Search

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/flknock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.Modulation of primary-ciliogenesis and downstream Hedgehog signaling controls Wolffian duct development.

In this study, Sudarshan et al. characterized the fusion protein produced by the EPC1-PHF1 translocations, which are highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs). Their results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.

Venous thromboembolism (VTE) is a frequent complication of childhood acute lymphoblastic leukemia (ALL).

MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain (CHD) recognizing methylation of lysine 36 on histone H3 (H3K36me3) in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing, and DNA repair by homologous recombination. To get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the CHD, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase, and PALB2–BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL (EP400 N-terminal like) that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3–binding CHD of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins–associated protein complexes, which are essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.

To adequately and permanently restore organ function after grafting, human tissue-engineered skin substitutes (TESs) must ultimately contain and preserve functional epithelial stem cells (SCs). It is therefore essential that a maximum of SCs be preserved during each in vitrostep leading to the production of TESs such as the culture process and the elaboration of a skin cell bank by cryopreservation. To investigate the presence and functionality of epithelial SCs within the human TESs made by the self-assembly approach, slow-cycling cells were identified using 5′-bromo-2′-deoxyuridine (BrdU) in the three-dimensional construct. A subset of basal epithelial cells retained the BrdU label and was positive for the SC-associated marker keratin 19 within TESs after a chase of 21 days in culture post-BrdU labeling. Moreover, keratinocytes harvested from TESs gave rise to SC-like colonies in secondary monolayer subcultures, indicating that SCs were preserved within TESs. To evaluate the effect of cryopreservation with dimethyl sulfoxide and storage in liquid nitrogen on SCs, human epithelial cells were extracted from skin samples, amplified in culture, and used to produce TESs, before cryopreservation as well as after thawing. We found that the proportion and the growth potential of epithelial SCs in monolayer culture and in TESs remained constant before and after cryopreservation. Further, the functionality of these substitutes was demonstrated by successfully grafting human TESs on athymic mice for 6 months. We conclude that human epithelial skin SCs are adequately preserved upon human tissue reconstruction. Thus, these TESs produced by the self-assembly approach are suitable for clinical applications.

Crystalline silica is known for its health hazards, and since 1997 has been listed as Group 1, Carcinogenic to Humans, by the International Agency for Research on Cancer. This issue is particularly important in the industrial environment, and there is still no method that allows quantification of the different polymorphs of crystalline silica. Many analytical methods have been proposed, and the major problem in almost all cases is attributable to the very large variety of matrixes encountered. This study evaluates the potential of X-ray diffraction techniques and an automated Rietveld analysis in order to overcome this problem and to adapt the quantitative analysis of quartz, the most prevalent crystalline silica polymorph, to routine analysis in the health and safety environment. Matrix simulations are done and many parameters are optimized. Sample preparation, the acquisition program, pattern treatment, and Rietveld refinement are evaluated, and a general procedure is determined. Automation of Rietveld refinement leads to a significant reduction in analysis time, but cannot be applied to every type of sample.

The antiapoptotic members of the Bcl-2 family of proteins play multiple roles in cancer. These membrane-integrated proteins inhibit the pro-apoptotic activity of oncogenes during oncogenesis, support the survival of established cancer cells, and increase resistance to chemotherapy. Hence, strategies aimed at inhibiting the expression or activity of Bcl-2 proteins are predicted to have therapeutic value. Several antisense oligonucleotides (AO), capable of reducing expression of either Bcl-2 or Bcl-XL, were shown to induce apoptosis in cancer cells, to inhibit tumour growth in certain mouse tumour models, and to sensitise cancer cells to chemotherapy. One such AO, oblimersen, is presently being evaluated in combination with standard therapy in patients with advanced cancers, including chronic lymphocytic leukaemia and multiple myeloma. Bcl-2 proteins are thought to inhibit apoptosis by interacting with the pro-apoptotic proteins Bax and Bak, and preventing their activation. Small molecules capable of inhibiting this interaction have been discovered and shown to induce apoptosis in cancer cells. Gossypol and chelerythrine are two such molecules that inhibit tumour growth in mouse tumour models. This review summarises the evidence supporting the role of Bcl-2 proteins in cancer and then examines patented therapeutic strategies that target Bcl-2 protein expression or activities.

