Beta Region Research Articles

T-Cell-Receptor Repertoire in Chronic Plaque-Stage Psoriasis Is Restricted and Lacks Enrichment of Superantigen-Associated Vβ Regions

Preferential usage of certain T-cell receptors by the lymphocytic infiltrate in psoriasis might indicate the involvement of an antigen in the pathogenesis of this disease. However, to date there are no data on the complete T-cell-receptor V alpha and V beta repertoire in psoriatic patients. We therefore compared the usage of T-cell-receptor variable regions in blood and skin of 10 patients with chronic plaque-stage psoriasis by means of semiquantitative polymerase chain reaction. Additionally, HLA class II alleles were analyzed by means of sequence-specific oligonucleotide typing. A considerable restriction of the T-cell-receptor repertoire was observed in the skin, where up to 20% of the variable regions present in the blood were not detectable. This was true for both alpha- and beta-chains. However, no interindividually constant pattern of T-cell-receptor restriction was deducible. Inconsistently, a certain preferential usage of some beta chains occurred within the cutaneous compartment. This report on the complete T-cell-receptor V alpha and V beta repertoire in psoriasis documents the restricted receptor repertoire of infiltrating T cells and a lack of enrichment of superantigen-associated V beta regions. Thus superantigens seem not to play a pathogenetically relevant role in chronic plaque-stage psoriasis.

Alpha particle heating at comet‐solar wind interaction regions

The satellite observations at comet Halley have shown strong heating of solar wind alpha particles over an extended region dominated by high‐intensity, low‐frequency turbulence. These waves are excited by the water group pickup ions and can energize the solar wind plasma by different heating processes. The alpha particle heating by the Landau damping of kinetic Alfven waves and the transit time damping of low‐frequency hydromagnetic waves in this region of high plasma beta are studied in this paper. The Alfven wave heating was shown to be the dominant mechanism for the observed proton heating, but it is found to be insufficient to account for the observed alpha particle heating. The transit time damping due to the interaction of the ions with the electric fields associated with the magnetic field compressions of magnetohydrodynamic waves is found to heat the alpha particles preferentially over the protons. Comparison of the calculated heating times for the transit time damping with the observations from comet Halley shows good agreement. These processes contribute to the thermalization of the solar wind by the conversion of its directed energy into the thermal energy in the transition region at comet‐solar wind interaction.

Selective expansion of T cells expressing V beta 2 in peanut allergy.

Peanuts are the most common cause of fatal and near-fatal food-induced anaphylaxis. The immune basis for susceptibility to peanut allergy is poorly understood. The current study examined the possibility that patients with peanut allergy, as compared to normals, use different T cell receptor variable beta regions (V beta) in the recognition of peanuts. The results demonstrate that stimulation of T cells from patients with peanut allergy results in the selective expansion of V beta 2+ T cells.

Analysis of clonal CD8+ T cell expansions in normal individuals and patients with rheumatoid arthritis.

In the course of studying the circulating TCR repertoire in humans, we noted several individuals with an increase in the percentage of CD8+ T cells expressing a particular V region. In some cases, these CD8 expansions were dramatic, occupying over 40% of the total CD8 repertoire. Using a panel of mAbs to different TCR V regions, we found that over 30% of healthy adults (> 35 years of age) harbor an expansion that alters the peripheral blood CD8 TCR repertoire. A wide range of V regions were expressed by these expansions. Considering that the mAbs used cover only a portion of the V beta repertoire, the data suggest that over 70% of adults are likely to harbor such expansions. Junctional region sequencing showed that the CD8 subset expansions were clonal, and serial studies as long as 4 years showed that they persisted indefinitely. Expansions were not identified in the CD4 population. Discordant expression of one large V beta 6.7+ clone was found in one identical twin set, suggesting the possibility that an environmental exposure is involved in their generation and/or expansion. In one large family, we found five family members with a large CD8 subset expansion. Remarkably similar usage of J beta regions was noted, and two individuals demonstrated V beta 3-expressing clones with homologous CDR3 regions, differing by only one major substitution. The repertoire data from this family suggest that the T cell clones have arisen in response to a common Ag. Studies of patients with rheumatoid arthritis found a significantly increased frequency of circulating CD8 subset expansions that expressed a different V region repertoire compared with the healthy individuals studied. Overall, our results emphasize a frequent alteration in the human CD8 TCR repertoire, most likely related to an environmental exposure, in both healthy individuals and patients with rheumatoid arthritis. The presence of these expansions will be important to consider in any study of human TCR repertoire, and their implication for health and disease will be important to understand.

