An Endoplasmic Reticulum Retention Signal Located in the Extracellular Amino-terminal Domain of the NR2A Subunit of N-Methyl-d-aspartate Receptors

N-Methyl-d-aspartate (NMDA) receptors play critical roles in complex brain functions as well as pathogenesis of neurodegenerative diseases. There are many NMDA isoforms and subunit types that, together with subtype-specific assembly, give rise to significant functional heterogeneity of NMDA receptors. Conventional NMDA receptors are obligatory heterotetramers composed of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits. When individually expressed in heterogeneous cells, most of the NR1 splice variants and the NR2 subunits remain in the endoplasmic reticulum (ER) and do not form homomeric channels. The mechanisms underlying NMDA receptor trafficking and functional expression remain uncertain. Using truncated and chimeric NMDA receptor subunits expressed in heterogeneous cells and hippocampal neurons, together with immunostaining, biochemical, and functional analyses, we found that the NR2A amino-terminal domain (ATD) contains an ER retention signal, which can be specifically masked by the NR1a ATD. Interestingly, no such signal was found in the ATD of the NR2B subunit. We further identified the A2 segment of the NR2A ATD to be the primary determinant of ER retention. These findings indicate that NR2A-containing NMDA receptors may undergo a different ER quality control process from NR2B-containing NMDA receptors.Ionotropic glutamate receptors (iGluRs)² mediate most of the excitatory neurotransmission in the central nervous system. They play key roles in complex brain functions as well as in the pathogenesis of neurodegenerative diseases. Based on pharmacological properties and sequence similarities, iGluRs can be grouped into three major subtypes: GluR1 to -4 subunits form α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, GluR5 to -7 and KA1 and -2 subunits make up kainate receptors, and NR1 together with NR2A to -D subunits comprise the NMDA receptors (1). All iGluR subunits share a unique membrane topology with a large extracellular NH₂-terminal domain, three transmembrane segments (TM1 (transmembrane domain 1), TM3, and TM4), a P-loop region, and a cytoplasmic COOH terminus (2, 3). Based on the sequence homology to bacterial periplasmic binding proteins, the NH₂-terminal domain of iGluRs can be divided into two domains in tandem: the amino-terminal domain (ATD), which includes the first 400 or so amino acids (4), and the following S1 domain preceding TM1, which forms the ligand-binding domain together with the extracellular loop between TM3 and TM4 (S2 domain) (5, 6).Among iGluRs, NMDA receptors are special in that conventional NMDA receptors are obligatory tetrameric membrane proteins composed of two glycine-binding NR1 and two glutamate-binding NR2 subunits. The NR1 subunit is essential for the formation of functional NMDA receptor channel, whereas the NR2 subunit modifies channel properties, such as current kinetics and channel conductance (1). The major NR1 splice variant and the NR2 subunits are retained in the ER when expressed alone in heterogeneous cells. Only when expressed together do they form functional receptors on the cell surface (7–9). In the last decade, enormous progress has been made in understanding the phenomenology and mechanisms of functional plasticity of NMDA receptors. However, much less is known about the mechanisms underlying the ER retention of NMDA receptor subunits. Previous studies focused on the COOH terminus have shown that the NR1a subunit contains an ER retention signal, RRR, in the C1 cassette, whereas a motif, HLFY, found in the NR2B subunit immediately following the TM4 (10) or, at least, the presence of any two amino acid residues after NR2 TM4 (11) is required for the export of NR1-NR2 complexes from the ER. Recently, novel ER retention signals were identified in the TM3 of both NR1 and NR2B subunits. In addition, TM3 of both NR1 and NR2B and TM4 of NR1 are necessary for masking ER retention signals found in TM3 (12).In the present study, we focused on the functional role of the ATD in the surface expression of NMDA receptors. Interestingly, we found an ER retention signal located in the ATD of the NR2A subunit but not in the corresponding domain of the NR2B. It is suggested that NR2A-containing NMDA receptors may undergo an ER quality control process different from that of NR2B-containing NMDA receptors.     

