gel filtration HPLC indicates a mass of about 387 kDa. These results suggest that A. aegypti saliva exerts a marked immunomodulatory influence on the environment at the bite site.

Variations of maternal care differentially influence 'fear' reactivity and regional patterns of cFos immunoreactivity in response to the shock-probe burying test.

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.

Differential modulation of murine host immune response by salivary gland extracts from the mosquitoes Aedes aegypti and Culex quinquefasciatus.

Mosquitoes (Diptera: Culicidae) are major vectors of numerous infectious agents. Compounds in mosquito saliva not only facilitate blood-feeding, but may also have an impact upon the immune system of vertebrate hosts. Consequently, the exposure to mosquito saliva may influence pathogen transmission, establishment and disease development. Using two medically important vector mosquitoes, Aedes aegypti (L.) and Culex quinquefasciatus Say, we examined the effects of mosquito saliva on immune cells of host mice. After antigen-specific or non-specific stimulation, murine splenocyte proliferation and production of both Th1 and Th2 cytokines were significantly reduced in the presence of salivary gland extract (SGE) from Ae. aegypti, but not SGE from Cx. quinquefasciatus. T cell populations were highly susceptible to this suppression, showing increased mortality and reduced division rates - judged by flow cytometric analyses. Evidently these two culicine mosquitoes differ in their host immunomodulatory activities.

The cholesterol-lowering drug probucol increases apolipoprotein E production in the hippocampus of aged rats: implications for Alzheimer's disease.

Several recent epidemiological studies have proposed that cholesterol-lowering drug Statin may provide protection against Alzheimer's disease (AD). Probucol is a non-Statin cholesterol-lowering drug and a potent inducer of apolipoprotein E (apoE) production in peripheral circulation. A recent clinical study using Probucol in elderly AD subjects revealed a concomitant stabilisation of cognitive symptoms and significant increases in apoE levels in the cerebral spinal fluid in these patients. To gain insight into the mechanisms underlying these effects, we treated a cohort of aged male rats (26-month-old) with oral dose of Probucol for 30 days. Specifically, we examined the effects of Probucol on apoE production and its receptors (low density lipoprotein receptor [LDLr] and low density lipoprotein receptor-related protein [LRP]), astroglial marker of cell damage (glial fibrillary acidic protein [GFAP]), markers of neuronal synaptic plasticity and integrity (synaptosomal associated protein of 25 kDa [SNAP-25] and synaptophysin) as well as cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase [HMGCoAr]) in the hippocampus. We report that Probucol induces the production of apoE and one of its main receptors, LRP, increases HMGCoAr (rate-limiting enzyme in cholesterol synthesis), substantially attenuates age-related increases in glial activation, and induces production of synaptic marker SNAP-25, a molecule commonly associated with synaptogenesis and dendritic remodeling. These findings suggest that Probucol could promote neural and synaptic plasticity to counteract the synaptic deterioration associated with brain aging through an apoE/LRP-mediated system. Consistent with the beneficial effects of other cholesterol-lowering drugs such as the Statin, Probucol could also offers additional benefits based on apoE neurobiology.

Apolipoprotein E knockout mice display procedural deficits in the Morris water maze: analysis of learning strategies in three versions of the task.

Apolipoprotein E knockout (apoEKO) mice have been shown to be impaired in the spatial Morris water maze (MWM). However, several groups failed to replicate this finding. One reason for this inconsistency may stem from variations in the experimental protocols and environment between laboratories. In the present study, we have tested if age and variations in protocol implementation that specifically affect salience of the visual extramaze cues influence performance and navigational strategies in the MWM. We tested three- and 12-month-old apoEKO and wild type mice in three versions of the MWM differing on the availability of visual extramaze cues: (1) salient cues, (2) diffuse cues, and (3) absence of cues. Our results show that the presence of salient cues enhances acquisition performance of wild type, but not apoEKO mice in the MWM. This effect was restricted to the acquisition phase since apoEKO mice reached a level of performance that was comparable to that of controls toward the end of the task. No significant differences were detected between apoEKO and controls in either the diffuse cues or absence of cues paradigms. Thigmotaxic tendencies were observed in apoEKO mice and correlated high latency scores. Thigmotaxis may have interfered with the initial ability to engage in a proficient navigational strategy. These findings suggest that, in contrast to what has been proposed in the past, apoEKO mice appear not to be impaired in spatial memory per se but are deficient in a procedural component of the MWM. Furthermore, the procedural deficit and corresponding thigmotaxic tendencies of apoEKO mice appeared to increase with age. Taken together, these findings confirm our hypothesis that age and variations in experimental protocols can influence MWM performances.

Kinetics and equilibria in ligand binding by nitrophorins 1-4: evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap.

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.

Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus.

Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), a 19-kDa protein isolated from the salivary gland of R. prolixus, was purified by strong cation exchange and reverse-phase high performance liquid chromatographies. Based on 49 amino-terminal amino acid sequences of RPAI-1, primers were produced to generate probes to screen an R. prolixus salivary gland cDNA library. A phage containing the full-length clone of RPAI-1 codes for a mature protein of 155 amino acids. RPAI-1 shows sequence homology to triabin and pallidipin, lipocalins from Triatoma pallidipennis. The cDNA sequence was cloned in Pet17B Escherichia coli expression vector, producing an active peptide. RPAI-1 inhibits human platelet-rich plasma aggregation triggered by low concentrations of ADP, collagen, arachidonic acid, thromboxane A(2) mimetics (U46619), and very low doses of thrombin and convulxin. Here we show that ADP is the target of RPAI-1 since (i) RPAI-1 inhibits ADP-dependent large aggregation formation and secretion triggered by U46619, without affecting Ca(2+) increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE(1)-induced increase of cAMP (which is antagonized by U46619 in an ADP-dependent manner) was restored by RPAI-1, (iv) RPAI-1 inhibits low concentrations of ADP-mediated responses of indomethacin-treated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin alpha(2)beta(1)- nor glycoprotein VI-mediated platelet responses. We conclude that RPAI-1 is the first lipocalin described that inhibits platelet aggregation by a novel mechanism, binding to ADP.

Brain lipoprotein metabolism and its relation to neurodegenerative disease.

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.

Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti.

The gene encoding sialokinin I, the principal vasodilatory peptide of Aedes aegypti, has been isolated and characterized. Degenerate oligonucleotide primers based on peptide amino acid sequence were used to amplify a gene fragment from messenger RNA (mRNA) isolated from female salivary glands. The amplification product was used to probe a salivary gland complementary DNA (cDNA) library, and a number of corresponding cDNAs were isolated and their primary sequence determined. Analysis of the conceptual translation product of a 406-bp cDNA indicates that sialokinin I is expressed as a preprosialokinin and is subsequently post-translationally processed to the active peptide. Northern analysis revealed a 490-bp transcription product expressed exclusively in female salivary glands, and hybridization in situ of probes to RNA in whole tissues localized gene expression to the medial lobe of female salivary glands. Screening of an Ae. aegypti genomic library with the cDNA resulted in the isolation of a clone containing the gene, designated Sialokinin I (Sia I). Comparison of the cDNA with the genomic clone reveals two introns of 62 bp and 833 bp. Primer extension analysis showed that several transcription initiation sites are present. Southern analysis of genomic DNA shows that Sia I is most probably a single-copy gene. Similarities of the Sia I gene product with other genes are confined to the region encoding the active decapeptide.

The crystal structure of nitrophorin 4 at 1.5 A resolution: transport of nitric oxide by a lipocalin-based heme protein.

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.

Analyses of cDNA and recombinant protein for a potent vasoactive protein in saliva of a blood-feeding black fly, Simulium vittatum.

A cDNA was cloned from the salivary glands of a blood-feeding black fly Simulium vittatum. The encoded protein has been given the name Simulium vittatum erythema protein or SVEP, because of its ability to increase blood perfusion in skin capillaries, resulting in the well-characterized erythema of black fly bites. The full-length cDNA contains 548 base pairs which encode 152 amino acid residues of the nascent protein. Post-translational processing produces a mature, secreted protein of 133 residues with a molecular mass of 15.4 kDa. Recombinant SVEP (rSVEP) was produced in a baculovirus expression system and purified by a one-step reversed-phase HPLC procedure. Analyses of physical properties and biological potency demonstrated fidelity of rSVEP to the native protein. Recombinant SVEP relaxed rabbit aorta preparations when preconstricted with 2 micromol l-1 phenylephrine or 25 mmol l-1 K+ but not with 60 mmol l-1 K+. Further, the rSVEP-induced relaxation response of phenylephrine-constricted aorta was inhibited by glibenclamide (10 micromol l-1), suggesting that at least part of its action to relax smooth muscle may result from the opening of ATP-dependent K+ channels. SVEP is a novel salivary-gland-derived vasoactive protein that may be essential for blood feeding by black flies and could potentially enhance transmission of filarial parasites.

CRFergic innervation of the paraventricular nucleus of the rat hypothalamus: a tract-tracing study.

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.

Crystal structures of a nitric oxide transport protein from a blood-sucking insect.

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.

Identification of a steroidogenic neurohormone in female mosquitoes.

In the female mosquito, Aedes aegypti, neurohormones are released from the brain in response to a blood meal and stimulate the ovaries to secrete ecdysteroid hormones, which modulate yolk protein synthesis in the fat body. Neuropeptides with this bioactivity were isolated from head extracts, and partial sequences from these peptides when aligned gave a 31-residue sequence at the amino terminus. Oligonucleotide primers for this sequence were used to amplify with the polymerase chain reaction a genomic DNA product that hybridized to a clone from a head cDNA library. The cDNA encodes a 149-residue preprohormone that is processed into an 86-residue peptide, as indicated by the mass value obtained from the native peptide, with the expected amino-terminal sequence. After modification, the cDNA for the putative neurohormone was expressed in a bacterial system, and the purified peptide had high specific activity in bioassays, thus confirming that it is a steroidogenic gonadotropin, the first to be identified for invertebrates.

Comparison of the performance of younger and older adults on three versions of a puzzle reproduction task.

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.

Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus.

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.