Altered Functional Properties Research Articles

A t(1;11) translocation linked to schizophrenia and affective disorders gives rise to aberrant chimeric DISC1 transcripts that encode structurally altered, deleterious mitochondrial proteins.

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a risk factor for psychiatric illness through its disruption by a balanced chromosomal translocation, t(1;11)(q42.1;q14.3), that co-segregates with schizophrenia, bipolar disorder and depression. We previously reported that the translocation reduces DISC1 expression, consistent with a haploinsufficiency disease model. Here we report that, in lymphoblastoid cell lines, the translocation additionally results in the production of abnormal transcripts due to the fusion of DISC1 with a disrupted gene on chromosome 11 (DISC1FP1/Boymaw). These chimeric transcripts encode abnormal proteins, designated CP1, CP60 and CP69, consisting of DISC1 amino acids 1–597 plus 1, 60 or 69 amino acids, respectively. The novel 69 amino acids in CP69 induce increased α-helical content and formation of large stable protein assemblies. The same is predicted for CP60. Both CP60 and CP69 exhibit profoundly altered functional properties within cell lines and neurons. Both are predominantly targeted to mitochondria, where they induce clustering and loss of membrane potential, indicative of severe mitochondrial dysfunction. There is currently no access to neural material from translocation carriers to confirm these findings, but there is no reason to suppose that these chimeric transcripts will not also be expressed in the brain. There is thus potential for the production of abnormal chimeric proteins in the brains of translocation carriers, although at substantially lower levels than for native DISC1. The mechanism by which inheritance of the translocation increases risk of psychiatric illness may therefore involve both DISC1 haploinsufficiency and mitochondrial deficiency due to the effects of abnormal chimeric protein expression.GenBank accession numbers: DISC1FP1 (EU302123), Boymaw (GU134617), der 11 chimeric transcript DISC1FP1 exon 2 to DISC1 exon 9 (JQ650115), der 1 chimeric transcript DISC1 exon 4 to DISC1FP1 exon 4 (JQ650116), der 1 chimeric transcript DISC1 exon 6 to DISC1FP1 exon 3a (JQ650117).

Loss of Stem Cell Quiescence and Impaired Stem Cell Function of CD34+/CD38low Cells One Year Following Autologous Stem Cell Transplantation

Abstract 1893Autologous stem cell transplantation (ASCT) allows the application of high-dose chemotherapy and is included in the standard treatment regimens for multiple myeloma and relapsing lymphoma. The application of ASCT has considerably improved treatment outcome but in 30–50% of the patients the underlying malignant disorder relapses. In that case treatment options are limited, in part due to a diminished capacity of the transplanted bone marrow to tolerate chemotherapy. The transplanted bone marrow seems to be more vulnerable to chemotoxic stress. This is supported by our recent observations that progenitor cells from post-ASCT bone marrow demonstrate altered phenotypic and functional properties (Woolthuis et al. BMT 2011:110-115). In the present study, a more detailed examination of the hematopoietic stem cell (HSC) compartment post-ASCT was performed. The examined bone marrow cells were obtained from patients with normal peripheral blood cell counts one year after ASCT. First it was studied whether stem cell quiescence had been changed due to the ASCT procedure. Therefore the percentage of cells in the G0 phase was measured by staining the cells with Hoechst and Pyronin Y followed by flowcytometric analysis. Both the stem cell (CD34+/CD38low) and progenitor (CD34+/CD38+) fractions were analyzed. Post-ASCT bone marrow cells (n=6) were compared with normal bone marrow cells (n=9) and mobilized peripheral blood stem cells (PBSC) (n=7). Interestingly, post-ASCT bone marrow contained a significantly lower percentage of quiescent cells in the CD34+/CD38low fraction compared to normal bone marrow (mean percentage 23.6% (95%CI: 6.5–40.8) vs. 48.6% (95%CI: 31.4–65.9), p=0.045). In contrast no differences were observed in the CD34+/CD38+ fraction. To examine whether the loss of stem cell quiescence is associated with loss of stem cell function, stem cell frequency was analyzed in vitro by the long-term colony-initiating cell assay. These analyses revealed a strongly decreased stem cell frequency in post-ASCT CD34+ cells compared to normal CD34+ bone marrow cells (mean frequency 0.0016 (95%CI: 0.0003–0.0028) vs. 0.0206 (95%CI: 0.0162–0.0250), p=0.002). Importantly, CD34+ PBSC cells demonstrated a stem cell frequency comparable with the frequency observed in normal bone marrow, suggesting that the decrease in stem cell frequency observed in post-ASCT bone marrow can not simply be explained by the previous chemotherapy and the mobilization procedure. To obtain more insight into the mechanisms explaining our observations, levels of reactive oxygen species (ROS) were measured by flowcytometry. Comparable ROS levels were observed in post-ASCT and normal bone marrow CD34+/CD38low cells while the CD34+/CD38+ post-ASCT cells demonstrated significantly higher ROS levels compared to normal bone marrow (p=0.01). In addition significant higher ROS levels were observed in CD34+/CD38low PBSC compared to normal bone marrow (p=0.043). To define pathways associated with altered quiescence and/or ROS production, gene expression analysis was performed comparing CD34+ cells from post-ASCT bone marrow (n=6), normal bone marrow (n=31) and PBSC (n=5). Preliminary analysis reveals an upregulation in post-ASCT CD34+ cells of genes involved in cell cycle compared to normal bone marrow. In contrast, cell cycle genes appeared to be downregulated in CD34+ PBSC cells. In conclusion, our data indicate that the diminished regenerative capacity of bone marrow post-ASCT is likely related to a loss of stem cell quiescence and enhanced ROS production by progenitor cells which will make the bone marrow cells more vulnerable for the effects of cytotoxic stress. Disclosures:No relevant conflicts of interest to declare.