Identification of Dopamine "D3'' and "D4'' Binding Sites, Labelled with [3H]2-Amino-6,7-Dihydroxy- 1,2,3,4- Tetrahydronaphthalene, as High Agonist Affinity States of the D1 and D2 Dopamine Receptors, Respectively

On the basis of affinity differences for spiperone, two binding sites for [3H](+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([3H]ADTN) in the rat brain could be distinguished: "D3" with a low and "D4" with a high affinity for spiperone. Evidence is provided that D3 and D4 sites are related to high agonist affinity states of the D1 and D2 dopamine receptors, respectively. Various well-known selective D1 and D2 agonists and antagonists showed potencies at these sites in agreement with this hypothesis. A comparison of the Bmax values for [3H]ADTN binding to D3 and D4 sites with the numbers of D1 receptors (labelled by [3H]SCH 23390) and of D2 receptors (labelled by [3H]spiperone), both in the striatum and in the mesolimbic system, indicated that under the conditions used for 3H-agonist binding experiments, both populations of D1 and D2 receptors were converted to their high agonist affinity states to a considerable, although different extent. In fact, when competition experiments with [3H]spiperone were performed under the conditions otherwise used for [3H]ADTN binding experiments (instead of the conditions usually used for antagonist binding), substantial shifts of the displacement curves of 3,4-dihydroxyphenylethylamine (dopamine) and ADTN toward higher affinities were observed. A comparison of the effects of various agonists and antagonists in the [3H]ADTN binding experiments and in functional tests revealed a significant correlation between their potencies at D4 binding sites and at D2 receptors modulating the release of [3H]acetylcholine from striatal slices. However, in the situation of the D1/D3 pair, when the measurement of adenylate cyclase activity was taken as a functional test for D1 receptors, agonists were more active in the binding than in the functional test, whereas for many antagonists the opposite was found. The results are discussed with regard to the classification and functional aspects of brain dopamine receptors.     

Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop

To obtain more information of the functional domains of the NPC1 protein, the mutational spectrum and the level of immunoreactive protein were investigated in skin fibroblasts from 30 unrelated patients with Niemann-Pick C1 disease. Nine of them were characterized by mild alterations of cellular cholesterol transport (the "variant" biochemical phenotype). The mutations showed a wide distribution to nearly all NPC1 domains, with a cluster (11/32) in a conserved NPC1 cysteine-rich luminal loop. Homozygous mutations in 14 patients and a phenotypically defined allele, combined with a new mutation, in a further 10 patients allowed genotype/phenotype correlations. Premature-termination-codon mutations, the three missense mutations in the sterol-sensing domain (SSD), and A1054T in the cysteine-rich luminal loop all occurred in patients with infantile neurological onset and "classic" (severe) cholesterol-trafficking alterations. By western blot, NPC1 protein was undetectable in the SSD missense mutations studied (L724P and Q775P) and essentially was absent in the A1054T missense allele. Our results thus enhance the functional significance of the SSD and demonstrate a correlation between the absence of NPC1 protein and the most severe neurological form. In the remaining missense mutations studied, corresponding to other disease presentations (including two adults with nonneurological disease), NPC1 protein was present in significant amounts of normal size, without clear-cut correlation with either the clinical phenotype or the "classic"/"variant" biochemical phenotype. Missense mutations in the cysteine-rich luminal loop resulted in a wide array of clinical and biochemical phenotypes. Remarkably, all five mutant alleles (I943M, V950M, G986S, G992R, and the recurrent P1007A) definitively correlated with the "variant" phenotype clustered within this loop, providing new insight on the functional complexity of the latter domain.     

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast

Background

The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified.

Methods

AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined.

Results

AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA? (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA? was confirmed by co-immunoprecipitation analysis.

Conclusion

The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates.

General significance

Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.     

Functional promiscuity correlates with conformational heterogeneity in A-class glutathione S-transferases

The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability.     

