Hes1 Increases the Invasion Ability of Colorectal Cancer Cells via the STAT3-MMP14 Pathway

The Notch pathway contributes to self-renewal of tumor-initiating cell and inhibition of normal colonic epithelial cell differentiation. Deregulated expression of Notch1 and Jagged1 is observed in colorectal cancer. Hairy/enhancer of split (HES) family, the most characterized targets of Notch, involved in the development of many cancers. In this study, we explored the role of Hes1 in the tumorigenesis of colorectal cancer. Knocking down Hes1 induced CRC cell senescence and decreased the invasion ability, whereas over-expression of Hes1 increased STAT3 phosphorylation activity and up-regulated MMP14 protein level. We further explored the expression of Hes1 in human colorectal cancer and found high Hes1 mRNA expression is associated with poor prognosis in CRC patients. These findings suggest that Hes1 regulates the invasion ability through the STAT3-MMP14 pathway in CRC cells and high Hes1 expression is a predictor of poor prognosis of CRC.     

Effects of dithiothreitol on protein activity unrelated to thiol-disulfide exchange: for consideration in the analysis of protein function with Cleland's reagent

Dithiothreitol (DTT) is widely used to reduce disulfide bonds in the analysis of protein structure and function. However, thiol-disulfide exchange is not the only mechanism whereby DTT can alter protein function. We observe that DTT diminishes the carbohydrate binding activity of a cysteineless mutant of pigpen as well as it inhibits the intact molecule. Lack of inhibition by threitol, a derivative of the four-carbon sugar threose, indicates that the thiol groups of DTT are required for inhibition, and also that DTT is not acting as a simple carbohydrate competitor. Moreover, inhibition of pigpen-carbohydrate binding is not likely due to metal chelation because pigpen binding to carbohydrate is insensitive to EDTA and 1, 10-phenanthroline, which would otherwise be expected to mimic the DTT effect. Our results suggest that DTT can interact with protein domains in the absence of cysteine residues, and that the biochemical reactivity of DTT is not necessarily one and the same with its assumed biochemical specificity.     

A C-terminal carbohydrate-binding domain in the endothelial cell regulatory protein, pigpen: new function for an EWS family member

The potential for encoding information in carbohydrate (CHO) structures has long been recognized. Selective CHO-binding proteins known as lectins and the biological events they mediate are well known. However, many lectins were originally discovered for biological activities other than saccharide binding, and only subsequently was it realized that one or more of their key functions were mediated by specific CHO recognition. Our previous observations suggested that the nuclear protein pigpen had an affinity for CHO structures. This would represent a new attribute for proteins of the EWS (Ewing's sarcoma) family, of which pigpen is a member. In this study we demonstrate that a CHO-binding domain resides in the C-terminus of the molecule and can be preferentially inhibited by saccharides, most notably N-acetyl-d-galactosamine (GalNAc) and the GalNAc-containing polysaccharide, chondroitin sulfate. Ligand blotting experiments were subsequently performed with fractionated, [(3)H]galactose-labeled cells to demonstrate the presence of chondroitin sulfate-inhibitable endogenous CHO ligands for pigpen in endothelial nuclei. Finally, microinjection of polysaccharide competitor into the nucleus of cultured endothelial cells resulted in a loss of pigpen focal accumulations, suggesting that the CHO-binding activity may be instrumental in subcellular localization of the protein. In summary, our results show ligand preference and domain specificity for pigpen's CHO affinity and provide initial evidence for physiological ligands and function. They may also shed new light on the mechanisms of oncogenic transformation involving EWS proteins.     

Coiled body heterogeneity induced by G(1) arrest with amiloride + bumetanide

Ki67 is a nuclear protein expressed in proliferating cells, but not in quiescent or G(0)-arrested cells. Similar to the proliferating cell nuclear antigen and several other well-characterized molecules, Ki67 exhibits a repeating pattern of regulated expression and redistribution during the cell cycle, making it a useful marker for cell cycle phase. In addition to other structures labeled, concentrated foci may be observed in the nucleus and sometimes the cytoplasm. We observed that these Ki67 foci can be found at any stage of the endothelial cell cycle. They are not coincident with coiled bodies (CB), as determined in double-label immunofluorescence experiments with anti-Ki67 and antibodies to the CB marker protein pigpen. However, arrest of BPA47 endothelial cells in G(1) with amiloride + bumetanide induces colocalization of pigpen and Ki67 in 40% of cells exhibiting Ki67 foci. We conducted a series of experiments to examine the possibilities that pigpen was exported from CB and into unique, Ki67-containing foci or that Ki67 was imported into pigpen-containing CB. Our results showed us that although CB typically contain both coilin and pigpen, amiloride + bumetanide-induced G(1) arrest reconfigured the CB compartment into three populations of foci: one containing pigpen without coilin, the second containing coilin without pigpen, and a third containing both pigpen and coilin together. Furthermore, G(1) arrest resulted in Ki67 redistribution into both coilin- and pigpen-containing foci. The results suggest that under certain conditions, "resident" CB proteins can be differentially redistributed, and proteins not previously recognized as resident in CB can be driven into that compartment. Our observations underscore the fluid nature of CB. They demonstrate that previously reported heterogeneity in the CB compartment can be amplified by a specific experimental manipulation. This may be useful in future analyses of protein trafficking within the CB compartment and between CB and other cellular compartments. Finally, the redistribution of Ki67 into CB represents a new finding for a widely expressed but poorly understood molecule, one that may be useful in elucidating function.     