The sentinel role of the airway epithelium in asthma pathogenesis

The adoption of the concept that asthma is primarily a disease most frequently associated with elaboration of T-helper 2 (Th2)-type inflammation has led to the widely held concept that its origins, exacerbation, and persistence are allergen driven. Taking aside the asthma that is expressed in non-allergic individuals leaves the great proportion of asthma that is associated with allergy (or atopy) and that often has its onset in early childhood. Evidence is presented that asthma is primarily an epithelial disorder and that its origin as well as its clinical manifestations have more to do with altered epithelial physical and functional barrier properties than being purely linked to allergic pathways. In genetically susceptible individuals, impaired epithelial barrier function renders the airways vulnerable to early life virus infection, and this in turn provides the stimulus to prime immature dendritic cells toward directing a Th2 response and local allergen sensitization. Continued epithelial susceptibility to environmental insults such as viral, allergen, and pollutant exposure and impaired repair responses leads to asthma persistence and provides the mediator and growth factor microenvironment for persistence of inflammation and airway wall remodeling. Increased deposition of matrix in the epithelial lamina reticularis provides evidence for ongoing epithelial barrier dysfunction, while physical distortion of the epithelium consequent upon repeated bronchoconstriction provides additional stimuli for remodeling. This latter response initially serves a protective function but, if exaggerated, may lead to fixed airflow obstruction associated with more severe and chronic disease. Dual pathways in the origins, persistence, and progression of asthma help explain why anti-inflammatory treatments fail to influence the natural history of asthma in childhood and only partially does so in chronic severe disease. Positioning the airway epithelium as fundamental to the origins and persistence of asthma provides a rationale for pursuit of therapeutics that increase the resistance of the airways to environmental insults rather than concentrating all effort on suppressing inflammation.