Cutting edge: functional characterization of the effect of the C3H/HeJ defect in mice that lack an Lpsn gene: in vivo evidence for a dominant negative mutation

A point mutation in the Tlr4 gene, which encodes Toll-like receptor 4, has recently been proposed to underlie LPS hyporesponsiveness in C3H/HeJ mice (Lpsd). The data presented herein demonstrate that F1 progeny from crosses between mice that carry a approximately 9-cM deletion of chromosome 4 (including deletion of LpsTlr4) and C3H/HeJ mice (i.e., Lps0 x Lpsd F1 mice) exhibit a pattern of LPS sensitivity, measured by TNF activity, that is indistinguishable from that exhibited by Lpsn x Lpsd F1 progeny and whose average response is "intermediate" to parental responses. Thus, these data provide clear functional support for the hypothesis that the C3H/HeJ defect exerts a dominant negative effect on LPS sensitivity; however, expression of a normal Toll-like receptor 4 molecule is apparently not required.     

The Global Sequence Signature algorithm unveils a structural network surrounding heavy chain CDR3 loop in Camelidae variable domains

Background

A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs — complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes.

Methods

An original algorithmic approach (Global Sequence Signature—GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences.

Results

Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation.

Conclusions

Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop.

General significance

In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.     

Nucleotide sequence of a 'truncated rRNA operon' of the Euglena gracilis chloroplast genome.

An extra 16S rRNA gene (s-16S rDNA) from the Euglena gracilis chloroplast genome and several hundred positions of its flanking regions have been sequenced. The structural part has 1486 positions and is to 98% homologous in its sequence with the 16S rRNA gene in functional chloroplast rRNA operons. Sequences of about 200 positions upstream and 15 positions downstream of the structural part of the s-16S rRNA gene region are highly homologous with corresponding parts in the functional operon. Neither tRNA genes (A1a, I1e) nor parts of the 23S and 5S rRNA genes are found within 557 positions after the 3' end of the s-16S rRNA gene, i.e., the 330 bp homology, observed in electron microscopic studies of heteroduplexes (4), between the s-16S rDNA downstream region and the 6.2 kb repeated segment containing the functional rRNA operon, must be due to a DNA stretch in the interoperon spacer. A structural model of the "truncated rRNA operon" is presented. Results from S-1 endonuclease analysis suggest that the s-16S rDNA region is probably not transcribed into stable s-16S rRNA.     

异色瓢虫法国种群对豆蚜的捕食效应

异色瓢虫Harmonia axyridis(Pallas)的法国种群具有飞行能力较低的特点,在生物防治方面可能更具利用价值。为了使其更好的在田间应用,本文进行了异色瓢虫的法国种群各龄幼虫及雌、雄成虫对豆蚜的捕食功能比较研究。结果表明:各龄幼虫及雌、雄成虫对豆蚜捕食功能反应符合Holling II模型,检验各圆盘方程理论值和实测值拟合较好。同一虫态在不同空间水平对蚜虫的捕食能力有显著差异,在广口瓶处理中(较大空间),1～4龄幼虫及雌、雄成虫的最大理论捕食量分别为4.51、83.33、111.12、166.67、190及200头;不同虫期及成虫的瞬间攻击率a为3龄>4龄>雄虫>雌虫>2龄>1龄,且1龄的处理时间Th最长(0.2225);在培养皿处理中(较小空间),1～4龄幼虫及雌、雄成虫的最大理论捕食量分别为6.17、19.23、45.45、100、142.86、111.11头,不同虫期的瞬间攻击率a为2龄>3龄>雄虫>4龄>1龄>雌虫,且4龄的处理时间Th最长(0.1624)。     

Investigation of brain functional connectivity in patients with mild cognitive impairment: A functional near‐infrared spectroscopy (fNIRS) study

