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H Kirby

Publications and source records attributed to H Kirby.

11 recordsLinked to original sources
PubMed2001

Generation of a minimal alpha5beta1 integrin-Fc fragment.

The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. (Stephens, P. E., Ortlepp, S., Perkins, V. C., Robinson, M. K., and Kirby, H. (2000) Cell. Adhes. Commun. 7, 377-390), a series of recombinant soluble human alpha(5)beta(1) integrin truncations have been expressed as Fc fusion proteins. These proteins were examined for their ligand-binding properties and for their expression of anti-integrin antibody epitopes. The shortest functional alpha(5)-subunit truncation contained the N-terminal 613 residues, whereas the shortest beta(1)-subunit was a fragment containing residues 121-455. Each of these minimally truncated integrins displayed the antibody binding characteristics of alpha(5)beta(1) purified from human placenta and bound ligand with the same apparent affinity as the native receptor.

Animals↗
PubMed2000

The activity of the Epstein-Barr virus BamHI W promoter in B cells is dependent on the binding of CREB/ATF factors.

The programme of Epstein-Barr virus (EBV) gene expression that leads to virus-induced growth transformation of resting B lymphocytes is initiated through activation of the BamHI W promoter, Wp. The factors regulating Wp, and the basis of its preferential activity in B cells, remain poorly understood. Previous work has identified a B cell-specific enhancer region which is critical for Wp function and which contains three binding sites for cellular factors. Here we focus on one of these sites and show, using bandshift assays, that it interacts with three members of the CREB/ATF family of cell transcription factors, CREB1, ATF1 and ATFa. A mutation which abrogates the binding of these factors reduces Wp reporter activity specifically in B cell lines, whereas a mutation which converts the site to a consensus CREB-binding sequence maintains wild-type promoter function. Furthermore Wp activity in B cell, but not in non-B cell, lines could be inhibited by cotransfection of expression plasmids expressing dominant negative forms of CREB1 and ATF1. Increasing the basal activity of CREB/ATF proteins in cells by treatment with protein kinase A or protein kinase C agonists led to small increases in Wp activity in B cell lines, but did not restore promoter activity in non-B cell lines up to B cell levels. We conclude that CREB/ATF factors are important activators of Wp in a B cell environment but require additional B cell-specific factors in order to mediate their effects.

Activating Transcription Factor 1↗
PubMed2000

The Epstein-Barr virus promoter initiating B-cell transformation is activated by RFX proteins and the B-cell-specific activator protein BSAP/Pax5.

Epstein-Barr virus (EBV)-induced B-cell growth transformation, a central feature of the virus' strategy for colonizing the human B-cell system, requires full virus latent gene expression and is initiated by transcription from the viral promoter Wp. Interestingly, when EBV accesses other cell types, this growth-transforming program is not activated. The present work focuses on a region of Wp which in reporter assays confers B-cell-specific activity. Bandshift studies indicate that this region contains three factor binding sites, termed sites B, C, and D, in addition to a previously characterized CREB site. Here we show that site C binds members of the ubiquitously expressed RFX family of proteins, notably RFX1, RFX3, and the associated factor MIBP1, whereas sites B and D both bind the B-cell-specific activator protein BSAP/Pax5. In reporter assays with mutant Wp constructs, the loss of factor binding to any one of these sites severely impaired promoter activity in B cells, while the wild-type promoter could be activated in non-B cells by ectopic BSAP expression. We suggest that Wp regulation by BSAP helps to ensure the B-cell specificity of EBV's growth-transforming function.

B-Lymphocytes↗
PubMed2000

Expression of a soluble functional form of the integrin alpha4beta1 in mammalian cells.

The integrin alpha4beta1(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human alpha4 and beta1 subunits were fused to the genomic DNA encoding the human gamma1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote alpha4beta1 heterodimer formation. The soluble alpha4beta1-Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent Kd for VCAM-1 binding were similar for both the soluble and native forms of alpha4beta1. In addition, the integrin-Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble alpha4beta1 should be generally applicable to a range of integrins.

Animals↗
PubMed1998

Characterisation of regulatory sequences at the Epstein-Barr virus BamHI W promoter.