Delineation of Tyrosine-Containing Epitopes within the beta Subunit of Bovine Thyrotropin

The epitopes recognised by two monoclonal antibodies (mAb 279 and mAb 299), specific for the beta subunit of bovine thyroid-stimulating hormone (bTSH), have been localised using a technique in which the tyrosine residues in the bTSH beta subunit were subjected to modification when the bTSH beta subunit was complexed with either mAb or in the free, unbound state. The epitope recognised by mAb 279 was localised to the C-terminal region of bTSH beta with the tyrosine residue Tyr104 protected from modification by the presence of this mAb. In addition, the experimental results indicate that the tyrosine residues Tyr18 and/or Tyr112 are also involved in the mAb 279 epitope. The epitope recognised by mAb 299 was localised to the region 59-74 of bTSH beta as both Tyr59 and Tyr74 were protected from modification by the presence of this mAb. Since both mAbs have been previously found to inhibit receptor binding, the sequence regions/amino acid positions recognised by these mAbs are likely to represent determinants for receptor binding. Moreover, these data indicate that the identified amino acid residues are located on the surface of the molecule, consistent with predictions of the tertiary structure of the bTSH beta subunit based on the recently elucidated X-ray crystal structure of human chorionic gonadotropin.

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.

Ontogeny of TCR V beta 1 expression revealed novel invariant alternative transcripts.

Analysis of TCR-beta gene recombination and expression was performed by quantitative PCR amplification technique throughout chicken embryogenesis and development. Our data demonstrated that TCR V beta 1 promoters were turned on by day 10 of embryogenesis, 2 days before detection of TCR-beta gene recombination. The V to D recombination step was first detected by day 11 of embryogenesis whereas DJ and V(D)J rearranged genes were detected 1 day later, on day 12 of embryogenesis. Thus, transcription of unrearranged TCR-beta genes in chickens precedes the expression of V(D)J recombinase activity as in mammals. In contrast, although TCR-beta rearrangement starts with the D to J recombination step in mammals, it can start either by the VD or the DJ step in chickens. Furthermore, reverse transcriptase-PCR amplification of TCR-beta transcripts revealed the presence of two kinds of alternative transcripts. These novel alternatively spliced products appeared in thymocytes from embryonic thymus during colonization periods and were absent in transformed T cell lines. Splicing sites are located in the middle of V beta 1 segments and lead to delta V beta 1-C beta and delta V beta 1-D beta-J beta-C beta transcripts. delta V beta 1-C beta transcripts might lead to synthesis of invariant truncated TCR beta-chains containing the aminoterminal portion of the V beta 1 region followed by the C beta region. Because this type of splicing can be generated by using all known V beta 1 members, these invariant forms could play a role in thymocyte development.

A new Turkish type of β‐thalassaemia major with homozygosity for two non‐consecutive 7.6 kb deletions of the Ψβ and β genes and an intact δ gene

In a 2.5-month-old infant with beta-thalassaemia major, DNA analysis of the gamma-beta region revealed homozygosity for two large deletions removing the entire psi beta and beta regions including their 5' promoter regions but leaving the delta gene intact. The downstream deletion was predicted to be 7.6 kb in length extending from a point 1.5 kb on the 3' side of the delta-globin gene to about 1.8 kb on the 3' side of the beta-globin gene. The upstream deletion, which was also about 7.6 kb, extended from a point 1.5 kb on the 5' side of the psi beta-globin gene to about 4.5 kb on the 3' of the psi beta gene. The delta-globin gene was intact. From the phenotypic expression of the disease it is concluded that removal of the psi beta gene probably prevents derepression of the gamma gene that has previously been observed in the absence of the promoter region of the beta gene and the switch mechanism from gamma to beta gene expression may take place earlier than expected.