This study examines brain functional connectivity in both cognitively normal seniors and patients with mild cognitive impairment (MCI) to elucidate prospective markers of MCI. A homemade four‐channel functional near‐infrared spectroscopy (fNIRS) system was employed to measure hemodynamic responses in the subjects' prefrontal cortex during a resting state, an oddball task, a 1‐back task, and a verbal fluency task. Brain functional connectivity was calculated as the Pearson correlation coefficients between fNIRS channels. The results show that during the verbal fluency task, while the healthy control (HC) group presents a significantly stronger inter‐hemispheric connectivity compared to intra‐hemispheric connectivity, there is no difference between the inter‐ and intra‐hemispheric connectivity in the MCI group. In addition, a comparison between the MCI and HC connectivity reveals that the MCI group has a statistically higher right and inter‐hemispheric connectivity during the resting state, but a significantly lower left and inter‐hemispheric connectivity during the verbal fluency test. These findings demonstrate the potential of fNIRS to study brain functional connectivity in neurodegenerative diseases.     

Common features in the functional surface of scorpion beta-toxins and elements that confer specificity for insect and mammalian voltage-gated sodium channels

Scorpion beta-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion beta-toxin, Css4, whose effects on rat brain Navs have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 A2 composed of the following: 1) a cluster of residues associated with the alpha-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion beta-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the beta2 and beta3 strands, which is likely to confer the specificity for mammalian Navs; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion beta-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.     

Re-evaluation of Chelichnus tazelwürmi,a non mammalian therapsid-grade track from the Upper Permian Arenaria di Val Gardena

In this paper, a revision of tracks referred to as Chelichnus tazelwürmi is reported. The performed analysis, consisting of a holistic approach by means of a mainly morphological analysis, and a secondarily functional one, led to the proposal of a new ichnogenus, named as Contiichnus tazelwurmi. The three dimensional morphology of the tracks allows for the inference of a complex cycle of locomotion by the trackmakers. The tracks were formed in the main phases (i.e. touch-down, weight-bearing and kick-off) by different axes of body load and transference, indicating that the whole fore autopod was involved in the cycle of locomotion and actively contacted the substrate, while for the hind autopod the functional prevalence was markedly centro-medial. Some track features suggest a therapsid-grade synapsid as potential trackmaker. However, the reconstructed autopodial structure does not correlate with known autopods from the Late Permian body fossil record. These observations stress the importance of tetrapod ichnology studies in improving knowledge in the field of vertebrate palaeontology. http://www.zoobank.org/urn:lsid:zoobank.org:pub:B4EB4D42-1A3B-48EC-B83F-6942F741AF30

http://www.zoobank.org/urn:lsid:zoobank.org:pub:B4EB4D42-1A3B-48EC-B83F-6942F741AF30     

Investigating the role of the human CIA2A-CIAO1 complex in the maturation of aconitase

Background

The CIA2A protein, in complex with CIAO1, has been proposed to be exclusively implicated in the maturation of cytosolic aconitase. However, how the CIA2A-CIAO1 complex generates active aconitase is still unknown and the available structural information has not provided any crucial insights into the molecular function of CIA2A.

BH3-mimetic gossypol-induced autophagic cell death in mutant BRAF melanoma cells with high expression of p21

Aims

The aim of the present study was to identify the potential therapeutic effects of BH3-mimetic gossypol on melanoma cells with acquired resistance to BRAF inhibitors.

Main methods

The IC₅₀ values of gossypol were determined using MTT assays in three melanoma cell lines with different resistances to BRAF inhibitor. The effects of gossypol on three melanoma cell lines were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. The functional role of autophagy in gossypol-induced growth inhibition was investigated using siRNA-mediated knockdown of Beclin-1.

Key findings

Gossypol retained its efficacy in BRAF-V600E melanoma clones with acquired resistance to BRAF inhibitors through a mechanism independent of MEK–ERK inhibition. Gossypol caused G₂/M arrest in both BRAF mutant A375P and A375P/Mdr cells with high expression of p21^Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21^Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells. In particular, alleviation of autophagy by knockdown of Beclin-1 partially caused a resistance to gossypol-induced cell cycle arrest at G₂/M in BRAF-V600E cells with a concomitant decreased induction of apoptosis.

