Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells

Background: Truncated variants of Ku86 protein have previously been detected in 86% to 100% of freshly isolated patient multiple myeloma (MM) cells. Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA-dependent protein kinase, we have been interested in the altered expression and function of Ku86 variant (Ku86v) proteins in genome maintenance of MM. Results: Although, a number of studies have suggested that truncated forms of Ku proteins could be artificially generated by proteolytic degradation in vitro in human lymphocytes, we now show using whole cell immunoblotting that the RPMI-8226 and SGH-MM5 human MM cell lines consistently express full-length Ku86 as well as a 69-kDa Ku86v; a C-terminus truncated 69-kDa variant Ku86 protein. In contrast, Ku86v proteins were not detected in the freshly isolated lymphocytes as was previously reported. Data also indicates that the Ku86v was not generated as a result of carbohydrate modification but that serine proteases may act on the full-length form of the protein. Conclusion: These data confirm that MM cells contain bona fide Ku86v proteins that were generated intracellularly by a post-transcriptional mechanism, which required proteolytic processing

Use of Phage Display to Isolate Specifi c Human Monoclonal Antibody Fragments Against a Potential Target for Multiple Myeloma

Introduction: Multiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM. Materials and Methods: Here, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86. Results: Anti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A450~1.1) showed a 1/3 increase in binding as compared to the first round scFvs (A450~0.4) with 100ug/mL of antigen (purified human Ku86). Subsequent selection and verifi cation of monoclonal antibodies using third round biopanning revealed 4 good affi nity binding clones ranging from A450~0.1 to A450~0.15 on 12.5ug/mL of antigen as compared to low binders (A450~0.07) and these antibodies bind to Ku86 in a specifi c and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1- 374. Conclusions: These studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents

Responses to gestational weight management guidance: a thematic analysis of comments made by women in online parenting forums

Background: The National Institute for Health and Clinical Excellence (NICE) published guidance on weight management in pregnancy in July 2010[1], and this received considerable press coverage across a range of media. This offered an opportunity to examine how gestational weight management guidance was received by UK women. Methods: A thematic analysis was conducted of 400 posts made in UK-based parenting internet forums in the week following the publication of the NICE guidance. This allowed us to examine the naturally occurring comments from 202 women who posted about the guidance on public forums. Results: Three main themes were identified and explored: i) Perceived control/responsibility ii) Risk perception iii) Confused messages. Conclusions: Women differed in their perceptions of the level of control that they had over being overweight with some feeling responsible and motivated to maintain a healthy lifestyle. Others felt there were multiple factors influencing their weight issues beyond their control. There were reports of feeling guilty about the impact of weight on the growing baby and experiencing significant obesity stigma from the public and health professionals. Information about the risks of overweight and obesity in pregnancy were difficult messages for women to hear, and for health professionals to deliver. Women reported being confused by the messages that they received. Health messages need to be delivered sensitively to women, and health professionals need support and training to do this. Risk information should always be accompanied with clear advice and support to help women to manage their weight in pregnancy. Keywords: internet-mediated research, gestational weight gain, parenting forums, NICE, women, views, risk perception</p

Pdl1 Is a Putative Lipase that Enhances Photorhabdus Toxin Complex Secretion

The Toxin Complex (TC) is a large multi-subunit toxin first characterized in the insect pathogens Photorhabdus and Xenorhabdus, but now seen in a range of pathogens, including those of humans. These complexes comprise three protein subunits, A, B and C which in the Xenorhabdus toxin are found in a 4∶1∶1 stoichiometry. Some TCs have been demonstrated to exhibit oral toxicity to insects and have the potential to be developed as a pest control technology. The lack of recognisable signal sequences in the three large component proteins hinders an understanding of their mode of secretion. Nevertheless, we have shown the Photorhabdus luminescens (Pl) Tcd complex has been shown to associate with the bacteria's surface, although some strains can also release it into the surrounding milieu. The large number of tc gene homologues in Pl make study of the export process difficult and as such we have developed and validated a heterologous Escherichia coli expression model to study the release of these important toxins. In addition to this model, we have used comparative genomics between a strain that releases high levels of Tcd into the supernatant and one that retains the toxin on its surface, to identify a protein responsible for enhancing secretion and release of these toxins. This protein is a putative lipase (Pdl1) which is regulated by a small tightly linked antagonist protein (Orf53). The identification of homologues of these in other bacteria, linked to other virulence factor operons, such as type VI secretion systems, suggests that these genes represent a general and widespread mechanism for enhancing toxin release in Gram negative pathogens