Epstein-Barr virus, a human gammaherpesvirus, possesses a unique set of latent genes whose constitutive expression in B cells leads to cell growth transformation. The initiation of this growth transforming infection depends on a viral promoter in BamHI W (Wp) whose regulation is poorly understood. Using Wp reporter constructs in in vitro transfection assays, we found that Wp was 11- to 190-fold more active in B cell than in non-B cell lines and that three regions of the promoter (termed UAS1, UAS2, and UAS3) contributed to transcriptional activation. The upstream regions UAS3 (-1168 to -440) and UAS2 (-352 to -264) both functioned in a cell lineage-independent manner and were together responsible for the bulk of Wp activity in non-B cells; mutational analysis indicated the importance of a YY1 binding site in UAS2 in that context. By contrast, UAS1 (-140 to -87) was B cell specific and was the key determinant of the promoter's increased activity in B cell lines. Mutational analysis of UAS1 sequences combined with in vitro bandshift assays revealed the presence of three binding sites for cellular factors in this region. When mutations that abolished factor binding in bandshift assays were introduced into a Wp reporter construct, the loss of any one of the three UAS1 binding sites was sufficient to reduce promoter activity by 10- to 30-fold in B cells. From sequence analysis, two of these appear to be novel transcription factor binding sites, whereas the third was identified as a cyclic AMP response element (CRE). Our data indicate that this CRE interacts with CREB and ATF1 proteins present in B cell nuclear extracts and that this interaction is important for Wp activity.

Activating Transcription Factors↗
PubMed1994

Harold Kirby's symbionts of termites: karyomastigont reproduction and calonymphid taxonomy.

Harold Kirby's brilliant principle of mastigont multiplicity is published here posthumously more than 40 years after it was written. He applies this principle to large multinucleate protist symbionts of termites in establishing the taxonomy of Calonymphids (Family Calonymphidae in Phylum Zoomastigina, Kingdom Protoctista). The nuclei and kinetosomes in these heterotrophic cells are organized into trichomonad-style mastigont units which reproduce independently of cytokinesis to generate nine new Calonympha and nineteen new Stephanonympha species. The total of six genera (Calonympha, Coronympha, Diplonympha, Metacoronympha, Snyderella and Stephanonympha, all symbionts of dry-wood-eating termites, Kalotermitidae) are recognized. With the aid of Michael Yamin, the distribution of all twenty-eight of Kirby's Calonympha and Stephanonympha species are tabulated. In italic type I have annotated this paper to be comprehensible to a wide readership of cell biologists, protistologists and those interested in insect symbionts. Although this extremely original and careful work was not finished when Kirby died suddenly in 1952, I deemed it important and complete enough to finally publish it so that it would not be lost to scientific posterity.

Animals↗
PubMed1990

Psychometric assessment of depression in an elderly general medical population. Over- or underassessment?

A total of 247 consecutively evaluated geriatric medical patients was administered a battery of neuropsychological and psychological tests as part of their diagnostic workup for unexplained deterioration in their functioning. Depression was assessed with a short form of the MMPI, the Brief Symptom Inventory, and the Geriatric Depression Scale. By Research Diagnostic Criteria, most suffered from major (59%) or minor (21%) depressions; some degree of cognitive impairment was seen in 80% of the patients, defining a population of "vulnerable" geriatric patients typical of referrals to a general medical hospital setting. Using both conventional score cutoff criteria and discriminant analyses, false-negative rates up to 53% for major depression and 100% for minor depression were found. Psychometric misrecognition of depression was not related to degree of dementia or education but on some measures was positively associated with verbal intelligence level and patient age. Contrary to previous suggestions that psychometric measures overestimate depression in the elderly, these findings suggest that there may be a subgroup of elderly in which treatable affective distress is not appreciated.

Aged↗
PubMed1989

Comments on "Speech Understanding and Aging".

The hypothesis advanced by the CHABA Working Group on Speech Understanding and Aging [J. Acoust. Soc. Am. 83, 859-895 (1986)] that the systematic decline in speech understanding with age might be explained by concomitant decline in extra-auditory cognitive factors was tested by examining speech audiometric findings in patients with dementia. The fact that performance was consistent with normal central auditory function in 12 of 23 such patients, in spite of deficits in immediate memory for spoken material, tolerance of distraction, mental tracking and sequencing, cognitive flexibility, and set shifting argues against the hypothesis that speech understanding deficits in the elderly can be explained as the simple consequence of cognitive decline.

Aged↗