Effect of saliva from horse fly Hybomitra bimaculata on kinetic properties of Na, K-ATPase: possible role in regulation of relaxation

The possible involvement of salivary gland extract (SGE) from horse flies in modifying hyperpolarization and relaxation via alterations in functional properties of sarcolemmal Na,K-ATPase in the host tissue was tested in vitro by application of various amounts of SGE from Hybomitra bimaculata.SGE in the amount of 3 µg proteins representing approximately the equivalent of one salivary gland of Hybomitra bimaculata induced a stimulatory effect on Na,K-ATPase at all ATP concentrations applied. This effect resulted from the improved ATP-binding site affinity in the Na,K-ATPase molecule, as implicated by the reduction in KM. Increasing the amount of SGE to 6.5 µg resulted in inhibition of the enzyme, which was characterized by reduction in Vmax and also KM. This suggests that in the presence of relatively high Hybomitra bimaculata SGE concentration some SGE components affect Na,K-ATPase, when ATP is already bound to the enzyme.Our results indicate that SGE from the horse fly Hybomitra bimaculata contain at least two different biologically active compounds modifying the acute recovery and maintenance of excitability during contractile activity in the host tissue by affecting Na,K-ATPase with opposite effects, depending on the ratio of SGE-proteins to proteins of the host tissue.

Altered Functional Properties of Eosinophils In Sickle Cell Anemia and Effects of Hydroxyurea Therapy

AbstractAbstract 2656The pathophysiology of sickle cell anemia (SCA) is now recognized to include a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion that characterize this disease. The vaso-occlusive process is a multi-step process that involves different cell types, such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. In vivo transgenic animal studies indicated that the neutrophil appears to be the most important leukocyte in vaso-occlusion in previous transgenic animal in vivo studies; however, eosinophils (EOs) may also participate in this phenomenon in humans. EOs are found in significantly elevated numbers in sickle cell disease individuals and, in static adhesion assays, demonstrate augmented adhesive properties suggesting an activated state of these cells. However, the role of EOs in the pathophysiology of SCD and the effect of hydroxyurea (HU) therapy on the functional properties of these cells are not well understood. The aim of this study was to evaluate EO adhesion, migration and degranulation in SCA patients and the effect of HU therapy on these cells. EOs were purified from the peripheral blood of healthy controls (CON) (n≥10), steady-state SCA patients (n≥12) and SCA patients on HU therapy (n≥15), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion was determined by static adhesion assays and degranulation was evaluated by measurement of eosinophil peroxidase activity. Migration of EOs was determined in 96-multiwell chemotaxis chamber assays. Plasma levels of Eotaxin-1, 2 and RANTES, important eosinophil attracting chemokines, were determined by ELISA. The absolute number of EOs in the peripheral blood of SCD patients not treated with HU was significantly higher compared to control individuals (0.504±0.09 vs 0.188±0.04; P=0.01, respectively). SCA patients taking HU presented significantly lower numbers of EOs (0.200±0.05, P = 0.01). Basal adhesion of EOs from SCA patients was significantly higher than in healthy controls (22.8±2.6 vs 13.7±1.7%, respectively, P = 0.004). Furthermore, the EOs from SCA patients on HU therapy demonstrated a significantly lower adhesion to fibronectin (12.5±2.0%, P=0.002). Spontaneous EO chemotaxis was significantly increased in SCA patients, compared to healthy controls (12.9±1.56 vs 6.97±0.8 × 105/ml, respectively, P=0.009). Interestingly, the chemotatic response of EOs isolated from SCA patients taking HU was also higher than that of control EOs (12.1± 1.9 × 105/ml). EO chemotaxis in response to RANTES, Eotaxin and IL5 was significantly augmented in all groups compared to spontaneous chemotaxis (RANTES: CON: 15.3± 2.3; SCA: 19.9±3.0; SCAHU: 25.0±4.7; Eotaxin: CON: 18.7±3.0; SCA: 24.5±4.2; SCAHU: 24.0±5.2; IL5: CON: 13.4±2.4; SCA: 18.8±3.0, SCAHU: 16.6±4.5, P<0.05). Baseline eosinophil peroxidase release was higher in SCA EOs, compared to CON EOs (0.4±0.04 vs 0.2±0.02, respectively, P=0.001), but patients on HU presented a lower EOs degranulation than SCA not on HU (0.19±0.03, P=0.003). Plasma levels of Eotaxin-1 and RANTES were significantly higher in SCA individuals, compared to CON individuals (124±7; 1057±125 vs 78±7; 540±50.2 pg/ml, P=0.0002; P=0.0009, respectively). HU therapy was not associated with any change in Eotaxin-1 and RANTES plasma levels (117.9±9.7; 1175±126.2 pg/ml, respectively). Eotaxin-2 plasma levels did not vary among groups (485±80.8, 450±34, 398±47.8 pg/ml for CON, SCA and SCAHU, respectively). EOs of patients with SCA demonstrate a higher capacity for spontaneous migration, stimulated migration and degranulation. Therapy with HU is associated with reduced adhesion and degranulation of EOs in these patients, but had no effect on the chemotactic ability of these cells or chemokine levels. The presence of relative eosinophilia along with altered functional properties in SCA patients warrants further investigation of the role of EOs in the vaso-occlusive process. In particular, complications associated with allergic reactions, such as acute thoracic syndrome and asthma in SCA children, in which EO adhesion and degranulation may be more important, should be studied. The characteristics of the effect of HU on EO also suggest that SCA events in which EO chemotaxis plays a role are likely to respond poorly to HU therapy, and should lead to the development of alternative therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Lesioning alters functional properties in isolated spinal cord hemisegmental networks