Selective gray matter staining of human brain slices: optimized use of cadaver materials

We report a novel staining technique for human brain slices that distinguishes clearly gray from white matter. Previously described techniques using either Prussian blue (Berlin blue) or phthalocyanine dyes usually have included a hot phenol pretreatment to prevent white matter staining. The technique we describe here does not require hot phenol pretreatment and allows the use of brains stored for postmortem periods of one to two years prior to staining. Our technique involves staining with copper(II) phthalocyanine-tetrasulfonic acid tetrasodium salt 1% in water for 2 h followed by acetic acid treatment; this produces excellent blue staining of gray matter with little white matter staining. The stained brain slices are excellent for teaching human brain anatomy and/or pathology, or for research purposes.     

Identification of pathogenic fungi in surgical and autopsy specimens by immunofluorescence

Summary A method is presented for the preparation of immune sera and detection by immunofluorescence ofC. immitis, S. schenckii, B. dermatitidis, C. neoformans, andC. albicans in surgical and autopsy material. Formalin fixation does not affect the antigens of the mycotic agents. There are no cross reactions except withC. immitis andC. neoformans, which can be differentiated by the site of the specific fluorescence in each organism.     

EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.     

Effect of Tetracosactid on Post Partum Cyclicity in Cows after Induction of Parturition with PGF2α

Parturition and retention of fetal membranes were induced with PGF_2α in 3 primiparous dairy cows. Starting on day 12 post partum (PP) the cows were treated with 500 μ g i.m. of ACTH-analogue (tetracosactid) every 6 h for 6 times. Changes in plasma concentrations of cortisol, progesterone and 15-ketodihydro-PGF_2α were evaluated immediately after treatment. The effects on the resumption of ovarian activity were evaluated by clinical and ultrasound examinations and by progesterone and 15-ketodihydro-PGF_2α analyses for 56 days after parturition. Treatment was able to induce a statistically significant (p < 0.01) similar increase in cortisol and progesterone after both the 1^st and the 6^th injections, in all cows. No changes in 15-ketodihydro-PGF_2αconcentrations were seen after any of the injections of ACTH-analogue. The first corpus luteum (CL) was seen on day 18 PP (cow A), and 28 (cow B) and in both cases it was followed by a normal ovarian cyclicity. No CL was observed during the whole period of study in cow C. Progesterone profiles confirmed these clinical and ultrasonographic findings. The steroid output, especially progesterone, induced by the ACTH-analogue might be a stimulus for the onset of ovarian cyclicity, since 2 of the 3 animals ovulated earlier than expected. These findings point to the fact that interference with the stress system might have a positive effect on ovarian cyclicity. The different pattern of response does however demand further studies.     

Cardiac and vascular phenotypes in the apolipoprotein E-deficient mouse

Cardiovascular death is frequently associated with atherosclerosis, a chronic multifactorial disease and a leading cause of death worldwide. Genetically engineered mouse models have proven useful for the study of the mechanisms underlying cardiovascular diseases. The apolipoprotein E-deficient mouse has been the most widely used animal model of atherosclerosis because it rapidly develops severe hypercholesterolemia and spontaneous atherosclerotic lesions similar to those observed in humans. In this review, we provide an overview of the cardiac and vascular phenotypes and discuss the interplay among nitric oxide, reactive oxygen species, aging and diet in the impairment of cardiovascular function in this mouse model.     

Nucleotide sequence of the D-loop region of the sperm whale (Physeter macrocephalus) mitochondrial genome

We have amplified, by the polymerase chain reaction, and have sequenced the D-loop region of the mitochondrial DNA from the sperm whale (Physeter macrocephalus). The sperm whale D-loop was aligned with D- loop sequences from four other cetaceans (Commerson's dolphin, orca, fin whale, and minke whale) and an out-group (cow). This alignment showed the sperm whale sequence to be larger than that of other cetaceans. In addition, some sequence blocks were highly conserved among all six species, suggesting roles in the functioning of mitochondrial DNA. Other blocks that were previously reported to be well conserved among cetaceans showed little sequence conservation with the sperm whale D-loop, which argues against the functional importance of these sequence blocks in cetaceans.