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified inSynechocystissp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway inEscherichia coli. Targeted inactivation of themenAand menBgenes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menAand menBmutants grow photoautotrophically under low light conditions (20 μE m−2s−1), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 μE m−2s−1). The menAandmenBmutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menAand menBmutant strains is identical to that of the wild type, but the level of active PS I is about 50–60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from themenAand menBmutant strains contain the full complement of polypeptides, show photoreduction of FAand FBat 15 K, and support 82–84% of the wild type rate of electron transfer from cytochrome c6to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menAand menBmutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A1site, where it functions as an efficient cofactor in electron transfer from A0to the iron-sulfur clusters.

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli. Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 microE m(-2) s(-1)), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 microE m(-2) s(-1)). The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50-60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F(A) and F(B) at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c(6) to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A(1) site, where it functions as an efficient cofactor in electron transfer from A(0) to the iron-sulfur clusters.

NF‐κB/Rel transcription factors participate in the activation of numerous genes involved in immune regulation/inflammation including cytokines, cell surface receptors, adhesion molecules, and acute phase proteins. NF‐κB activity is controlled by inhibitory proteins, IκBs, that maintain the DNA‐binding forms of NF‐κB in an inactive state in the cytoplasm. Many viruses, including the human retroviruses HIV‐1 and HTLV‐1, also utilize the NF‐κB/IκB pathway to their transcriptional advantage during viral infection. Our recent studies have focused on the IκBα inhibitor and have characterized several protein interactions that modulate the functional activity of IκBα during human retrovirus infection. In this article, we summarize recent studies demonstrating that (1) chronic HIV‐1 infection of human myelomonoblastic PLB‐985 cells leads to constitutive NF‐κB activity, activated in part due to enhanced IκBα turnover and increased NF‐κB/Rel production; (2) HTLV‐1 Tax protein physically associates with the IκBα protein in vivo and in vitro and also mediates a 20‐ to 40‐fold stimulation of NF‐κB DNA binding activity mediated via an enhancement of NF‐κB dimer formation; (3) casein kinase II phosphorylates IκBα at multiple sites in the C‐terminal PEST domain and regulates IκBα function; (4) transdominant forms of IκBα, mutated in critical Ser or Thr residues required for inducer‐mediated (S32A,S36A) and/or constitutive phosphorylation block HIV LTR trans‐activation and also effectively inhibit HIV‐1 multiplication in a single cycle infection model; and (5) the amino‐terminal 55aa of IκBα (NIK) interacts with the human homologue of dynein light chain 1, a small 9‐kDa human homologue of the dynein light chain protein involved in microtubule and cytoskeletal dynamics. Together, our results highlight a number of intriguing molecular interactions between IκBα and cellular or viral proteins that modulate transcription factor activity and nuclear‐cytoplasmic flow of host proteins. J. Leukoc. Biol. 62: 82–92; 1997.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains two binding sites for the NF-kappa B/Rel family of transcription factors which are required for the transcriptional activation of viral genes by inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1. In the present study, we examined the effect of transdominant mutants of I kappa B alpha on the synergistic activation of the HIV-1 LTR by TNF-alpha and the HIV-1 transactivator, Tat, in Jurkat T cells. The synergistic induction of HIV-1 LTR-driven gene expression represented a 50- to 70-fold stimulation and required both an intact HIV-1 enhancer and Tat-TAR element interaction, since mutations in Tat protein (R52Q, R53Q) or in the bulge region of the TAR element that eliminated Tat binding to TAR were unable to stimulate LTR expression. Coexpression of I kappa B alpha inhibited Tat-TNF-alpha activation of HIV LTR in a dose-dependent manner. Transdominant forms of I kappa B alpha, mutated in critical serine or threonine residues required for inducer-mediated (S32A, S36A) and/or constitutive (S283A, T291A, T299A) phosphorylation of I kappa B alpha were tested for their capacity to block HIV-1 LTR transactivation. I kappa B alpha molecules mutated in the N-terminal sites were not degraded following inducer-mediated stimulation (t1/2, > 4 h) and were able to efficiently block HIV-1 LTR transactivation. Strikingly, the I kappa B alpha (S32A, S36A) transdominant mutant was at least five times as effective as wild-type I kappa B alpha in inhibiting synergistic induction of the HIV-1 LTR. This mutant also effectively inhibited HIV-1 multiplication in a single-cycle infection model in Cos-1 cells, as measured by Northern (RNA) blot analysis of viral mRNA species and viral protein production. These experiments suggest a strategy that may contribute to inhibition of HIV-1 gene expression by interfering with the NF-kappa B/Rel signaling pathway.