Hemisegmental networks produced by longitudinal lesions of the spinal cord midline are able to generate rhythmic bursting activity. This has led to the suggestion that hemisegmental networks can independently burst in the intact spinal cord. Previous analyses in the lamprey spinal cord failed to show hemisegmental bursting in NMDA. This was subsequently attributed to the failure to wait sufficient time for NMDA-evoked hemisegmental activity to recover after being abolished by the lesion, which can take tens of minutes to hours. The reason for this delay in the onset of NMDA-evoked activity was not previously addressed. We have investigated it here by examining two hypotheses: that hemisegmental networks intrinsically burst under normal conditions but that NMDA-evoked bursting was temporarily silenced by lesion-induced transmitter release; or that lesioning altered functional properties in the hemisegment that subsequently led to the development of bursting. We found no evidence to support transmitter-induced silencing of ongoing NMDA-evoked hemisegmental activity, but did find evidence for significant changes in the cellular and synaptic properties of motor neurons and premotor excitatory interneurons in lesioned hemisegmental networks. These results thus suggest that there are lesion-induced changes in functional properties in hemisegmental networks. As the interpretation of lesion studies rests on the assumption that the functional properties of hemisegmental components are not altered, further work is needed before conclusions can be made about the function of the intact system.

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K(d)=0.60 microM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.

Defining the Structural Consequences of Mechanism-Based Inactivation of Mammalian Cytochrome P450 2B4 Using Resonance Raman Spectroscopy

In view of the potent oxidizing strength of cytochrome P450 intermediates, it is not surprising that certain substrates can give rise to reactive species capable of attacking the heme or critical distal-pocket protein residues to irreversibly modify the enzyme in a process known as mechanism-based (MB) inactivation, a result that can have serious physiological consequences leading to adverse drug-drug interactions and toxicity. While methods exist to document the attachment of these substrate fragments, it is more difficult to gain insight into the structural basis for the altered functional properties of these modified enzymes. In response to this pressing need to better understand MB inhibition, we here report the first application of resonance Raman spectroscopy to study the inactivation of a truncated form of mammalian CYP2B4 by the acetylenic inhibitor 4-(tert-butyl)phenylacetylene, whose activated form is known to attach to the distal-pocket T302 residue of CYP2B4.

A Biophysical-Biochemical Comparison of Hemoglobins from Mammoth, Asian Elephant, and Human