Validity of the Katz Index to assess activities of daily living by informants in neuropathological studies

Abstract OBJECTIVE To analyze the evidences of construct validity of the Katz Index for the retrospective assessment of activities of daily living (ADL) by informants, to assist neuropathological studies in the elderly. METHOD A cross-sectional study analyzed the functional ability of ADL measure by the Katz Index, of 650 cases randomly selected from the Brazilian Brain Bank of the Ageing Brain Study Group (BBBABSG) database. Sample was divided in two subsamples for the analysis (N=325, each) and then stratified according to cognitive decline assessed by the Clinical Dementia Rating Scale (CDR). Factor analyses with calculations of internal consistency and invariance were performed. RESULTS Factor analysis evidenced a unidimensional instrument with optimal internal consistency, in all subgroups. Goodness of fit indices were obtained after two treatments of covariance, indicating adequacy of the scale for assessing ADL by informants. The scale is invariant to cognitive decline meaning that it can be used for subjects with or without cognitive impairment. CONCLUSION Katz Index is valid for the retrospective assessment of basic ADL by informants, with optimal reliability

Protein Mapping and Characterization of Post-Translational Modifications of Ku86 Protein Variants in Multiple Myeloma Cell Lines.

Abstract A single biological event, immunoglobulin heavy chain (IgH) isotype class switch recombination (CSR) is thought to mark the onset of multiple myeloma (MM). The DNA repair enzyme, DNA-dependent protein kinase (DNA-PK), which consists of a heterodimeric regulatory subunit (Ku70/Ku86) and its catalytic subunit (DNA-PKcs), is the principal mediator of IgH isotype CSR. Studies in Ku70 and Ku86 knockout mice have suggested that Ku proteins could have a role to play in carcinogenesis. Since we have previously detected truncated variants of Ku86 (Ku86v) in 86% to 100% of freshly isolated patient MM cells, we therefore hypothesize that Ku86v could be a putative oncoprotein. Two variants of Ku86 are predominantly found in MM cells: a 69 kDa variant truncated at the C-terminus (Ku86v-N), which has lost its DNA repair function; and a 56 kDa N-terminus truncated variant (Ku86v-C), which is not translocated into the nucleus and aberrantly expressed on the cell membrane. Our prior studies using Northern blotting, whole cell polyacrylamide gel electrophoresis (PAGE) and electrophoretic mobility shift assays (EMSA) have demonstrated that Ku86vs in MM cells are not the result of alternative RNA splicing; or in vitro degradation, as has been described in human lymphocytes. Moreover, since many proteins in MM cells are known to be heavily glycosylated, and deglycosylation could lead to shorter forms of the protein, we now show using Endo H digestion that Ku86vs are also not the result of N-linked deglycosylation. Rather, Ku86vs are formed by in vivo proteolytic cleavage via a trypsin-like serine protease. Since the proteasome degradation pathway is highly active in MM cells; and protease digestion of DNA-PK holoenzyme is enhanced by proteasome inhibition (i.e. bortezomib treatment), these data support the notion that in vivo activation of putative Ku86v oncoproteins within MM cells could arise via a constitutively active post-translational proteolytic process. To define a functional role for Ku86v, we first demonstrate that Ku86v is physically associated with the most abundant anti-apoptotic protein in MM cells, BCL2. We then used cyanogen bromide immunoprecipitation to purify BCL2-bound Ku86vs followed by 2-dimensional gel electrophoresis (2DGE) to resolve these purified proteins, and demonstrate that only the 56 kDa Ku86v-C (pI 5.0) bound to BCL2. Both full length Ku86 and Ku86v-N were not detected. We then isolated Ku86v-C from the 2D gel and preliminary mass spectrometric analyses suggest that the N-terminus truncation retains the Ku autoantigen related protein 1 (KARP1) motif of Ku86. The significance of this is still under investigation. In conclusion, we have found that Ku86v-C (56 kDa pI 5.0) is formed by post-translational proteolytic cleavage. Since, Ku86v-C is significantly associated with BCL2 oncoprotein, we therefore speculate that Ku86v-C could possibly potentiate BCL2’s anti-apoptotic function. Accordingly, Ku86v-C could be considered as a potential target for therapeutic intervention in MM.</jats:p