EEG power and coherence in presenile and senile depression. Characteristic findings related to differences between anxiety type and retardation type

Quantitative EEG analysis was done on 29 medicated right-handed depressive patients at age over 45 and age and sex matched right-handed 20 normal controls. The patients were all diagnosed as major depression with melancholia by DSM-III-R and were divided into anxiety type (n = 17) and retardation type (n = 12) evaluated with the Hamilton rating scale for depression. Eye closed resting EEGs were recorded on a data recorder from the 16 electrode leads (10-20 system). Artifact rejected 3 minutes EEGs were analyzed by off-line with Fast Fourier Transform from the bilateral frontal (F3, F4), parietal (P3, P4) and occipital (O1, O2) regions. The mean values of absolute amplitude power (microV) and the mean Z scores of inter-, and intrahemispheric coherence were obtained in theta 1 (4.0-6.0 Hz), theta 2 (6.0-8.0 Hz), alpha 1 (8.0-10.5 Hz), alpha 2 (10.5-13.0 Hz), beta 1 (13.0-20.0 Hz), and beta 2 (20.0-40.0 Hz) frequency bands respectively. The main findings were: 1) Beta 1 and beta 2 power were greater in patients with anxiety type depression than in normal controls, and the differences were statistically significant over the parietal and occipital regions in beta 1 and the frontal region in beta 2. The anxiety type was distinguished from the retardation type with the increase of beta 2 power. The retardation type showed higher alpha 1 power over the frontal region and lower alpha 2 power over the occipital region than normal controls. 2) Both types showed greater frontal predominant ratio to the parietal in beta 2 power than did normal controls. The retardation type was distinguished from the anxiety type with the right predominance in beta 1 power over the frontal region. 3) Both groups of patients showed lower frontal interhemispheric coherences than normal controls in each band. In particular, the anxiety type showed significantly lower alpha 2 band and lower beta 1 and beta 2 bands. The parietal interhemispheric coherence was lower in the anxiety type but higher in the retardation type than in normal controls in each band, and this difference was prominent in theta 2 band. 4) The two groups of patients showed higher fronto-parietal intrahemispheric coherences in both hemispheres than the normal controls in each band. Especially, the anxiety type showed higher in theta two and alpha 1 bands.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue‐Specific variation in the length of the 5′ untranslated region of the βa‐inhibin mRNA in sheep

The 5' untranslated region (UTR) of beta A inhibin mRNA was compared in a variety of sheep tissues, using primer extension. Considerable variation in the length and number of 5' extended products were noted between tissues. Specific bands were noted in ovarian follicular RNA, which were also present in samples from corpora lutea, stroma, and placental cotyledon RNA. Other extended products were observed in RNA from corpora lutea, stroma, cotyledon, pituitary, bone marrow, frontal cortex, medial basal hypothalamus, adrenal, liver, and kidney, which were not present or weakly represented in follicular RNA. Additional tissue-specific bands were noted in testis and bone marrow RNA. No specific differences in the lengths of the 5' UTR of the beta A inhibin mRNA were observed in sheep homozygous for the Booroola fecundity gene FecB, in any tissue studied. The coding region of ovine beta A inhibin cDNA was sequenced and a genetic polymorphism confirmed within or close to the ovine beta A inhibin gene. We conclude that the beta A inhibin gene is expressed widely in the sheep. Furthermore there is variation in the length of the 5' UTR of beta A inhibin mRNA between male and female gonads and other tissues, implying that expression of this gene is differentially controlled. However, the FecB mutation does not affect mRNA splicing events or the initiation site used in ovarian transcription. The mechanism by which the FecB mutation influences the amounts of beta A inhibin mRNA, follicle-stimulating hormone (FSH) secretion and ovulation rate has still to be elucidated.

Translational control of activin in Xenopus laevis embryos.

Activin is a potent mesoderm inducing factor present in embryos of Xenopus laevis. Recent evidence has implicated activin in the inhibition of neural development in addition to the well-established induction of mesoderm in ectodermal explants. These diverse effects are critically dependent on the concentration of activin yet little is known about the mechanisms regulating the level of activin in the embryo. We report that the 3' untranslated region (3' UTR) of activin beta B mRNA inhibits the translation of activin in embryos. Micro-injection of activin mRNA from which the 3' UTR has been deleted is 8-10-fold more potent in inducing mesoderm than mRNA containing the 3' UTR. Truncation of the 3' UTR also leads to a marked enhancement of activin protein levels in embryos but has no effect when the truncated mRNA is translated in vitro. The 3' UTR also confers translational inhibition on a heterologous mRNA. These data show that a maternal factor(s) present in X. laevis regulates the translation of injected activin beta B mRNA. This factor(s) could be responsible for regulating the levels of endogenous activin beta B protein during mesoderm induction and the specification of ectodermal derivatives such as neural and epidermal tissues.

Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25-35 region to aggregation and neurotoxicity.

The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of beta-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of beta-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of beta-amyloid, beta 25-35, and the full-length protein, beta 1-42. We examine the effects of amino acid residue deletions and substitutions on the ability of beta-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant beta-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of beta 25-35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the beta 33-35 region. In beta 1-42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of beta-sheet secondary structure in aggregating, toxic beta-amyloid peptides but not in nonaggregating, nontoxic beta-amyloid peptides. Together, these data further define the primary and secondary structures of beta-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.

Detection of T cell receptors in early rheumatoid arthritis synovial tissue.

Synovial tissue is rarely available from patients with early synovitis, with the exception of synovial biopsies. However, T cell populations early in the development of synovitis may be enriched in antigen-specific cells and critical to disease pathogenesis. To investigate the T cell repertoire in early synovitis, we utilized a PCR protocol for detection of T cell receptor (TCR) transcripts present in small amounts of synovial tissue. To expand the substrate for PCR, preamplification of cDNA was performed with a 3' constant region primer plus either a mixture of variable region primers or random hexanucleotides. Utilizing this method improved the sensitivity of detection. This technique is applied here to the analysis of TCR transcripts in synovial biopsies from individuals with early rheumatoid arthritis (RA) and non-RA synovitis. TCR alpha-chain transcripts were detectable in 5/5 RA and 4/4 non-RA specimens evaluated, with beta-chain transcripts detected in 4/5 early RA and 4/4 non-RA specimens evaluated. Confirmation of transcripts by sequencing of cloned PCR products verified the specificity of amplification. The most frequently expressed TCR V region families in early RA synovitis were V alpha 11, V alpha 14, V alpha 28, V beta 7, V beta 9 and V beta 17. Several of these V regions have previously been implicated in studies of chronic RA synovitis. J alpha and J beta region usage was similar to that seen in chronic RA, and conserved N region motifs were apparent. We conclude that it is possible to detect TCR transcripts in small synovial biopsies from individuals with early arthritis.(ABSTRACT TRUNCATED AT 250 WORDS)

A Lymnaea stagnalis gene, with sequence similarity to that of mammalian beta 1–>4-galactosyltransferases, encodes a novel UDP-GlcNAc:GlcNAc beta-R beta 1–>4-N-acetylglucosaminyltransferase.

A cDNA encoding a novel glycosyltransferase, that may be involved in a variant pathway for the synthesis of complex type oligosaccharide chains, was cloned from the pond snail Lymnaea stagnalis. By heterologous hybridization, using bovine beta 1-->4-galactosyltransferase cDNA as probe, a genomic clone from a snail library was isolated. This genomic clone was subsequently used to clone the corresponding cDNA from a prostate gland library. The isolated cDNA encodes a polypeptide of 490 amino acids with a type II membrane protein topology typical for glycosyltransferases. The carboxyl-terminal part, encoding the putative catalytic domain, reveals considerable sequence similarity with the corresponding region of mammalian beta 1-->4-galactosyltransferases, suggesting an evolutionary relationship. Expression of this cDNA in COS cells and insect cells revealed that the encoded enzyme transfers GlcNAc, rather than Gal or GalNAc, from the corresponding nucleotide sugars to several beta-N-acetylglucosaminides. Structural characterization by 1H NMR spectroscopy of products formed in vitro demonstrated that the enzyme can be identified as a UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyl-transferase. A new family of glycosyltransferases has hereby been discovered, consisting of enzymes that act on acceptor substrates with a terminal beta-linked GlcNAc residue and establish a beta 1-->4-linkage, but have a different nucleotide sugar requirement.

