Beta Region Research Articles

Transcription activation by the human estrogen receptor subtype beta (ER beta) studied with ER beta and ER alpha receptor chimeras.

We have studied the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and chimeric constructs with ER alpha and ER beta to examine the bioactivities of these receptors and their responses to estrogen and antiestrogen ligands. Transcriptional activity of ER beta is highly dependent on cell/promoter context and on the nature of the ligand. ER beta activated significant levels of transcription in response to estrogens in certain cell types, but showed only moderate activity compared with ER alpha in others. Antiestrogens such as tamoxifen and 2-phenylbenzofuran, which show some agonistic activity with ER alpha, exhibit no agonistic activity with ER beta. Alteration of the amino-terminal A/B receptor domain can result in a dramatic change in cell type- and ligand-specific transcriptional activity of ER beta. Upon replacing the A/B domain of ER beta with the A/B domain of ER alpha, this receptor chimera not only exhibits an improved transcriptional response to estrogens, but also is now able to activate transcription upon treatment with these antiestrogens. As antiestrogen agonism was lacking in ER beta and the ER beta/alpha chimera containing the amino-terminal A/B domain of ER beta fused to domains C through F of ER alpha, but was restored in an ER alpha/beta chimera containing the A/B domain of ER alpha, antiestrogen agonism was shown to depend on the A/B domain (activation function-1-containing region) of ER alpha. Together, these results indicate that the differences in the amino-terminal regions of ER alpha and ER beta contribute to the cell- and promoter-specific differences in transcriptional activity of these receptors, and their ability to respond to different ligands, thus providing a mechanism for differentially regulated transcription by these two ERs.

Accessibility control of variable region gene assembly during T-cell development.

T-cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an alpha beta or gamma delta T-cell antigen receptor (TCR). The genes that encode the TCR beta and gamma chains lie in distinct loci, whereas the genes that encode the TCR alpha and delta chains lie in a single locus (TCR alpha/delta locus). Assembly of TCR variable region genes is mediated by a site-specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR beta, gamma and delta variable region genes are assembled prior to TCR alpha variable region genes. Furthermore, assembly of TCR beta variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement and expression of genes within the TCR alpha/delta locus presents a complicated problem. TCR alpha and delta variable region genes are assembled at different stages of T-cell development, and fully assembled TCR alpha and delta variable region genes must be expressed in distinct lineages of T cells, alpha beta and gamma delta, respectively. We have developed several experimental approaches to assess the role of cis-acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR beta and TCR alpha/delta loci during T-cell development.

Function in vivo of separate segments of the beta subunit of Escherichia coli RNA polymerase.

Transcription of genetic material is catalysed by the enzyme DNA-dependent, RNA polymerase. The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed beta and beta', are conserved throughout nature. The beta subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme. Several lines of evidence have suggested that the function of the beta subunit is not dependent upon the contiguity of the sequence blocks. In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the beta subunit of RNA polymerase. To this end, the beta structural gene rpoB was separated into four near-equal, non-overlapping segments (as well as 'half' genes) on the basis of 'split' genes in nature, known functional organization and sequence conservation. These segments were used to prepare sequence-specific antibodies against the four individual regions, as well as being expressed in vivo from a tight, lac-controlled high-copy number vector. Immunological probing of the holoenzyme in vitro suggested that the amino-terminal half of the beta polypeptide is buried within the enzyme complex. Of the four segments expressed in vivo, the extreme C-terminal segment was trans-dominant lethal (of the effect of large N-terminal amber fragments on cellular growth; Nene & Glass 1982) and this isolated region was shown to bind the translational elongation factor EF-Tu in vivo. These in vivo and in vitro studies, in conjunction with recent in vitro work (Severinov et al. 1995), unambiguously demonstrate that individual regions of beta may adopt structurally and functionally competent forms, and underline the possibility of in vivo investigation of separate regions of this massive polypeptide chain. A model is presented for the role of EF-Tu in stringent control.