The Biochemical Characterization of Three Forms of Ku86 Protein in Multiple Myeloma Cell Lines.

Abstract Karyotypic analysis of tumor cells from patients with multiple myeloma (MM), as well as MM cell lines, frequently demonstrates numerous complex chromosomal abnormalities. Moreover, new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32), often heralds transformation to more aggressive MM. Since DNA double stranded break repair (DSBR) is important in mediating these processes, these data suggest that abnormalities in DSBR could ultimately lead to genomic instability, clonal evolution and disease progression in MM. Truncated variants of Ku86 protein (i.e. Ku86v) have previously been detected in 86% to 100% of freshly isolated patient MM cells. Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA protein kinase (DNA-PK), we and others have been interested in the altered expression and function of Ku86v proteins in genome maintenance in MM. Although a number of studies have suggested that truncated forms of Ku proteins could be artificially generated by proteolytic degradation in vitro in B cells and the K562 chronic myeloid leukemia cell line, we now show using whole cell Western immunoblotting that the RPMI 8226 and SGH-MM5 human MM cell lines consistently express full-length Ku86 as well as at least 2 forms of Ku86v - a C-terminus truncated 69 kDa variant Ku86 protein (Ku86v-N); and an N-terminus truncated 56 kDa Ku86v (Ku86v-C). Expression of full-length Ku86 and Ku86v proteins was confirmed using electrophoretic mobility shift assays (EMSA) that incorporate a Ku86-specific DNA probe. In contrast, Ku86v proteins were not detected in the non-MM K562 cell line, by neither whole cell Western blotting nor EMSA, as was previously reported. These data confirm that MM cell lines contained bona fide Ku86v proteins that were generated intracellularly. However, the expected shorter mRNA transcripts of Ku86v’s were not detected using Northern blotting, indicating that Ku86v’s could have been generated by enzymatic cleavage, i.e. post-translational modification, rather than by alternative splicing. Since protease digestion of DNA protein kinase (DNA-PK) and Ku proteins is enhanced by proteasome inhibition (i.e. bortezomib treatment) in MM cell lines; these data taken in aggregate further suggest that proteolytic enzymes that are capable of digesting Ku proteins are constitutively activated, and possibly accumulate and/or become further activated under proteasome inhibition in MM cells. In order to characterize the functional role for Ku86v, we demonstrate using EMSA that both full-length Ku86 and Ku86v-N, but not Ku86v-C, are capable of binding DNA. Since the DNA binding motifs of Ku86 are located in the N-terminus, and the functional domains are located in the C-terminus, these data support the notion that whilst Ku86v-N binds DNA, it is in fact incapable of regulating DNA repair. By contrast, although Ku86v-C does not bind DNA, it may be capable of regulating other biological processes. Accordingly, we demonstrate that Ku86v-C binds to CDK4, E2F-4, BAX, Bcl2 and p53; suggesting at least a possible role for Ku86v proteins in regulating the growth and survival of MM cells. In conclusion, this study confirms that MM cells generate at least 3 forms of Ku86 protein, and that the processes of genome maintenance and/or myelomagenesis could be functionally regulated by these abnormal Ku86v proteins.</jats:p