Monitoring of monoclonal gammopathies: Rational use of densitometry and rate nephelometry

The follow-up of patients with monoclonal gammopathies at our institution includes serial serum protein electrophoresis (SPE) with densitometry and IgG, IgA, and IgM quantitative immunoglobulin (QIG) determinations by rate nephelometry. This retrospective audit compares monoclonal protein concentration as estimated by SPE versus QIG in 456 serial serum specimens from 105 patients to determine whether any of the tests provide redundant information. A comparison of the methods demonstrated good correlation between SPE ( x-axis) and QIG ( y-axis) quantitation for each immunoglobulin class: IgG had a slope of 1.45 and an intercept of 1.60 ( S y x = 7.46, r = 0.96, n = 250 ); IgA had a slope of 1.30 and an intercept of −1.37 ( S y x = 6.85, r = 0.96, n = 78 ); and IgM had a slope of 1.95 and an intercept of 2.06 ( S y x = 5.16, r = 0.98, n = 128 ). The data for individual patients showed similar good correlations. Exceptions included IgA peaks “buried” in the beta region of the SPE (resulting in invalid SPE estimates of monoclonal protein concentration), and IgG peaks of less than 10 g/L (when background polyclonal IgG immunoglobulin skews the QIG estimate of monoclonal protein concentration). An algorithm is proposed whereby monoclonal protein concentration is measured by the specific QIG ( i.e., IgG, IgA, or IgM) determination for the routine monitoring of patients, except for those with IgG peaks of less than 10 g/L that are followed by SPE.

Evidence for Active Hotspots on Venus from Analysis of Magellan Gravity Data

The 500-Myr average crater retention age for Venus has raised questions about the present-day level of tectonic activity. In this study we examine the relationship between the gravity and topography of four large volcanic swells, Beta, Atla, Bell, and Western Eistla Regiones, for clues about their stage of evolution. The Magellan line-of-sight gravity data are inverted using a point mass model of the anomalous mass to solve for the local vertical gravity field. Spectral admittance calculated from both the local gravity inversions and a spherical harmonic model is compared to three models of compensation: local compensation, a "flexural" model with local and regional compensation of surface and subsurface loads, and a "hotspot" model of compensation that includes top loading by volcanoes and subsurface loading due to a deep, low density mass anomaly. The coherence is also calculated in each region, but yields an elastic thickness estimate only at Bell Regio. In all models, the long wavelengths are compensated locally. The long-wavelength estimates of the deep compensation depth for Bell, Atla, Western Eistla, and Beta Regiones are 125, 175, 200, and 225 km, respectively, with an error of approximately ± 35 km. The flexural model is rejected primarily because it gives values of effective elastic thickness of approximately 100 km, which is at least a factor of two larger than estimates of effective elastic thickness obtained from the coherence spectra at Bell Regio and from other studies that model the topographic expression of flexure at Bell Regio and in other tectonic settings globally. Assuming a crustal thickness of 30 km, the fit to the hotspot model at Atla Regio gives an effective elastic thickness of 30 ± 5 km. Atla Regio is interpreted as an active hotspot because of the deep compensation depth and a strong subsurface loading signature. At Bell Regio, effective elastic thickness is 30 ± 5 km at short wavelengths and 50 ± 5 km at long wavelengths, using a crustal thickness of 10 km. The 30-km value is interpreted as the effective elastic thickness at the time when the volcanoes are emplaced; the 50-km value is believed to reflect the present-day effective elastic thickness. Bell Regio has a relatively shallow compensation depth, 125 km, and a weak bottom-to-top-loading ratio determined from the coherence spectra. These results may indicate a relatively old, possibly inactive plume. The data at Beta and Western Eistla Regiones are of relatively poor quality and do not permit detailed interpretations. These areas are interpreted as active hotspots because of their large compensation depths, greater than typical of the rest of the planet.

Surface expression of functional T cell receptor chains formed by interlocus recombination on human T lymphocytes.

Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire.

Beta II-spectrin (fodrin) and beta I epsilon 2-spectrin (muscle) contain NH2- and COOH-terminal membrane association domains (MAD1 and MAD2).