Mutations in and Monoclonal Antibody Binding to Evolutionary Hypervariable Region of Escherichia coli RNA Polymerase β′ Subunit Inhibit Transcript Cleavage and Transcript Elongation

A 190 amino acid-long region centered around position 1050 of the 1407-amino acid-long beta' subunit of Escherichia coli RNA polymerase (RNAP) is absent from homologues in eukaryotes, archaea and many bacteria. In chloroplasts, the corresponding region can be more than 900 amino acids long. The role of this hypervariable region was studied by deletion mutagenesis of the cloned E. coli rpoC, encoding beta'. Long deletions mimicking beta' from Gram-positive bacteria failed to assemble into RNAP. Mutants with short, 40-60-amino acid-long deletions spanning beta' residues 941-1130 assembled into active RNAP in vitro. These mutant enzymes were defective in the transcript cleavage reaction and had dramatically reduced transcription elongation rates at subsaturating substrate concentrations due to prolonged pausing at sites of transcriptional arrest. Binding of a monoclonal antibody, Pyn1, to the hypervariable region inhibited transcription elongation and intrinsic transcript cleavage and, to a lesser degree, GreB-induced transcript cleavage, but did not interfere with GreB binding to RNAP. We propose that mutations in and antibody binding to the hypervariable, functionally dispensable region of beta' inhibit transcript cleavage and elongation by distorting the flanking conserved segment G in the active center.

Identification of effector binding sites on S100 beta: studies with guanylate cyclase and p80, a retinal phosphoprotein.

S100 beta is a calcium-binding protein, which regulates the activities of several enzymes and inhibits the phosphorylation of a variety of protein kinase C substrates in a calcium-dependent manner. The protein was recently found to activate a retinal membrane guanylate cyclase, and in this paper, we report that it inhibits the phosphorylation of an 80 kDa retinal protein (p80). Structurally, S100 beta consists of two EF-hands connected by a hinge region. In view of its small size, wide distribution in a variety of tissues, and regulation of many different proteins, it is of interest to identify the sites on the protein that interact with the effectors, and to determine if the same sites are responsible for interaction with different effectors. We addressed these questions with the use of synthetic peptides with sequences corresponding to different regions of S100 beta and testing their effects on the protein's activation of guanylate cyclase, and inhibition of p80 phosphorylation. Peptides with sequences corresponding to effector interaction sites were anticipated to either block or simulate the effects of S100 beta. The results show that two regions of S100 beta interact with effectors: the C-terminal region of Thr81-Glu91 and the hinge region of Leu32-Leu40. The synthetic peptide containing the latter sequence blocked the S100 beta activation of guanylate cyclase and inhibition of p80 phosphorylation, while the peptide containing the former sequence blocked cyclase activation and simulated S100 beta in inhibiting p80 phosphorylation. By determining the effects of including or excluding dithiothreitol in the assays, we observed that the cysteine residue in the C-terminal region of S100 beta (Cys84) participates in the regulation of guanylate cyclase but not of p80 phosphorylation. We conclude from these results that the C-terminal and hinge regions of S100 beta are important in the regulation of effector proteins and that Cys84 is essential for interaction with only specific effectors.

Bovine F1-ATPase covalently inhibited with 4-chloro-7-nitrobenzofurazan: the structure provides further support for a rotary catalytic mechanism.