This study is aimed at investigating the molecular basis of environmental adaptation of hemoglobin from tropical climate to low temperature in the Arctic region. We have carried out a biochemical-biophysical characterization of the structural and functional properties of hemoglobins from woolly mammoth (Hb WM) and Asian elephant (Hb AE) and compared those to human hemoglobins (Hb A and Hb A2) in 0.1 M phosphate buffer. Hb A consists of two α and two β subunits. Hb AE was found to contain two α subunits and two β/δ fusion subunits. Hb WM was expressed by inserting Asian elephant α-like and β/δ-like cDNA into our E. coli Hb plasmid (pHE2), and then introducing the mammoth-specific residue differences (αK5N, β/δT12A, β/δA86S, and β/δG101Q) into the Asian elephant plasmid. Since Hb AE and Hb WM contain β/δ fusion chains, we have also compared them to Hb A2, which contains δ chains instead of the β subunits present in Hb A. Oxygen affinity, Bohr effect, and cooperativity of oxygenation were measured at different temperatures and pH and 1H-NMR spectra were obtained for structural comparisons for each Hb. Our results show: (i) Hb AE has the higher O2 affinity as compared to Hb WM, Hb A2, and Hb A; (ii) the effect of an allosteric effector, inositol hexaphosphate (IHP), is the most prominent on Hb A2 as compared to Hb A, Hb AE, and Hb WM. 1H NMR results indicates that the α1β/δ1 and α1β/δ2 interfaces are perturbed in both Hb AE and Hb WM, whereas only the α1δ1 interface is perturbed in Hb A2 compared to Hb A. Hb AE and Hb WM have structural features that are very different from that of Hb A2 and Hb A, consistent with the altered functional properties.

The epileptic human hippocampal cornu ammonis 2 region generates spontaneous interictal-like activity in vitro

The dentate gyrus, the cornu ammonis 2 region and the subiculum of the human hippocampal formation are resistant to the cell loss associated with temporal lobe epilepsy. The subiculum, but not the dentate gyrus, generates interictal-like activity in tissue slices from epileptic patients. In this study, we asked whether a similar population activity is generated in the cornu ammonis 2 region and examined the electrophysiological and neuroanatomical characteristics of human epileptic cornu ammonis 2 neurons that may be involved. Hippocampal slices were prepared from postoperative temporal lobe tissue derived from epileptic patients. Field potentials and multi-unit activity were recorded in vitro using multiple extracellular microelectrodes. Pyramidal cells were characterized in intra-cellular records and were filled with biocytin for subsequent anatomy. Fluorescent immunostaining was made on fixed tissue against the chloride-cation cotransporters sodium-potassium-chloride cotransporter-1 and potassium-chloride cotransporter-2. Light and electron microscopy were used to examine the parvalbumin-positive perisomatic inhibitory network. In 15 of 20 slices, the hippocampal cornu ammonis 2 region generated a spontaneous interictal-like activity, independently of population events in the subiculum. Most cornu ammonis 2 pyramidal cells fired spontaneously. All cells fired single action potentials and burst firing was evoked in three cells. Spontaneous excitatory postsynaptic potentials were recorded in all cells, but hyperpolarizing inhibitory postsynaptic potentials were detected in only 27% of the cells. Two-thirds of cornu ammonis 2 neurons showed depolarizing responses during interictal-like events, while the others were inhibited, according to the current sink in the cell body layer. Two biocytin-filled cells both showed a pyramidal-like morphology with axons projecting to the cornu ammonis 2 and cornu ammonis 3 regions. Expression of sodium-potassium-chloride cotransporter-1 and potassium-chloride cotransporter-2 was reduced in some cells of the epileptic cornu ammonis 2 region, but not to an extent corresponding to the proportion of cells in which hyperpolarizing postsynaptic potentials were absent. Numbers of parvalbumin-positive inhibitory cells and axons were shown to be decreased in the epileptic tissue. Electron microscopy showed the preservation of somatic inhibitory input of cornu ammonis 2 cells, and confirmed the loss of parvalbumin from the interneurons rather than their death. An extra excitatory input (partly coming from sprouted mossy fibres) was demonstrated to innervate cornu ammonis 2 cell bodies. Our results show that the cornu ammonis 2 region of the sclerotic human hippocampus can generate an independent epileptiform activity. Inhibitory and excitatory signalling were functional but modified in epileptic cornu ammonis 2 pyramidal cells. Overexcitation and the altered functional properties of perisomatic inhibitory network, rather than a modified chloride homeostasis, may account for the perturbed gamma-aminobutyric acid-ergic signalling and the generation of interictal-like activity in the human epileptic cornu ammonis 2 region.

