Functional outcome of De Quervain's tenosynovitis with longitudinal incision in surgically treated patients.

INTRODUCTION: De Quervain described tenosynovitis of first dorsal compartment more than 120 years ago. Women, particularly of 4th-5th decades, are at more risk of developing disease. Steroid injection has been described as first line of management over many decades, but it is associated with some significant complications like depigmentation of skin, atrophy of subcutaneous tissue, suppurative tenosynovitis and even tendon rupture. Animal studies have also reported increased risk of peritendinous adhesions with steroid injection. MATERIALS AND METHODS: We prospectively managed 46 cases of De Quervain's tenosynovitis with longitudinal incision at tertiary care hospital from 2014 to 2016. There were totally 40 patients with 9 males and 31 females between age group of 28 and 62 years. All patients were evaluated using DASH and VAS scores preoperatively and post-operatively. RESULTS: The mean preoperative DASH score was 42.26 which reduced to 5.37 post-operatively. The mean preoperative VAS score was 7.30 which reduced to 2.33 post-operatively. Intraoperatively, we found peritendinous adhesions in 8 patients and ganglion arising from first dorsal compartment in one patient. Post-operatively, we found hypertrophic scar in 3 patients and persistent numbness to first dorsal web space due to injury to superficial radial nerve in 2 patients. Six patients had recurrent symptoms and required revision surgery. CONCLUSION: Surgical release of De Quervain's tenosynovitis remains the gold standard treatment, and longitudinal incision offers advantage of easy identification of compartment, more complete releases of tendon sheath and peritendinous adhesions and less risk of palmar subluxation of tendons.

ACK1/TNK2 tyrosine kinase: molecular signaling and evolving role in cancers.

Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase (non-RTK), ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. Although early studies focused on ACK1 as a cytosolic effector of activated transmembrane RTKs, wherein it shuttles between the cytosol and the nucleus to rapidly transduce extracellular signals from the RTKs to the intracellular effectors, recent data unfold a new aspect of its functionality as an epigenetic regulator. ACK1 interacts with the estrogen receptor (ER)/histone demethylase KDM3A (JHDM2a) complex, which modifies KDM3A by tyrosine phosphorylation to regulate the transcriptional outcome at HOXA1 locus to promote the growth of tamoxifen-resistant breast cancer. It is also well established that ACK1 regulates the activity of androgen receptor (AR) by tyrosine phosphorylation to fuel the growth of hormone-refractory prostate cancers. Further, recent explosion in genomic sequencing has revealed recurrent ACK1 gene amplification and somatic mutations in a variety of human malignancies, providing a molecular basis for its role in neoplastic transformation. In this review, we will discuss the various facets of ACK1 signaling, including its newly uncovered epigenetic regulator function, which enables cells to bypass the blockade to major survival pathways to promote resistance to standard cancer treatments. Not surprisingly, cancer cells appear to acquire an 'addiction' to ACK1-mediated survival, particularly under stress conditions, such as growth factor deprivation or genotoxic insults or hormone deprivation. With the accelerated development of potent and selective ACK1 inhibitors, targeted treatment for cancers harboring aberrant ACK1 activity may soon become a clinical reality.

SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation.

Activated Cdc42-associated kinase 1 (ACK1) is a nonreceptor tyrosine kinase linked to cellular transformation. The aberrant regulation of ACK1 promotes tumor progression and metastasis. Therefore, ACK1 is regarded as a valid target in cancer therapy. Seven in absentia homolog (SIAH) ubiquitin ligases facilitate substrate ubiquitinylation that targets proteins to the proteasomal degradation pathway. Here we report that ACK1 and SIAH1 from Homo sapiens interact in a yeast two-hybrid screen. Protein-protein interaction studies and protein degradation analyses using deletion and point mutants of ACK1 verify that SIAH1 and the related SIAH2 interact with ACK1. The association between SIAHs and ACK1 depends on the integrity of a highly conserved SIAH-binding motif located in the far C-terminus of ACK1. Furthermore, we demonstrate that the interaction of ACK1 with SIAH1 and the induction of proteasomal degradation of ACK1 by SIAH1 are independent of ACK1's kinase activity. Chemical inhibitors blocking proteasomal activity corroborate that SIAH1 and SIAH2 destabilize the ACK1 protein by inducing its proteasomal turnover. This mechanism apparently differs from the lysosomal pathway targeting ACK1 after stimulation with the epidermal growth factor. Our data also show that ACK1, but not ACK1 mutants lacking the SIAH binding motif, has a discernable negative effect on SIAH levels. Additionally, knockdown approaches targeting the SIAH2 mRNA uncover specifically that the induction of SIAH2 expression, by hormonally-induced estrogen receptor (ER) activation, decreases the levels of ACK1 in luminal human breast cancer cells. Collectively, our data provide novel insights into the molecular mechanisms modulating ACK1 and they position SIAH ubiquitin ligases as negative regulators of ACK1 in transformed cells.

Association of terminal deoxynucleotidyl transferase with Ku.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein-protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.

Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis.

BACKGROUND: The caspase-mediated proteolysis of many cellular proteins is a critical event during programmed cell death or apoptosis. It is important to determine which caspases are activated in mammalian cells, and where and when activation occurs, upon receipt of specific death stimuli. Such information would be useful in the design of strategies to regulate the activation of caspases during apoptosis. RESULTS: We developed two novel fluorescent substrates that were specifically cleaved by caspase-1 or caspase-3. For in vitro studies, four-amino-acid recognition sequences, YVAD for caspase-1 and DEVD for caspase-3, were introduced between blue fluorescent protein (BFP) and green fluorescent protein (GFP), expressed in bacteria and purified. For in vivo studies, YVAD and DEVD were introduced between cyan fluorescent protein and yellow fluorescent protein, and expression was monitored in live mammalian cells. The proximity between fluorophores was determined using fluorescence resonance energy transfer. Purified substrates were cleaved following exposure to purified caspase-1 and caspase-3. In Cos-7 cells, caspase-1 and caspase-3 substrates were cleaved upon induction of apoptosis with staurosporine, a protein-kinase inhibitor, whereas caspase-3 but not caspase-1 substrate was cleaved upon treatment of cells with the DNA-damaging agent mitomycin c. CONCLUSIONS: These substrates allow the spatial activation of specific members of the caspase family to be deciphered during the initiation and execution phase of programmed cell death, and allow activation of specific caspases to be monitored both in vivo and in vitro. This technology is also likely to be useful for high-throughput screening of reagents that modulate caspase activity.

Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer.

It has been hypothesized that interaction of Bcl-2 and Bax may regulate apoptosis. The spatial and temporal interaction of Bcl-2 and Bax at the single cell level has not, however, been demonstrated. To achieve this goal, we have developed two-fusion FRET (fluorescence resonance energy transfer). Using green fluorescent protein (GFP)-Bax and blue fluorescent protein (BFP)-Bcl-2 fusion proteins coexpressed in the same cell, we demonstrate a direct interaction between Bcl-2 and Bax in individual mitochondria. Mitochondrially localized cytochrome c-GFP and BFP-Bcl-2 showed little or no FRET, while nuclear-localized GFP-human papillomavirus E6 and BFP-Bcl-2 did not interact when coexpressed in the same cell. These findings indicate that two-fusion FRET provides an opportunity to examine the interaction between two different proteins coexpressed in single intact mammalian cells.

Nucleotide sequence and expression in E. coli of the complete P4 type VP4 from a G2 serotype human rotavirus.

The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 in E. colic has not been achieved. In this study we present successful expression in E. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.