F1-ATPase is the globular domain of F1F0-ATP synthase that catalyses the hydrolysis of ATP to ADP and phosphate. The crystal structure of bovine F1-ATPase has been determined previously to 2.8 A resolution. The enzyme comprises five different subunits in the stoichiometry alpha 3 beta 3 gamma delta epsilon; the three catalytic beta subunits alternate with the three alpha subunits around the centrally located single gamma subunit. To understand more about the catalytic mechanisms, F1-ATPase was inhibited by reaction with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and the structure of the inhibited complex (F1-NBD) determined by X-ray crystallography. In the structure the three beta subunits adopt a different conformation with different nucleotide occupancy. NBD-Cl reacts with the phenolic oxygen of Tyr311 of the beta E subunit, which contains no bound nucleotide. The two other catalytic subunits beta TP and beta DP contain bound adenylyl-imidodiphosphate (AMP-PNP) and ADP, respectively. The binding site of the NBD moiety does not overlap with the regions of beta E that form the nucleotide-binding pocket in subunits beta TP and beta DP nor does it occlude the nucleotide-binding site. Catalysis appears to be inhibited because neither beta TP nor beta DP can accommodate a Tyr311 residue bearing an NBD group. The results presented here are consistent with a rotary catalytic mechanism of ATP synthesis and hydrolysis, which requires the sequential and concerted participation of all three catalytic sites. NBD-Cl inhibits the enzyme by preventing the modified subunit from adopting a conformation that is essential for catalysis to proceed.

An immunological based study of monoclonal gammopathies among suspected individuals in Kashmir region

Serum and urine samples from 513 patients clinically suspected of monoclonal gammopathies over a period of five years (1992-97) were subjected to various immunological procedures viz, electrophoresis, immunoelectrophoresis and immunoglobulin estimations. Laboratory investigations confirmed gammopathies in 10.33%. It was observed that overall age of incidence for monoclonal gammopathies in both sexes was between 42-72 years with a male to female ratio of 1.4∶1. Predominant paraprotein detected was IgG type (75.47%) followed by IgA (16.98%) and Bence Jones proteins (7.55%). Amongst positive patients, 64.16% were having kappa (k) type light chains and 35.84% lambda (δ) type light chains. 69.39% patients with serum M component (IgG and IgA) had Bence Jones proteinuria. Densitometric scanning revealed that majority of IgG type paraprotein was found in the slow gamma globulin region and majority of IgA type paraprotein was found equally distributed between beta and fast gamma globulin regions. Both types had decreased albumin and alpha-2-globulin concentrations as compared to normal controls. Immunoglobulin levels in patients with paraprotein had very high levels of serum IgG (6467.0 mg%) and IgA (2714.0 mg%) in respective types of monoclonal gammopathies; the rest of immunoglobulin classes were either at normal or decreased levels.

Detection and Characterization of Monoclonal Componentsin Serum and Urine

Detection of a monoclonal component (the immunoglobulin product of a clone of plasma cells) in the serum and/or urine is an important part of the laboratory evaluation of a patient with a plasma cell or B-lymphocyte proliferative disease. Usually, large quantities of monoclonal components are present in the serum of patients with multiple myeloma or Waldenstrom’s macroglobulinemia. Small quantities of monoclonal components in serum may signify light chain disease, a B-lymphocytic lymphoma or leukemia, amyloid AL (amyloidosis associated with immunoglobulin light chains), a neuropathy, or the predisposition to develop a malignant plasma cell/B-lymphocyte proliferative disorder in a patient with no currently evident clinical problem (monoclonal gammopathy of undetermined significance–MGUS). About 25% of individuals who fall into the MGUS category develop myeloma or a related B-lymphocyte proliferative disorder after 20 to 35 years of follow-up (1). Because of this, annual follow-up is prudent, even for patients with relatively small monoclonal components in their serum. For the past half-century, protein electrophoresis using either cellulose acetate or agarose gels has been the mainstay in screening serum and urine for monoclonal components. Most of the currently used electrophoretic techniques require a considerable amount of manual labor. Thus, although the reagents are relatively inexpensive, the time involved in applying the samples, fixing, staining, and washing the gels, and performing the densitometric scans of the gels make electrophoresis rather costly. Furthermore, there is considerable variation in the resolution of the major protein fractions from one manufacturer’s product to another. Some relatively simple and inexpensive techniques allow one to recognize only five bands that often have fuzzy borders. Those techniques make it difficult to detect small monoclonal components or those that migrate in the beta region, where transferrin, C3, and β-lipoprotein may obscure a subtle monoclonal band. Not surprisingly, techniques that provide a high-resolution electrophoretic pattern with …

