52K Protein Research Articles

Anti-TCP1 Antibody Is a Potential Biomarker for Diagnosing Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease caused by autoantibodies. Serum samples from patients with SLE (n = 10) were compared with those from normal controls (NCs, n = 5) using 21K protein chip analysis to identify a biomarker for SLE, revealing 63 SLE-specific autoantibodies. The anti-chaperonin-containing t-complex polypeptide-1 (TCP1) antibody exhibited higher expression in patients with SLE than in NCs. To validate the specificity of the anti-TCP1 antibody in SLE, dot blot analysis was conducted using sera from patients with SLE (n = 100), rheumatoid arthritis (RA; n = 25), Behçet's disease (BD; n = 28), and systemic sclerosis (SSc; n = 30) and NCs (n = 50). The results confirmed the detection of anti-TCP1 antibodies in 79 of 100 patients with SLE, with substantially elevated expression compared to both NCs and patients with other autoimmune diseases. We performed an enzyme-linked immunosorbent assay to determine the relative amounts of anti-TCP1 antibodies; markedly elevated anti-TCP1 antibody levels were detected in the sera of patients with SLE (50.1 ± 17.3 arbitrary unit (AU), n = 251) compared to those in NCs (33.9 ± 9.3 AU), RA (35 ± 8.7 AU), BD (37.5 ± 11.6 AU), and SSc (43 ± 11.9 AU). These data suggest that the anti-TCP1 antibody is a potential diagnostic biomarker for SLE.

BioPlexR and BioPlexPy: integrated data products for the analysis of human protein interactions.

The BioPlex project has created two proteome scale, cell-line-specific protein-protein interaction (PPI) networks: the first in 293T cells, including 120k interactions among 15k proteins; and the second in HCT116 cells, including 70k interactions between 10k proteins. Here, we describe programmatic access to the BioPlex PPI networks and integration with related resources from within R and Python. Besides PPI networks for 293T and HCT116 cells, this includes access to CORUM protein complex data, PFAM protein domain data, PDB protein structures, and transcriptome and proteome data for the two cell lines. The implemented functionality serves as a basis for integrative downstream analysis of BioPlex PPI data with domain-specific R and Python packages, including efficient execution of maximum scoring sub-network analysis, protein domain-domain association analysis, mapping of PPIs onto 3D protein structures and analysis of BioPlex PPIs at the interface of transcriptomic and proteomic data. The BioPlex R package is available from Bioconductor (bioconductor.org/packages/BioPlex), and the BioPlex Python package is available from PyPI (pypi.org/project/bioplexpy). Applications and downstream analyses are available from GitHub (github.com/ccb-hms/BioPlexAnalysis).