Altered functional properties of satellite glial cells in compressed spinal ganglia

The cell bodies of sensory neurons in the dorsal root ganglion (DRG) are enveloped by satellite glial cells (SGCs). In an animal model of intervertebral foraminal stenosis and low-back pain, a chronic compression of the DRG (CCD) increases the excitability of neuronal cell bodies in the compressed ganglion. The morphological and electrophysiological properties of SGCs were investigated in both CCD and uninjured, control lumbar DRGs. SGCs responded within 12 h of the onset of CCD as indicated by an increased expression of glial fibrillary acidic protein (GFAP) in the compressed DRG but to lesser extent in neighboring or contralateral DRGs. Within 1 week, coupling through gap junctions between SGCs was significantly enhanced in the compressed ganglion. Under whole-cell patch clamp recordings, inward and outward potassium currents, but not sodium currents, were detected in individual SGCs. SGCs enveloping differently sized neurons had similar electrophysiological properties. SGCs in the compressed vs. control DRG exhibited significantly reduced inwardly rectifying potassium currents (Kir), increased input resistances and positively shifted resting membrane potentials. The reduction in Kir was greater for nociceptive medium-sized neurons compared to non-nociceptive neurons. Kir currents of SGCs around spontaneously active neurons were significantly reduced 1 day after compression but recovered by 7 days. These data demonstrate rapid alterations in glial membrane currents and GFAP expression in close temporal association with the development of neuronal hyperexcitability in the CCD model of neuropathic pain. However, these alterations are not fully sustained and suggest other mechanisms for the maintenance of the hyperexcitable state.

Altered functional properties of a TRPM2 variant in Guamanian ALS and PD

Two related neurodegenerative disorders, Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD), originally occurred at a high incidence on Guam, in the Kii peninsula of Japan, and in southern West New Guinea more than 50 years ago. These three foci shared a unique mineral environment characterized by the presence of severely low levels of Ca(2+) and Mg(2+), coupled with high levels of bioavailable transition metals in the soil and drinking water. Epidemiological studies suggest that genetic factors also contribute to the etiology of these disorders. Here, we report that a variant of the transient receptor potential melastatin 2 (TRPM2) gene may confer susceptibility to these diseases. TRPM2 encodes a calcium-permeable cation channel highly expressed in the brain that has been implicated in mediating cell death induced by oxidants. We found a heterozygous variant of TRPM2 in a subset of Guamanian ALS (ALS-G) and PD (PD-G) cases. This variant, TRPM2(P1018L), produces a missense change in the channel protein whereby proline 1018 (Pro(1018)) is replaced by leucine (Leu(1018)). Functional studies revealed that, unlike WT TRPM2, P1018L channels inactivate. Our results suggest that the ability of TRPM2 to maintain sustained ion influx is a physiologically important function and that its disruption may, under certain conditions, contribute to disease states.

Fourth Conference on Epileptogenesis, May 23–26, 2007, Pisa, Italy

Fourth Conference on Epileptogenesis, May 23–26, 2007, Pisa, Italy

Nuclear Exclusion of the HIV-1 Host Defense Factor APOBEC3G Requires a Novel Cytoplasmic Retention Signal and Is Not Dependent on RNA Binding

Human APOBEC3G (hA3G) is a host factor that defends against HIV-1 as well as other exogenous retroviruses and endogenous retroelements. To this end, hA3G is restricted to the cytoplasm of T lymphocytes where it interacts with viral RNA and proteins to assemble with viral particles causing a post-entry block during reverse transcription. hA3G also exhibits a mechanism to inhibit the reverse transcription of retroelements by RNA binding and sequestration into mRNA processing centers in the cytoplasm. We have determined that the molecular basis for this specialized property of hA3G is a novel cytoplasmic retention signal (CRS) that is necessary and sufficient to restrict wild-type hA3G and chimeric constructs to the cytoplasm. The CRS resides within amino acids 113-128 and is embedded within a basic flanking sequence and does not require RNA binding to retain hA3G in the cytoplasm. Paralogs of hA3G that have nuclear or cytoplasmic distributions differ from hA3G within the region encompassing the CRS motif with respect to charge and amino acid composition. We propose that the CRS enables hA3G to interact with cytoplasmic factors, and thereby enables hA3G to serve in host cell defense by restricting an antiviral sentinel to the cytoplasm. The CRS lies in a region involved in both Gag and Vif interactions; therefore, identification of this motif has important implications for the design of therapeutics that target HIV-1 while maintaining antiviral and cellular functions.

