Immunohistochemical analysis of HNF4A and β-catenin expression to predict low-grade dysplasia in the colitis-neoplastic sequence

Animal studies indicated that P1 promoter–driven hepatocyte nuclear factor 4 alpha (HFN4A) prevents carcinogenesis in colitis. But the function of total HNF4A protein has not been fully investigated, and it was assumed to be involved in the colitis-neoplastic sequence. The aim of this study was to determine the clinical value of total P1-/P2-driven HNF4A combined with β-catenin in the colitis-neoplastic sequence. A total of 69 samples, including 4 normal colon tissues, 16 sporadic colorectal cancer (CRC) tissues, 35 inflammatory bowel disease (IBD) tissues, and 14 IBD-associated low-grade dysplasia tissues, were collected to assess P1-/P2-driven HNF4A and β-catenin expressions by immunohistochemical assay. In addition, a colonic epithelial cell line Caco2 with stable P1-/P2-driven HNF4A knockdown was constructed. β-Catenin expression and skeleton structure were determined in the transfected cells by western blot analysis and immunofluorescence assay respectively. Increased expression of nuclear P1-/P2-driven HNF4A was observed in the colitis-associated colorectal neoplasm and sporadic CRC samples, compared with that in colitis samples. The parallel alterations between cytoplasmic β-catenin and nuclear P1-/P2-driven HNF4A were also verified. Silencing of P1-/P2-driven HNF4A expression in Caco2 cells decreased β-catenin expression and F-actin formation. Our results confirmed the elevated expressions of nuclear P1-/P2-driven HNF4A and cytoplasmic β-catenin in the colitis-neoplastic sequence, and both of them may be used as potential biomarkers to predict low-grade dysplasia.     

Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes

Larval Galleria melonella(L.)hemocytes form microaggregates in response to stimulation by Gram-positive bacteria Hemocyte adhesion to foreign materials is mediated by the CAMP/protein kinase A pathway and the B-subunit of cholera toxin using a cAMP-independent mechanism.Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide,implying integrins may be part of the mechanism.Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses ars,ory,Ai,and B3 subunits occred on both granular cell and plasmatocyte hemocyte subtypes.A fluorescent band representing the binding of rabbit as-integrin subunit antibodies occurred between adhering heterotypic hemocytes.The frequency of the bands was increased by cholera toxin.The as andβrabbit integrin subunit antibodies inhibited removal of Bacillus subtilis(Cohn)from the hemolymph in vivo,A as ir-specific synthetic peptide blocker similarly diminished hemocyte function whereas the 0v Bs-specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities.Western blots revealed several proteins reacting with a given integrin-antibody subtype.Thus integrin-antibody reactive proteins(which may include integrins)with possible as and B epitopes modulate immediate hemocyte function.Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followved by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.     

Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65

To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E. coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.     

Structural basis of interaction between protein tyrosine phosphatase PCP-2 and β-catenin

PCP-2 is a member of receptor-like protein tyrosine phosphatase of the MAM domain family. To investigate which part of PCP-2 was involved in its interaction with β-catenin, we constructed various deletion mutants of PCP-2. These PCP-2 mutants and wild-type PCP-2 were co-transfected into BHK-21 cells with β-catenin individually. An in vivo binding assay revealed that the expression of wild-type PCP-2, PCP-2 DC1C2 (deleted PCP-2 without both PTP domains) and PCP-2 ΔC2 (deleted PCP-2 without the second PTP domain) could be immunoprecipitated by anti-catenin antibody in every co-transfection, but PCP-2 EXT (deleted PCP-2 without the juxtamembrane region and both PTP domains) was missing, which implied that PCP-2 and b-catenin could associate directly and the juxtamembrane region in PCP-2 was sufficient for the process.     

AKINβ1 is Involved in the Regulation of Nitrogen Metabolism and Sugar Signaling in Arabidopsis

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been located at the heart of the control of metabolism and development in plants. The active SnRK1 form is usually a heterotrimeric complex. Subcellular localization and specific target of the SnRK1 kinase are regulated by specific beta subunits. In Arabidopsis, there are at least seven genes encoding beta subunits, of which the regulatory functions are not yet clear. Here, we tried to study the function of one beta subunit, AKINβ1. It showed that AKINβ1 expression was dramatically induced by ammonia nitrate but not potassium nitrate, and the investigation of AKINβ1 transgenic Arabidopsis and T-DNA insertion lines showed that AKINβ1 negatively regulated the activity of nitrate ruductase and was positively involved in sugar repression in early seedling development. Meanwhile AKINβ1 expression was reduced upon sugar treatment (including mannitol) and did not affect the activity of sucrose phos-phate synthase. The results indicate that AKINβ1 is involved in the regulation of nitrogen metabolism and sugar signaling.     

ARRB2 promotes colorectal cancer growth through triggering WTAP

Colorectal cancer (CRC) is one of the most lethal cancers worldwide. The expression of β-arrestin2 (β-Arr2, ARRB2) in CRC has been well investigated;however, its exact mechanism causing the cancer progression remains unclear. In this study, we discovered that the expression level of ARRB2 was significantly upregulated in CRC as compared to the normal tissues by employing the Cancer Genome Atlas (TCGA) data, western blot analysis, and immunohistochemistry. Furthermore, the level of ARRB2 was correlated with the patients’ overall survival by Kaplan–Meier analysis. The higher expression of ARRB2 promoted CRC cell growth, enhanced the cell motility, and blocked cell apoptosis, which is crucial for tumor growth. Lastly, the suppression of ARRB2 expression was enough to attenuate the progression of CRC induced by azoxymethane/dextran sodium sulfate. Interestingly, we also found that the knockdown of ARRB2 decreased several cancer pathways mediated by the expression of Wilms tumor 1 associated protein (WTAP), which led to the inhibition of cell proliferation and migration. Altogether, our results demonstrated that ARRB2 promoted the growth and migration of CRC cells by regulating the WTAP expression.     