Significance

Taken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21^Cip1 is a prerequisite for a positive response to its clinical application.     

Predictores de mala salud autopercibida en una población de personas mayores

Introduction

Self-Rated Health is part of the comprehensive concept of Quality of Life and is a valid measurement of health status. The main objective of this study was to test the predictive value of some different variables on the poor Self-Rated Health among elders.

Material and methods

We performed a cross-sectional study on a sample consisting of 140 participants. Measurements: age, gender, level of education, environment, cognitive status, physical impairment, diseases, health perception and social support. The influence of the studied variables on the poor Self-Rated Health was performed with a logistic regression analysis and a ROC curve to establish the cut-off values for these variables with the best sensitivity and specificity to predict the poor Self-Rated Health.

Results

A poor Self-Rated Health was significantly associated with age, comorbidity, and the perception of poor functional social support, whereas no association was found with gender, environment and educational level.

Conclusions

Old age, the number of diagnosed diseases, and functional social support are Self-Rated Health risk factors, while the characteristics and repercussions of the diseases should not be considered.     

Time- and order-dependent changes in functional and NO-mediated dilation during exercise training

Lash, Julia M., and H. Glenn Bohlen. Time- andorder-dependent changes in functional and NO-mediated dilation during exercise training. J. Appl. Physiol.82(2): 460-468, 1997.Arterial vessel responses to sodiumnitroprusside (SNP) and acetylcholine (ACh) were measured in thespinotrapezius muscle of sedentary (Sed) and treadmill-trained (Tr)rats to determine whether these endothelium-dependent (ACh) and-independent (SNP) mechanisms contribute to thetraining-induced increase in functional vasodilation previouslyobserved. Control and maximal vessel diameters were similar between Sedand Tr. After 8 wk of training, functional dilation (2-, 4-, and 8-Hzcontractions) was enhanced in all orders of vessels studied[terminal feed artery (FA), largest arterioles (1A), andintermediate-sized arterioles (2A)], but responses to SNP wereincreased only in FA. Responses to ACh were not significantly increasedin any vessel order. After 16 wk of training, functional dilation hadregressed in Tr such that only the FA response to 4 Hz wassignificantly elevated relative to Sed. However, the FA and 1Aresponses to SNP were significantly greater in Tr than in Sed, as werethe 1A and 2A responses to ACh. These results show a dissociation offunctional dilation and SNP- or ACh-mediated responses, as well asage-dependent interactions, a time-dependent progression, and vesselorder specificity in the adaptations to training.

     

El anciano con enfermedad avanzada de órgano. Consideraciones desde la geriatría,la medicina paliativa y la bioética

Objective

To analyze the literature as regards the knowledge, skills and attitudes that these disciplines can provide in improving technical, ethical and human quality health care in the elderly with advanced organ failure, multimorbidity, frailty and progressive dementia.

Material and methods

A comprehensive review focused on available references on the interrelationship between geriatric medicine and palliative medicine, education in bioethics, prognostic tools, functional status, and the humanization of health care.

Results

Advance care planning, comprehensive geriatric assessment, the study of the values of the patient and their introduction in decision-making process, as well as the need to promote moral, care, and healthcare organizational ethics, are essential elements to achieve this objective.

Conclusions

Practitioners and healthcare organizations should seek excellence as a moral requirement. To achieve this, there is a priority to acquire virtues of care and fundamental concepts of geriatric and palliative medicine, assessing functional status, advance care planning and patient/family needs as essential issues to protect, care for and promote them in all care settings.     