Corona Associations and Their Implications for Venus

Geologic mapping principles were applied to determine genetic relations between coronae and surrounding geomorphologic features within two study areas in order to better understand venusian coronae. The study areas contain coronae in a cluster versus a contrasting chain and are (1) directly west of Phoebe Regio (quadrangle V-40; centered at latitude 15°S, longitude 250°) and (2) west of Asteria and Beta Regiones (between latitude 23°N, longitude 239° and latitude 43°N, longitude 275°). Results of this research indicate two groups of coronae on Venus: (1) those that are older and nearly coeval with regional plains, and occur globally; and (2) those that are younger and occur between Beta, Atla, and Themis Regiones or along extensional rifts elsewhere, sometimes showing systematic age progressions. Mapping relations and Earth analogs suggest that older plains coronae may be related to a near-global resurfacing event perhaps initiated by a mantle superplume or plumes. Younger coronae of this study that show age progression may be related to (1) a tectonic junction of connecting rifts resulting from local mantle upwelling and spread of a quasi-stationary hotspot plume, and (2) localized spread of post-plains volcanism. We postulate that on Venus most of the young, post-resurfacing coronal plumes may be concentrated within an area defined by the bounds of Beta, Atla, and Themis Regiones.

Enhanced Three-Body Decay of the Charged Higgs Boson

If the charged Higgs boson H+ exists with mh < mt + mb, the conventional expectation is that it will decay dominantly into c s-bar and tau+ nu-tau. However, the three-body decay mode H+ --> W+ b b-bar is also present and we show that it becomes very important in the low tan(beta) region for mh >~ 140 GeV. We explore its phenomenological implications for the charged-Higgs-boson search in top-quark decay.

Biology of the Interleukin-2 Receptor

Studies of the biology of the IL-2 receptor have played a major part in establishing several of the fundamental principles that govern our current understanding of immunology. Chief among these is the contribution made by lymphokines to regulation of the interactions among vast numbers of lymphocytes, comprising a number of functionally distinct lineages. These soluble mediators likely act locally, within the context of the microanatomic organization of the primary and secondary lymphoid organs, where, in combination with signals generated by direct membrane-membrane interactions, a wide spectrum of cell fate decisions is influenced. The properties of IL-2 as a T-cell growth factor spawned the view that IL-2 worked in vivo to promote clonal T-cell expansion during immune responses. Over time, this singular view has suffered from increasing appreciation that the biologic effects of IL-2R signals are much more complex than simply mediating T-cell growth: depending on the set of conditions, IL-2R signals may also promote cell survival, effector function, and apoptosis. These sometimes contradictory effects underscore the fact that a diversity of intracellular signaling pathways are potentially activated by IL-2R. Furthermore, cell fate decisions are based on the integration of multiple signals received by a lymphocyte from the environment; IL-2R signals can thus be regarded as one input to this integration process. In part because IL-2 was first identified as a T-cell growth factor, the major focus of investigation in IL-R2 signaling has been on the mechanism of mitogenic effects in cultured cell lines. Three critical events have been identified in the generation of the IL-2R signal for cell cycle progression, including heterodimerization of the cytoplasmic domains of the IL-2R beta and gamma(c) chains, activation of the tyrosine kinase Jak3, and phosphorylation of tyrosine residues on the IL-2R beta chain. These proximal events led to the creation of an activated receptor complex, to which various cytoplasmic signaling molecules are recruited and become substrates for regulatory enzymes (especially tyrosine kinases) that are associated with the receptor. One intriguing outcome of the IL-2R signaling studies performed in cell lines is the apparent functional redundancy of the A and H regions of IL-2R beta, and their corresponding downstream pathways, with respect to the proliferative response. Why should the receptor complex induce cell proliferation through more than one mechanism or pathway? One possibility is that this redundancy is an unusual property of cultured cell lines and that primary lymphocytes require signals from both the A and the H regions of IL-2R beta for optimal proliferative responses in vivo. An alternative possibility is that the A and H regions of IL-2R beta are only redundant with respect to proliferation and that each region plays a unique and essential role in regulating other aspects of lymphocyte physiology. As examples, the A or H region could prove to be important for regulating the sensitivity of lymphocytes to AICD or for promoting the development of NK cells. These issues may be resolved by reconstituting IL-2R beta-/-mice with A-and H-deleted forms of the receptor chain and analyzing the effect on lymphocyte development and function in vivo. In addition to the redundant nature of the A and H regions, there remains a large number of biochemical activities mediated by the IL-2R for which no clear physiological role has been identified. Therefore, the circumstances are ripe for discovering new connections between molecular signaling events activated by the IL-2R and the regulation of immune physiology. Translating biochemical studies of Il-2R function into an understanding of how these signals regulate the immune system has been facilitated by the identification of natural mutations in IL-2R components in humans with immunodeficiency and by the generation of mice with targeted mutations in these gen