Central to spectrin's function is its association with the plasma membrane. The linking proteins ankyrin and protein 4.1 partly mediate this association, and their interactions with spectrin are well understood. Both beta I (erythrocyte) and beta II (fodrin, beta G) spectrin also associate with unknown protein receptors in crude membrane preparations by ankyrin and protein 4.1 independent mechanisms. As a first step to understanding this interaction, kinetic and equilibrium assays have been used to monitor which regions of beta I and beta II spectrin inhibit the binding of purified 125I-labeled bovine brain spectrin to demyelinated and NaOH-stripped bovine brain membranes. A series of 19 recombinant proteins spanning the entire sequence of beta II spectrin, including an alternatively spliced NH2-terminal isoform (beta II epsilon 2 spectrin), were prepared as glutathione S-transferase fusion proteins. Also prepared were peptides representing the alternatively spliced COOH-terminal domain found in beta I epsilon 2 spectrin ("muscle spectrin"). Two distinct sequence motifs inhibited the binding of native brain spectrin. Membrane association domain 1 (MAD1) was represented in all fusion peptides that included spectrin repeat 1. These peptides slowed the kinetics of brain spectrin binding and inhibited up to 46% of the maximal binding under the conditions of these assays (apparent Ki < or = 0.2 microM). Peptides representative of repeats 2-17 of beta II spectrin were devoid of inhibitory activity. The second membrane association domain (MAD2) was identified in penultimate COOH-terminal sequences (domain III) of both beta II and beta I epsilon 2 spectrin. These sequences were absent in beta I epsilon 1 (erythrocyte) spectrin. MAD2 competitively inhibited over 80% of brain spectrin binding in these assays, with an apparent Ki < or = 0.1 microM. Direct binding studies confirmed that both MAD1 and MAD2 peptides associated with membranes with affinities comparable to their inhibition constants. Sequence comparisons suggest that MAD1 is created by the insertion of two non-homologous sequence motifs into repeat 1, extending it from 106 to 122 amino acids. Similarly, MAD2 encompasses a putative site of beta gamma-heterotrimeric G-protein binding called the pleckstrin homology domain, and MAD2 may in fact be the pleckstrin homology domain although this has not been rigorously proven. Collectively these studies identify two novel functional motifs in spectrin that mediate ankyrin independent association with membranes. We hypothesize that these motifs and their still to be discovered ligands play a primary role in the nascent assembly and stabilization of an ordered and polarized spectrin skeleton.

The Relationship between Topography and Gravity on Earth and Venus

The measurement of the admittance between the topography and the gravity of a planet as a function of wavelength provides important constraints on its interior processes. A detailed discussion of the influence of noise on such measurements shows that at long wavelengths the admittance is best determined in the frequency domain, using gravity as input and topography as output. This procedure is used to estimate the admittance in the Pacific and Indian Oceans. The observed values are reduced by volcanism above plumes, which produces a contribution to the long-wavelength-compensated topography. When this effect is taken into account there is little evidence that the admittance is reduced by the presence of a low viscosity zone in the upper mantle. A similar study of Venus shows an excellent correlation between gravity g and topography h for wavelengths greater than 700 km, with an admittance of about 50 mgal km -1. This value agrees well with that expected from numerical axisymmetric models of plumes and can be used to construct maps of residual topography by calculating h - g/50, where h is in kilometers and g in milligals. The power spectrum of the gravity field and its skewness, with positive anomalies covering a smaller area, but being larger, than the negative ones, are also in agreement with those of the numerical models. Of the major topographic features, only Aphrodite and Ishtar Terrae behave differently, with a value of admittance of about one-third of other large structures. This difference requires their topography to be partly supported by variations in crustal thickness. By analogy with similar structures on Earth, both are likely to be supported by volcanically thickened crust on top of rising plumes, though the observed relationships do not rule out other models. The behavior of the admittance at long wavelengths is important, because it can constrain the thickness of the convecting layer. At wavelengths of more than 3000 km the gravity data provides some evidence that the layer depth is about 1000 km. Over the center of Beta and Phoebe Regiones there are short-wavelength anomalies of residual topography, produced by crust that is thinner than that elsewhere, flanked by positive anomalies of thicker crust. The regions of thin crust occur beneath rift zones. This behavior can be explained if crust flows down the flanks of elevated regions above plumes, and can generate coronae when the plume support disappears.

Rol genes of Agrobacterium rhizogenes cucumopine strain: sequence, effects and pattern of expression

By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA--which we refer to as rol alpha, beta and gamma--were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5' non-coding region of rol alpha and rol beta was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol beta in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol alpha-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.