Dimerization of three Id proteins (Id1, Id2, and Id3) with the four class A E proteins (E12, E47, E2-2, and HEB) and two groups of class B proteins, the myogenic regulatory factors (MRFs: MyoD, myogenin, Myf-5 and MRF4/Myf-6), and the hematopoietic factors (Scl/Tal-1, Tal-2, and Lyl-1) were tested in a quantitative yeast 2-hybrid assay. All three Ids bound with high affinity to E proteins, but a much broader range of interactions was observed between Ids and the class B factors. Id1 and Id2 interacted strongly with MyoD and Myf-5 and weakly with myogenin and MRF4/Myf-6, whereas Id3 interacted weakly with all four MRFs. Similar specificities were observed in co-immunoprecipitation and mammalian 2-hybrid analyses. No interactions were found between the Ids and any of the hematopoietic factors. Each Id was able to disrupt the ability of E protein-MyoD complexes to transactivate from a muscle creatine kinase reporter construct in vivo. Finally, mutagenesis experiments showed that the differences between Id1 and Id3 binding map to three amino acids in the first helix and to a small cluster of upstream residues. The Id proteins thus display a signature range of interactions with all of their potential dimerization partners and may play a role in myogenesis which is distinct from that in hematopoiesis.An increasingly important role is ascribed to protein-protein interactions in the regulation of cellular growth and differentiation pathways. Dimerization serves to convert inactive monomeric molecules into transcriptionally active dimeric complexes at specific times during cellular development. Deletional analysis has identified a number of evolutionarily conserved regions that mediate these interactions. One such region, commonly associated with transcription factors involved in a range of proliferative and differentiation pathways, is the basic-helixloop-helix (bHLH) 1 (1). This domain is conserved from yeast to mammals and is composed of a positively charged basic region followed by two amphipathic ␣-helices separated by a spacer loop. Dimers are stabilized by a series of hydrophobic and electrostatic interactions between the helices of compatible molecules (2-5). The juxtaposition of two basic regions resulting from dimerization forms a DNA binding interface able to insert into the major groove in a sequence-specific manner (2, 6). Although bHLH proteins have no discernible DNA binding activity as monomers, dimers recognize a consensus DNA sequence (CANNTG), termed the E box (4, 7-9).bHLH transcription factors can be broadly placed into two categories (reviewed in Ref. 10). The class A factors, or E proteins, (E2-2, HEB, and the E2A gene products E12 and E47) are expressed in a virtually ubiquitous pattern and are able to dimerize efficiently with tissue-restricted class B factors to activate gene expression (1,(11)(12)(13). Because each factor contributes a specific DNA recognition half-site, class A and class B heterodimeric complexes and class A hetero-or homodimers theoretically provide distinct combinatorial E b...

BackgroundAcne is a common disorder of the human pilosebaceous unit, yet the mechanisms underlying hyperkeratinisation and subsequent inflammation (comedogenesis) remain to be determined, although cutaneous pathogens are implicated. Previously, it was reported that the release of the cytokine interleukin-1α (IL-1α) by keratinocytes of the sebaceous duct was pivotal in the life cycle of the comedone, mediating both its development and its spontaneous resolution. Toll-like receptors are a family of molecules that recognise pathogen associated molecular patterns (PAMPs) presented by microorganisms, initiating a signalling cascade terminating in the release of antimicrobial compounds and cytokines.MethodsWe used ex vivo sebaceous gland and primary monolayer keratinocyte culture, alongside ELISAs, immunohistochemistry, Western blotting and RT-PCR to investigate the contribution of TLR activation to acne pathogenesis.ResultsWe found TLR2 to be expressed in basal and infundibular keratinocytes, and sebaceous glands, and its activation provoked the release of IL-1α from primary human keratinocytes in vitro. The exposure of microdissected human sebaceous glands to PAMPs specific for TLR2 in vitro resulted in a pattern of IL-1α like cornification after seven days of exposure.ConclusionsTLR activation and secretion of IL-1α from keratinocytes may be initiating steps in comedogenesis and, therefore, critical to the pathophysiology of acne.

Summary. We report a largely retrospective analysis of minimal residual disease (MRD) in a cohort of 66 children suffering from acute lymphoblastic leukaemia (ALL). All patients lacked high-risk features at diagnosis, i.e. the presenting white cell count was <50 × 10 9 /l, age 1-16 years and translocations t(9;22) and t(4;11) were not present. All were treated according to either the MRC protocols UKALL X or XI. PCR of IgH, TCRd and TCRg gene rearrangements and allele-specific oligoprobing were employed for the detection of MRD. Sensitivity was at least 10 ¹4 in 78/82 (93%) probes examined. A total of 33 patients relapsed (seven on therapy and 26 off) and 33 remain in continuing complete remission (CCR) (median follow-up 69 months from diagnosis). Of those who remain in CCR, MRD was present in the bone marrow in 32%, 10% and 0% at 1, 3 and 5 months into therapy respectively. This is in marked contrast to the presence of MRD at these times in 82%, 60% and 41% of patients who relapsed (P<0·001, P<0·005 and P<0·005). These results provide further evidence of a strong correlation between clearance of MRD early in therapy and clinical outcome in childhood ALL.

BackgroundTexture within biological specimens may reveal critical insights, while being very difficult to quantify. This is a particular problem in histological analysis. For example, cross-polar images of picrosirius stained skin reveal exquisite structure, allowing changes in the basketweave conformation of healthy collagen to be assessed. Existing techniques measure gross pathological changes, such as fibrosis, but are not sufficiently sensitive to detect more subtle and progressive pathological changes in the dermis, such as those seen in ageing. Moreover, screening methods for cutaneous therapeutics require accurate, unsupervised and high-throughput image analysis techniques.ResultsBy analyzing spectra of images post Gabor filtering and Fast Fourier Transform, we were able to measure subtle changes in collagen fibre orientation intractable to existing techniques. We detected the progressive loss of collagen basketweave structure in a series of chronologically aged skin samples, as well as in skin derived from a model of type 2 diabetes mellitus.ConclusionsWe describe a novel bioimaging approach with implications for the evaluation of pathology in a broader range of biological situations.

Investigators require intuitive tools to rationalize complex datasets generated by transcriptional profiling experiments. Pathway analysis methods, in which differentially expressed genes are mapped to databases of reference pathways to facilitate assessment of relative enrichment, lead investigators more effectively to biologically testable hypotheses. However, once a set of differentially expressed genes is isolated, pathway analysis approaches tend to ignore rich gene expression information and, moreover, do not exploit relationships between transcripts. In this article, we report the development of a new method in which both pathway topology and the magnitude of gene expression changes inform the scoring system, thereby providing a powerful filter in the enrichment of biologically relevant information. When four sample datasets were evaluated with this method, literature mining confirmed that those pathways germane to the physiological process under investigation were highlighted by our method relative to z-score overrepresentation calculations. Moreover, non-relevant processes were downgraded using the method described herein. The inclusion of expression and topological data in the calculation of a pathway regulation score (PRS) facilitated discrimination of key processes in real biological datasets. Specifically, by combining fold-change data for those transcripts exceeding a significance threshold, and by taking into account the potential for altered gene expression to impact upon downstream transcription, one may readily identify those pathways most relevant to pathophysiological processes.