Cytotoxic T lymphocyte clone specific for autologous human hepatocellular carcinoma cell line SUHC-1.

We established a cytotoxic T lymphocyte (CTL) clone directed against an autologous hepatocellular carcinoma (HCC) cell line SUHC-1 which had been established in our department from a patient with HCC associated with hepatitis C virus infection. The CTL clone lysed autologous SUHC-1 cells but did not lyse autologous Epstein-Barr (EB) virus-transformed B cells, natural killer (NK) cell-sensitive erythroleukaemia cell line K562, the NK-resistant B cell line Daudi, or allogeneic HCC cell lines, Hep-G2, Hep-3B, Mahlavu and PLC/PRF/5. The CTL clone expressed CD3 and CD8 molecules. The cytotoxic activity of the clone was inhibited by anti-CD3, anti-CD8 and anti-histocompatibility antigen (HLA) class I monoclonal antibodies. These results indicated that the CTL clone recognized HCC tumour antigen in an HLA class I-restricted manner. Furthermore, we investigated the T cell receptor (TCR) gene usage of the CTL clone. The CTL clone expressed TCR alphabeta. We searched for expression of TCR variable (V) alpha and beta regions and sequenced complementary determining region (CDR) 3 of the clone. The clone expressed V alpha14, junctional (J) region alpha9.7 and V beta7, J beta2.1. The amino acid sequence of the N region of the of chain was S-P-G-G-G-G-A-D-G-L-T and of the N-D-N region of the beta chain was S-W-T-G-A-S-T-D-T-Q-Y. These results suggested that HLA class I-restricted CTL play an important role in the elimination of human HCC cells.

Photoinduced Inactivation of Protein Kinase C by Dequalinium Identifies the RACK-1-binding Domain as a Recognition Site

1,1'-Decamethylenebis-4-aminoquinaldinium diiodide (DECA; dequalinium) is an anti-tumor agent and protein kinase C (PKC) inhibitor whose mechanism of action with PKC is unknown. This study reports that with human PKC alpha, DECA exhibited competitive inhibition (Ki = 11.5 +/- 5 microM) with respect to RACK-1 (receptor for activated C kinase-1), an adaptor protein that has been proposed to bind activated PKC following translocation (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180-24187). When exposed to UV light, DECA covalently modified and irreversibly inhibited PKC (alpha or beta), with IC50 = 7-18 microM. UV/DECA treatment of synthetic peptides modeled after the RACK-1-binding site in the C2 region of PKC beta induced modification of Ser218-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr226, but not of a control peptide. This modification occurred at a tryptophan residue (Trp223) that is conserved in all conventional PKC isoforms. In overlay assays with native RACK-1 that had been immobilized on nitrocellulose, UV-treated control PKC alpha bound well to RACK-1, whereas UV/DECA-inactivated PKC alpha had reduced binding activity. The significance of these findings is shown with adenocarcinoma cells, which, when pretreated with 10 microM DECA and UV light, exhibited diminished 12-O-tetradecanoylphorbol-13-acetate-induced PKC alpha translocation. Overall, this work identifies DECA as a tool that prevents PKC translocation by inhibiting formation of the PKC.RACK-1 complex.