Identification of several functional subgroups of HLA-B27 by restriction of the activity of antiviral T killer lymphocytes

Anti-Epstein-Barr virus and antiinfluenza A cytotoxic T lymphocytes (CTL) have been used to study the restriction of human antiviral responses by HLA-B27 antigens. Three functional subgroups of HLA-B27 have been clearly distinguished by this restriction-typing assay. No cross-reaction could be detected between the three subgroups either at the CTL level or at the level of antigen-presenting cells. The cells of subgroup 1 are always positive [M2(+)] when tested in immunofluorescence with a monoclonal B27-specific antibody which divides HLA-B27 into a major M2(+) and a minor M2(–) subgroup. These M2(+) group 1 cells are apparently also HLA-B27W as previously shown by Ivanyi and co-workers using anti-HLA-CTL. Subgroup 2 includes only M2(–) cells. A comparison between this group and the previously described HLA-B27K is not fully conclusive, since two typing cells which were clearly HLA-B27K apparently did not belong to group 2. Only two donors, both of Oriental origin, have been included in subgroup 3. Both of them were M2 intermediate. These results demonstrate (1) the existence of several functional subgroups of HLA-B27 with an interesting correlation with the M2(+), M2(–), or M2 intermediate phenotypes, and (2) the possibility of using the restriction-typing assay to define such functional subgroups not detected by classical allosera.Abbreviations used in this paper CTL cytolytic T lymphocyte - EBV Epstein-Barr virus - AS ankylosing spondylarthritis - PBL peripheral blood leukocytes - MSL medium for separation of lymphocyte - UV ultra violet - PHA-M phytohemagglutinin M - LCL lymphoblastoid cells lines - FCS fetal calf serum - CRT chromium release test - SCR specific chromium release - LU lytic units - MoAb monoclonal antibodies.     

Enhancement of natural killer cell activity in mice by treatment with a thymic factor

Summary The administration of a thymic factor, thymostimulin (TP-1), to mice resulted in considerable augmentation of natural killer (NK) cell activity as measured in a short-term assay against ⁵¹Cr-labeled YAC-1 target cells. Conditions suitable for detection of the thymostimulin-induced boosting of NK included multiple daily exposures to TP-1 (50 g/kg), and peak levels of reactivity were observed at 2–4 days after discontinuation of treatment. A strict age-dependency of the effect was also observed, with optimal augmentation of NK-cell activity when TP-1 was administered to mice at 4–6 weeks of age. The effect was not limited to TP-1 treatment but was also observed on administration of another thymic factor (thymosin ₁). The activated cells responsible for the increased natural cell-mediated cytotoxicity appeared to be typical murine NK cells, judging by both functional and antigenic criteria.     

Structural and functional differences between pheromonotropic and melanotropic PK/PBAN receptors

Background

The pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) plays a major role in regulating a wide range of physiological processes in insects. The ubiquitous and multifunctional nature of the PK/PBAN peptide family raises many questions regarding the mechanisms by which these neuropeptides elicit their effects and the nature of the receptors that mediate their functions.

Methods

A sex pheromone gland receptor of the PK/PBAN family from Heliothis peltigera female moth and a Spodoptera littoralis larval receptor were cloned and stably expressed, and their structural models, electrostatic potentials and cellular functional properties were evaluated.

Results

Homology modeling indicated highly conserved amino-acid residues in appropriate structural positions as experimentally shown for class A G-protein coupled receptors. Structural differences could be proposed and electrostatic potentials of the two receptor models revealed net charge differences. Calcium mobilization assays demonstrated that both receptors were fully functional and could initiate extracellular calcium influx to start PK/PBAN signal transduction. Evaluation of the signaling response of both receptors to PBAN and diapause hormone (DH) revealed a highly sensitive, though differential response. Both receptors responded to PBAN whereas only Spl-PK/PBAN-R exhibited a high response toward DH.

Conclusions

The structural, electrostatic and cellular functional differences indicate that different PK/PBAN in vivo functions may be mediated by different PK/PBAN receptors and elicited by different peptide(s).

General significance

The results advance our understanding of the mode of action of the PK/PBAN family, and might help in exploring novel high-affinity receptor-specific antagonists that can serve as a basis for the development of new families of insect-control agents.