Co-expression of neuropeptide Y Y1 and Y5 receptors results in heterodimerization and altered functional properties

Centrally administered neuropeptide Y (NPY) produces anxiolytic and orexigenic effects by interacting with Y1 and Y5 receptors that are colocalized in many brain regions. Therefore, we tested the hypothesis that co-expression of Y1 and Y5 receptors results in heterodimerization, altered pharmacological properties and altered desensitization. To accomplish this, the carboxyl-termini of Y1 and Y5 receptors were fused with Renilla luciferase and green flourescent protein and the proximity of the tagged receptors assessed using bioluminescent resonance energy transfer. Under basal conditions, cotransfection of tagged Y1 receptor and Y5 produced a substantial dimerization signal that was unaffected by the endogenous, nonselective agonists, NPY and peptide YY (PYY). Selective Y5 agonists produced an increase in the dimerization signal while Y5 antagonists also produced a slight but significant increase. In the absence of agonists, selective antagonists decreased dimerization. In functional studies, Y5 agonists produced a greater inhibition of adenylyl cyclase activity in Y1/Y5 cells than cells expressing Y5 alone while NPY and PYY exhibited no difference. With PYY stimulation, the Y1 antagonist became inactive and the Y5 antagonist exhibited uncompetitive kinetics in the Y1/Y5 cell line. In confocal microscopy studies, Y1/Y5 co-expression resulted in increased Y5 signaling following PYY stimulation. Addition of both Y1 and Y5 receptor antagonists was required to significantly decrease PYY-induced internalization. Therefore, Y1/Y5 co-expression results in heterodimerization, altered agonist and antagonist responses and reduced internalization rate. These results may account for the complex pharmacology observed when assessing the responses to NPY and analogs in vivo.

β‐strand recombination in tricalbin evolution and the origin of synaptotagmin‐like C2 domains

Two protein families involved in membrane traffic, tricalbins and synaptotagmins, contain several copies of C2 domains and are related based on their sequence and domain architecture. Paradoxically, tricalbin and synaptotagmin C2 domains belong to different structural types with apparent circular permutation of terminal beta-strands. To understand whether a topological switch took place, we analyzed tricalbin and synaptotagmin-like C2 domains using two-dimensional structural analysis. We found that yeast tricalbins contain five to six C2 domains. One of these C2 domains possesses many features of synaptotagmin-like C2 domains and also carries a conserved C-terminal strand that is similar to its structural equivalent in synaptotagmin-like C2 domains, suggesting a structural permutation event. Indeed, among higher eukaryotes, animal tricalbins have evolved a C2 domain with synaptotagmin-like topology indicating that the structural conversion has taken place. Investigation of plant synaptotagmins, however, proves that they are direct tricalbin orthologs. Our analysis shows that beta-strand recombination is a possible evolutionary mechanism to generate new structural topologies with altered functional properties.

Influence of pH on the solubility and conformational characteristics of muscle proteins from mullet ( Mugil cephalus)

Mullet ( Mugil cephalus) muscle homogenates were adjusted to different pH ranging from 2 to12 and the proteins extracted were evaluated for changes in solubility and conformational characteristics viz. surface hydrophobicity and reactive sulphydryl groups. Altering the pH of muscle homogenate to acidic or alkaline increased protein solubility. The hydrophobicity of the proteins increased on exposure to extreme pH indicating unfolding. The reactive sulphydryl groups decreased at acidic and alkaline pH with the lowest at pH 4. When the pH of the muscle homogenates was brought back to the original pH (6.3), the protein solubility was found to decrease. Reactive sulphydryl groups and ANS hydrophobicity of the proteins increased on readjusting the pH resulting in a molten-globule state. The electrophortogram of the samples corresponded well with the observations. Alterations in functional properties of these modified proteins are an area of interest for commercial application.