Interaction between the Retinoid X Receptor and Transcription Factor IIB Is Ligand-dependent in Vivo

The retinoid X receptor (RXR) influences gene activation through heterodimeric and homodimeric association with DNA and associates with TATA binding protein, TAF110, and cAMP response element-binding protein-binding protein; yet the molecular mechanisms responsible for gene activation by RXRs remain incompletely defined. Since the general transcription factor IIB (TFIIB) is a common target of sequence-specific transcriptional activators, we suspected that RXR might regulate target genes via an interaction with TFIIB. Coimmunoprecipitation, far Western analysis, and glutathione S-transferase binding studies indicated that murine RXR beta (mRXR beta) was capable of binding to human TFIIB in vitro. Functional analysis with a dual-hybrid yeast system and cotransfection assays revealed the interaction of mRXR beta with TFIIB to be ligand-dependent in vivo. Truncation experiments mapped the essential binding regions to the carboxyl region of mRXR beta (amino acids (aa) 254-389) and two regions in the carboxyl region of TFIIB (aa 178-201 and aa 238-271). Furthermore, the delta 390-410 mRXR beta mutant bound to TFIIB in vitro but was not active in the dual-hybrid yeast system, suggesting that the extreme carboxyl region of RXR was required for in vivo interaction with TFIIB. These data indicate that interaction of mRXR beta with TFIIB is specific, direct, and ligand-dependent in vivo and suggest that gene activation by RXR involves TFIIB.

T-cell receptor variable region genes in cutaneous T-cell lymphomas.

The genes encoding the T-cell receptor (TCR) variable beta (TCRBV) regions were studied in skin biopsy samples from 24 patients with cutaneous T-cell lymphomas (CTCL), i.e. mycosis fungoides (n = 7), Sézary syndrome (n = 4), lymphomatoid papulosis (n = 3) and large cell CTCL with (n = 3) or without (n = 7) CD30 expression. A panel of 24 primers specific for TCRBV families 1-24 was designed and applied to cDNA using a semiquantitative reverse transcriptase coupled polymerase chain reaction (PCR) method. Three patients showed restricted expression of a limited number (1-3) of TCRBV families. In the remaining patients, an average of 17 (range: 13-22) different families was expressed. All patients showed elevated (> 10%) expression of individual families significantly higher than that seen in normal blood lymphocytes, but no preferential usage of particular gene families was observed. Direct sequence analysis of more than 60 PCR products revealed clonal TCR transcripts in 18 patients. A single T-cell clone, constituting 9-100% (mean: 26%) of the TCRBV mRNA, was present in 12 patients; two T-cell clones, constituting 13-72% (mean: 21.5%) of the TCRBV mRNA, were present in five patients; and three T-cell clones, accounting for < 0.5-13% of the TCRBV mRNA, were present in one patient. In the remaining six patients, clonal TCR transcripts could not be identified. It is concluded that most CTCL contain both clonal and non-clonal (reactive) T cells, and that the latter cells are more numerous than anticipated. These cells may be engaged in tumour immune reactions, although prospective studies and/or serial investigations are needed to elucidate this issue fully.

A β4 Isoform-specific Interaction Site in the Carboxyl-terminal Region of the Voltage-dependent Ca2+ Channel α1A Subunit

The voltage-gated calcium channel beta subunit is a cytoplasmic protein that stimulates activity of the channel-forming subunit, alpha 1, in several ways. Complementary binding sites on alpha 1 and beta have been identified that are highly conserved among isoforms of the two subunits, but this interaction alone does not account for all of the functional effects of the beta subunit. We describe here the characterization in vitro of a second interaction, involving the carboxyl-terminal cytoplasmic domain of alpha 1A and showing specificity for the beta 4 (and to a lesser extent beta 2a) isoform. A deletion and chimera approach showed that the carboxyl-terminal region of beta 4, poorly conserved between beta isoforms, contains the interaction site and plays a role in the regulation of channel inactivation kinetics. This is the first demonstration of a molecular basis for the specificity of functional effects seen for different combinations of these two channel components.