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the partially purified MoFe protein (Av 1) preparation was obtained from Azotobacter vinelandii Lipmann OP by chromatography on DEAE-cellulose (DE52) and Sephacryl S-200 columns and analyzed by PAGE and matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Ay 1 band, the preparation was shown to have three other main bands that migrated slower than Avl. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase (PGI). In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaCl concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Identification of binding epitope of a monoclonal antibody (Z12) against human TNF-α using computer modeling and deletion mutant technique

The genes of the heavy and light chain variable region (VH, VL) of Z12 antibody against hTNF-α were cloned, and according to the translated sequence of amino acids, the spatial structures of VH and VL domains were modeled by using homology-based modeling method, followed by constructing the whole three-dimensional structure of Fv fragment. The complex model of Fv interacting with hTNF-α was gained with computer-guided molecular docking method, based on which, it was predicted that the epitope recognized by Z12 was from 141 to 146 of hTNF-α. hTNF-α molecule was divided into two fragments of N-terminal region from 1 to 91 and C-terminal region from 92 to 157 with prokaryotic expression. The measured results suggested that the antigenic epitope recognized by Z12 antibody was located in the C-terminal region 92-157 of hTNF-α, proving the predicted result reliable preliminarily. Further experimental results showed that after hTNF-α 141-146 residues were deleted, Z12 antibody almost lost the ability to recognize the mutant, suggesting that the amino acid residues from 141 to 146 of hTNF-α were specially recognized by Z12 antibody.     

Endothelial K(+) channel lacks the Ca(2+) sensitivity-regulating beta subunit.

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.     

beta(1)-Subunit of BK channels regulates arterial wall[Ca(2+)] and diameter in mouse cerebral arteries.

Mice with a disrupted beta(1) (BK beta(1))-subunit of the large-conductance Ca(2+)-activated K(+) (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca(2+) sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca(2+) concentration ([Ca(2+)](i)) and its regulation by Ca(2+) sparks and BK channel subunits. We utilized a BK beta(1) knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca(2+)](i) and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 microM) or iberiotoxin (100 nM) increased [Ca(2+)](i) by approximately 75 nM and constricted +/+ BK beta(1) wild-type arteries (pressurized to 60 mmHg) with myogenic tone by approximately 10 microm. In contrast, ryanodine (10 microM) or iberiotoxin (100 nM) had no significant effect on [Ca(2+)](i) and diameter of -/- BK beta(1)-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca(2+) sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca(2+) sparks reduces the voltage-dependent Ca(2+) influx and [Ca(2+)](i) through tonic hyperpolarization. Deletion of BK beta(1) disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca(2+)](i).     

The beta1 subunit enhances oxidative regulation of large-conductance calcium-activated K+ channels

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) alpha subunits alone. Because native BKCa channel complexes may include the auxiliary subunit beta1, we investigated whether beta1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with beta1 present shifted the half-activation voltage much further in the hyperpolarizing direction (-75 mV) as compared with that with alpha alone (-30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of beta1. Oxidation of cysteine and methionine residues within beta1 was not involved in these potentiated effects because expression of mutant beta1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type beta1. Unlike the results with alpha alone, oxidation by Ch-T caused a significant acceleration of channel activation only when beta1 was present. The beta1 M177 mutation disrupted normal channel activation and prevented the Ch-T-induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming alpha subunit are greatly amplified by the presence of beta1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within beta1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with beta1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.     

Modes of operation of the BKCa channel beta2 subunit

The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.     

The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states

Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.     

(Xeno)estrogen sensitivity of smooth muscle BK channels conferred by the regulatory beta1 subunit: a study of beta1 knockout mice

Estrogen and xenoestrogens (i.e. agents that are not steroids but possess estrogenic activity) increase the open probability (P(o)) of large conductance Ca(2+)-activated K(+) (BK) channels in smooth muscle. The mechanism of action may involve the regulatory beta1 subunit. We used beta1 subunit knockout (beta1-/-) mice to test the hypothesis that the regulatory beta1 subunit is essential for the activation of BK channels by tamoxifen, 4-OH tamoxifen (a major biologically active metabolite), and 17beta-estradiol in native myocytes. Patch clamp recordings demonstrate BK channels from beta1-/- mice were similar to wild type with the exception of markedly reduced Ca(2+)/voltage sensitivity and faster activation kinetics. In wild type myocytes, (xeno)estrogens increased NP(o) (P(o) x the number of channels, N), shifted the voltage of half-activation (V(12)) to more negative potentials, and decreased unitary conductance. These effects were non-genomic and direct, because they were rapid, reversible, and observed in cell-free patches. None of the (xeno)estrogens increased the NP(o) of BK channels from beta1-/- mice, but all three agents decreased single channel conductance. Thus, (xeno)estrogens increase BK NP(o) through a mechanism involving the beta1 subunit. The decrease in conductance did not require the beta1 subunit and probably reflects an interaction with the pore-forming alpha subunit. We demonstrate regulation of smooth muscle BK channels by physiological (steroid hormones) and pharmacological (chemotherapeutic) agents and reveal the critical role of the beta1 subunit in these responses in native myocytes.