Programmable type III-A CRISPR-Cas DNA targeting modules.

The CRISPR-Cas systems provide invader defense in a wide variety of prokaryotes, as well as technologies for many powerful applications. The Type III-A or Csm CRISPR-Cas system is one of the most widely distributed across prokaryotic phyla, and cleaves targeted DNA and RNA molecules. In this work, we have constructed modules of Csm systems from 3 bacterial species and heterologously expressed the functional modules in E. coli. The modules include a Cas6 protein and a CRISPR locus for crRNA production, and Csm effector complex proteins. The expressed modules from L. lactis, S. epidermidis and S. thermophilus specifically eliminate invading plasmids recognized by the crRNAs of the systems. Characteristically, activation of plasmid targeting activity depends on transcription of the plasmid sequence recognized by the crRNA. Activity was not observed when transcription of the crRNA target sequence was blocked, or when the opposite strand or a non-target sequence was transcribed. Moreover, the Csm module can be programmed to recognize plasmids with novel target sequences by addition of appropriate crRNA coding sequences to the module. These systems provide a platform for investigation of Type III-A CRISPR-Cas systems in E. coli, and for introduction of programmable transcription-activated DNA targeting into novel organisms.

Enforced SOCS1 and SOCS3 expression attenuates Lck-mediated cellular transformation.

Lck is an Src family protein tyrosine kinase with predominant T cell expression. Aberrant expression or activation of Lck kinase has been reported in both lymphoid and non-lymphoid malignancies. We showed previously that the signal transduction pathway involving Janus kinase (JAK) and signal transducer and activator of transcription (STAT) is constitutively activated and contributes to Lck-mediated oncogenesis. Under normal physiological conditions, active STAT proteins induce the expression of suppressor of cytokine signaling (SOCS) family proteins to inhibit further JAK/STAT signaling. It is not fully understood whether and how SOCS-mediated negative feedback control is dysregulated in Lck-transformed cells. Here we report that two SOCS family members, SOCS1 and SOCS3, are not expressed in Lck-transformed LSTRA leukemia. While SOCS1 gene is silenced by DNA hypermethylation, loss of SOCS3 expression is through a mechanism independent of epigenetic silencing by DNA methylation. Furthermore, ectopic expression of SOCS1 or SOCS3 leads to reduced cell proliferation and increased apoptosis in Lck-transformed cells. This is consistent with the attenuation of Lck kinase activity by exogenous SOCS1 or SOCS3 expression. Downstream STAT5 activity is also inhibited as shown by reduced STAT5 tyrosine phosphorylation and in vitro DNA binding. All together, our data highlight the importance of silencing multiple SOCS genes in tumorigenesis and support the roles of SOCS1 and SOCS3 as tumor suppressors toward oncogenic Lck kinase.

Yin yang 1 regulates the expression of snail through a distal enhancer.

Expression of the Snail gene is required for the epithelial-mesenchymal transitions that accompany mammalian gastrulation, neural crest migration, and organ formation. Pathologic expression of Snail contributes to the migratory capacity of invasive tumors, including melanomas. To investigate the mechanism of Snail up-regulation in human melanoma cells, a conserved enhancer located 3' of the Snail gene was analyzed. An overlapping Ets and yin yang 1 (YY1) consensus sequence, in addition to a SOX consensus sequence, was required for full enhancer activity. Proteins specifically binding these sequences were detected by electrophoretic mobility shift assay. The Ets/YY1 binding activity was purified by DNA-affinity chromatography and identified as YY1. Although ubiquitously expressed, YY1 was bound at the Snail 3' enhancer in vivo in Snail-expressing cells but not in cells that did not express Snail. Knockdown of YY1 in A375 cells led to decreased Snail expression. These results identify a role for YY1 in regulating transcription of Snail in melanoma cells through binding to the Snail 3' enhancer.

A constitutively active Lck kinase promotes cell proliferation and resistance to apoptosis through signal transducer and activator of transcription 5b activation.

Lck is a Src family protein tyrosine kinase and is expressed predominantly in T cells. Aberrant expression or activation of Lck kinase has been reported in both lymphoid and nonlymphoid malignancies. However, the mechanisms underlying Lck-mediated oncogenesis remain largely unclear. In this report, we establish a tetracycline-inducible system to study the biochemical and biological effects of a constitutively active Lck mutant with a point mutation at the negative regulatory tyrosine. Expression of the active Lck kinase induces both tyrosine phosphorylation and DNA-binding activity of signal transducer and activator of transcription 5b (STAT5b), a STAT family member activated in a variety of tumor cells. The active Lck kinase interacts with STAT5b in cells, suggesting that Lck may directly phosphorylate STAT5b. Expression of the constitutively active Lck mutant in interleukin-3 (IL-3)-dependent BaF3 cells promotes cell proliferation. In addition, the active Lck kinase protects BaF3 cells from IL-3 withdrawal-induced apoptotic death and leads to IL-3-independent growth. These transforming properties of the oncogenic Lck kinase can be further augmented by expression of exogenous wild-type STAT5b but attenuated by a dominant-negative form of STAT5b. All together, our results suggest the potential involvement of STAT5b in Lck-mediated cellular transformation.