An 8 kb nucleotide sequence at the 3' flanking region of the sspC gene (184 degrees) on the Bacillus subtilis 168 chromosome containing an intein and an intron.

As part of the Bacillus subtilis genome sequencing project, we determined the complete nucleotide sequence of an 8000-bp fragment downstream of the sspC gene (184 degrees) of the B. subtilis 168 chromosome. The sequence analysis shows that the sspC gene is located inside of the SP beta region, which differs from the current genetic map of B. subtilis 168. This region contains 12 putative ORFs (yojQ through yojZ and sspC). A homology search for the deduced products of the ORFs shows significant similarities to enzymes involved in deoxyribonucleotide metabolism: ribonucleotide reductase (Nrd) E, NrdF, thioredoxin and dUTPase. Interestingly, this DNA fragment includes two split genes, yojP containing conserved motifs of an intein and yojQ and yojS with an 808-bp intervening sequence for a putative intron structure. In addition, the yojR gene includes a putative new DNA replication terminator.

Kawasaki Disease and the T-Cell Antigen Receptor

ABSTRACT: We investigated the evidence for an infectious etiology of Kawasaki disease (KD), an acute vasculitis of unknown etiology, by assessing the effects of KD on the T cell antigen receptor variable beta region families (Vβ). Using 3-color flow cytometry, we studied KD patients pre- and post-intravenous gamma globulin (IVIG) therapy and at >40 days post therapy, additionally comparing them to matched pediatric control patients (PCC) and their own healthy parents (one parent/KD child). Of all the Vβ families examined, only V β2 exhibited statistically significant differences, between the pre- and postIVIG samples and preIVIG and parent samples. No associations were found between V β2 findings and T cell memory, activation, or adhesion markers. For 2 KD patients, 4 parents, and 1 PCC participant, >15% of resting CD8+ lymphocytes and >15% of blastic CD8+ lymphocytes expressed a single Vβ family, which varied by individual, without similar expansions in the CD4+ cell populations. One of the participants with this abnormality was the only one with significant cardiac abnormalities. For all participants with the Vβ abnormality, other T-cell abnormalities were extensive and involved both CD4+ and CD8+ cells. We suggest that V β2 changes do occur in KD, as previously reported. However, these may not be involved in disease pathogenesis. Other Vβ changes also occur. Those occurring in parents may reflect asymptomatic reinfection with an infectious agent causing KD. Further, some KD patients may have restricted cytotoxic T-cell responses to that as yet unidentified agent; this restricted response may be associated with more severe cardiac involvement. Published by Elsevier Science Inc.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel.

Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta-ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 310 helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-I, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Structural basis of integrin-mediated signal transduction.

Integrins are a family of alpha/beta heterodimers of cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions. Both alpha and beta subunits have a large extracellular domain and a short cytoplasmic domain. The alpha subunit has seven sequence repeats of 60-70 residues in its N-terminal region. The beta-propeller model, in which seven four-stranded beta-sheets are arranged in a torus around a pseudosymmetry axis, has been proposed as a structural model of these seven repeats. Several predicted loops critical for ligand binding have been identified in the upper face of the proposed beta-propeller model. Several alpha subunits (e.g., alpha 2, alpha L and alpha M) have I-domains of about 200 residues inserted between their second and third repeats. These I-domains adopt a Rossman-fold structure and have major ligand and cation binding sites (the MIDAS site) on their surfaces. The beta subunit has an I-domain-like structure in its N-terminal region. This structure includes multiple sequences/conserved oxygenated residues critical for ligand binding (e.g., Asp-119 in beta 3), and non-conserved residues critical for ligand specificities. Several "activation-dependent" epitopes have been identified in the Cys-rich (stalk) region of beta 1. It has yet to be determined how these multiple ligand binding sites in the alpha and beta subunits are involved in ligand binding, and how conformational changes on activation/ligand occupancy relate to signal transduction.