Quasispecies and its impact on viral hepatitis

Quasispecies dynamics mediates adaptability of RNA viruses through a number of mechanisms reviewed in the present article, with emphasis on the medical implications for the hepatitis viruses. We discuss replicative and non-replicative molecular mechanisms of genome variation, modulating effects of mutant spectra, and several modes of viral evolution that can affect viral pathogenesis. Relevant evolutionary events include the generation of minority virus variants with altered functional properties, and alterations of mutant spectrum complexity that can affect disease progression or response to treatment. The widespread occurrence of resistance to antiviral drugs encourages new strategies to control hepatic viral disease such as combination therapies and lethal mutagenesis. In particular, ribavirin may be exerting in some cases its antiviral activity with participation of its mutagenic action. Despite many unanswered questions, here we document that quasispecies dynamics has provided an interpretation of the adaptability of the hepatitis viruses, with features conceptually similar to those observed with other RNA viruses, a reflection of the common underlying Darwinian principles.

Purification, crystallization and X-ray diffraction analyses of the T. elongatus PSII core dimer with strontium replacing calcium in the oxygen-evolving complex

The core complex of photosystem II (PSII) was purified from thermophillic cyanobaterium Thermosynechococcus elongatus grown in Sr 2+-containing and Ca 2+-free medium. Functional in vivo incorporation of Sr 2+ into the oxygen-evolving complex (OEC) was confirmed by EPR analysis of the isolated and highly purified SrPSII complex in agreement with the previous study of Boussac et al. [J. Biol. Chem. 279 (2004) 22809–22819]. Three-dimensional crystals of SrPSII complex were obtained which diffracted to 3.9 Å and belonged to the orthorhombic space group P2 12 12 1 with unit cell dimensions of a = 133.6 Å, b = 236.6 Å, c = 307.8 Å. Anomalous diffraction data collected at the Sr K-X-ray absorption edge identified a novel Sr 2+-binding site which, within the resolution of these data (6.5 Å), is consistent with the positioning of Ca 2+ in the recent crystallographic models of PSII [Ferreira et al. Science 303 (2004) 1831–1838, Loll et al. Nature 438 (2005) 1040–1044]. Fluorescence measurements on SrPSII crystals confirmed that crystallized SrPSII was active in transferring electrons from the OEC to the acceptor site of the reaction centre. However, SrPSII showed altered functional properties of its modified OEC in comparison with that of the CaPSII counterpart: slowdown of the Q A-to-Q B electron transfer and stabilized S 2Q A − charge recombination.

Characterization of wheat puroindoline proteins

Puroindoline proteins were purified from selected UK-grown hexaploid wheats. Their identities were confirmed on the basis of capillary electrophoresis mobilities, relative molecular mass and N-terminal amino acid sequencing. Only one form of puroindoline-a protein was found in those varieties, regardless of endosperm texture. Three allelic forms of puroindoline-b protein were identified. Nucleotide sequencing of cDNA produced by RT-PCR of isolated mRNA indicated that these were the 'wild-type', found in soft wheats, puroindoline-b containing a Gly-->Ser amino acid substitution (position 46) and puroindoline-b containing a Trp-->Arg substitution (position 44). The latter two were found in hard wheats. Microheterogeneity, due to short extensions and/or truncations at the N-terminus and C-terminus, was detected for both puroindoline-a and puroindoline-b. The type of microheterogeneity observed was more consistent for puroindoline-a than for puroindoline-b, and may arise through slightly different post-translational processing pathways. A puroindoline-b allele corresponding to a Leu-->Pro substitution (position 60) was identified from the cDNA sequence of the hard variety Chablis, but no mature puroindoline-b protein was found in this or two other European varieties known to possess this puroindoline-b allele. Wheats possessing the puroindoline-b proteins with point mutations appeared to contain lower amounts of puroindoline protein. Such wheats have a hard endosperm texture, as do wheats from which puroindoline-a or puroindoline-b are absent. Our results suggest that point mutations in puroindoline-b genes may confer hard endosperm texture through accumulation of allelic forms of puroindoline-b proteins with altered functional properties and/or through lower amounts of puroindoline proteins.