Nontuberculous mycobacterial adenitis: effectiveness of chemotherapy following incomplete excision.

BACKGROUND: Management of lymphadenopathy caused by nontuberculous mycobacteria (NTM) is primarily surgical. Where this cannot achieve sufficient clearance of infected nodes, chemotherapy is often given. AIM: This study compared results of surgery alone with surgery followed by chemotherapy in instances where there was incomplete surgical removal of diseased tissue. METHODS: Chemotherapy comprised azithromycin 10 mg/kg and rifabutin 6 mg/kg both given once daily for 6 mo. Ninety-eight children with NTM infection were seen in the period 1990-2004. Sixty-eight cases with adenopathy where "time to healing" (discharge stopped and inflammation settled) was known were available to compare response to treatment. RESULTS: The median (range) "time to healing" in weeks for 43 patients who had surgery alone was: incision and drainage (I&D)/curettage 6 (1-72) (n = 10); excision 3 (1-28) (n = 22); and from the last operation of multiple (repeat) surgery 3 (1-40) (n = 11). For 25 patients who required chemotherapy in addition to surgery, the median (range) "time to healing" in weeks was I&D/curettage 10 (1-40) (n = 17), excision 14 (8-20) (n = 2) and multiple surgery 29 (2-88) (n = 6). CONCLUSION: In children with adenitis due to NTM, where surgical resection is followed by continued discharge and inflammation, chemotherapy should be considered before further surgery is undertaken.

Characterization of STAT5B phosphorylation correlating with expression of cytokine-inducible SH2-containing protein (CIS).

Cytokine-inducible SH2-containing protein (CIS) is the first identified member of genes encoding for the suppressor of cytokine signaling (SOCS). CIS is also a well-known target gene of signal transducer and activator of transcription 5 (STAT5) pathways, providing normal negative feedback control of signaling by cytokines and growth factors. Three other SOCS genes, SOCS1, SOCS2, and SOCS3, can be silenced by DNA hypermethylation in human cancers, suggesting a potential mechanism for constitutive STAT activation. However, it is not known whether CIS expression is similarly perturbed in tumor cells. We report here the absence of CIS expression in T lymphoma LSTRA that overexpresses the Lck protein tyrosine kinase and exhibits elevated STAT5 activity. Pervanadate-induced CIS expression and STAT5 binding to the CIS promoter in vivo over a short time course implies that mechanisms other than DNA hypermethylation may contribute to defective CIS expression in LSTRA cells. Comparison with cytokine-dependent BaF3 cells stimulated with interleukin-3 (IL-3) further reveals that CIS induction correlates with specific STAT5b post-translational modifications. It exhibits as the slowest migrating form through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This distinctly modified STAT5b is the predominant form that binds to the consensus STAT5 sites in the CIS promoter and accumulates in the nucleus. In vitro phosphatase assays and phosphoamino acid analysis suggest the involvement of phosphorylation on residues other than the highly conserved tyrosine and serine sites in this distinct STAT5b mobility shift. All together, our results provide a novel link between incomplete STAT5b phosphorylation and defective SOCS gene expression in cancer cells.

Eminectomy and plication of the posterior disc attachment following arthrotomy for temporomandibular joint internal derangement.

AIM: The aim of this study was to assess the success of a single surgical procedure (eminectomy and meniscal plication) in patients with internal derangement of the temporomandibular joint. PATIENTS: A retrospective survey of 119 joints (92 patients), that had undergone eminectomy +/- meniscal plication for internal derangement of the temporomandibular joint, over a 10 year period, was undertaken. METHODS: The same surgeon undertook all surgery and clinical evaluation. Assessment of joint pain, noise/click, and mobility were assessed pre- and post-operatively. Clinical assessment at 24 months postoperatively and patient evaluation (average 59 months postoperatively) formed the basis for the results. RESULTS: Clinical assessment and patient evaluation revealed an improvement in pain (65%), noise (63%) and mobility (71%). The outcome of surgery as assessed by the patients and clinicians showed that both have similar expectations and evaluations of the results of temporomandibular joint surgery, which were reasonably good. Arthrography, which was used as a diagnostic technique prior to surgery, was shown to be unreliable with relatively low sensitivity and specificity.