Enhancing the Interaction Between MAS and ETB Receptors is Vasoprotective

Introduction The ET-1 system plays an important role in the pathophysiology of pulmonary hypertension (PH). We previously demonstrated that treatment of hypoxic mice (an experimental model of PH) with Ang(1-7) lead to amelioration of right ventricle hypertrophy and systolic pressure, vascular remodelling and hypercontractility to ET-1. The protective effects of Ang(1-7) were associated with an increase in ETBR expression. Peptide array analysis identified that the Ang(1-7) MAS receptor physically interacts with the ETBR. In this study, we evaluated the vascular effects of such interaction and whether enhancers of MAS/ETBR would modulate endothelial and vascular function. Methods Human aortic endothelial cells (HAEC) were used and stimulated with Ang(1-7) (0.1 μM) in the presence/absence of A779 (MAS antagonist, 1μM), BQ788 (ETBR antagonist, 1μM) and MAS/ETBR peptide disruptor (10μM). Protein expression and signalling activation was assessed by immunoblotting, NO production by DAF-FM fluorescence and vascular function by wire myography. Results In HAECs, ETBR expression was increased after stimulation with Ang(1-7) (Control=100% vs. 2h=147%±6.8, 4h=146.1%±5.5 and 24h=176%±21.9) in a MAS receptor dependent manner (+A779 - 2h= 88.2%±1.5, 4h= 84.3%±5.1 and 24h =86.7%±3.6) (p<0.05). Ang(1-7) also increased AKT (40%) and eNOS (35%) phosphorylation; followed by an increase in NO production (Control=100% vs. 10min= 242.5%±25.4) (p<0.05). The effects on endothelial cell signalling and No production were inhibited by MAS and ETBR antagonists, as well as, a MAS/ETBR interaction peptide disruptor. In a high through put screening of a 20K protein:protein interaction modulator library, we discovered 23 potential enhancers of the MAS/ETBR interaction, where we tested 4 in HAECs (Enh1-4: 10μM). From all enhancers, only enhancers 3 and 4 increased eNOS activation (Vehicle=100% vs. En3 10min= 202%±18.2, Enh4 10min= 194.7%±19.6) and NO production (Vehicle=100% vs. En3 10min= 121.2%±7.4, Enh4 10min= 123.6%±8.2). Mesenteric resistance arteries were pre-incubated with Enh3-4 for 30 minutes prior to acetylcholine (Ach) curves, to assess endothelium-dependent relaxation. Only Enh4 increased ACh-induced relaxation (Emax: 96.7±4.6 vs Emax control: 70.4±3.3), p<0.05. Conclusion Our data demonstrates that the MAS/ETBR interaction has protective effects in the endothelium. Enhancing MAS/ETBR crosstalk leads to NO production and improvement of endothelial function, demonstrating potential in the treatment of vascular dysfunction and associated diseases.

ENHANCING INTERACTIONS BETWEEN MASR AND ETBR AS A NEW VASOPROTECTIVE STRATEGY

Objective: We demonstrated that the MAS and ETB receptors physically interact and may be involved in the protective actions of Ang(1–7) in endothelial cells (ECs). We assessed the biological role of MAS/ETBR interaction in the vasculature, focusing on the NO pathway. Design and method: Human ECs were stimulated with Ang(1–7) (10-7 M) in the presence/absence of A779 (MAS antagonist, 10-5 M), BQ788 (ETBR antagonist, 10-5 M) and MAS/ETBR peptide disruptor (10-5 M). ETBR expression and AKT/eNOS activation were evaluated by immunoblotting and NO production by DAF-FM fluorescence. Results: Ang(1–7) increased ETBR expression (50%), an effect blocked by A779 (p &lt; 0.05 vs control). Ang(1–7) also induced AKT (40%) and eNOS (35%) activation; followed by an increase in NO production (2 fold) (p &lt; 0.05 vs control). These effects were inhibited by A779, BQ788 and the MAS/ETBR disruptor. In a high throughput screening of a 20K protein:protein interaction modulator library, we discovered 23 potential enhancers of the MAS/ETBR interaction, where we tested 4 in ECs (Enh1–4: 10–5 M). All four MAS/ETBR enhancers increased eNOS activation with variable responses (Enh1: 44.2%; Enh2: 65.4%; Enh3: 80.7%; Enh4: 94.9% vs control, p &lt; 0.05), but only treatment with Enh1 and Enh2 increased NO production (Enh1: 109.4%; Enh2: 46.5% vs control, p &lt; 0.05) in ECs. Mesenteric resistance arteries were pre-incubated with Enh1–4 for 30 minutes prior to acetylcholine (Ach) curves, to assess endothelium-dependent relaxation. Enh1 decreased ACh-induced relaxation (Emax: 52.8 ± 2.9 vs Emax control: 70.4 ± 3.3), while Enh4 increased ACh-induced relaxation (Emax: 96.7 ± 4.6 vs Emax control: 70.4 ± 3.3), p &lt; 0.05. Conclusions: Promoting the MAS/ETBR interaction with specific enhancers leads to eNOS activation and vasorelaxation, where Enh1–4 have variable effects. Current studies are focusing on identifying the most sensitive enhancer that will provide vasoprotection, and potentially new strategies in the treatment of vascular disease.

Domains of bovine adenovirus-3 protein 22K involved in interacting with viral protein 52K and cellular importins α-5/α-7

The L6 region of bovine adenovirus-3 (BAdV-3) encodes unspliced and spliced proteins named 22K and 33K, respectively. Earlier, anti-22K sera detected two proteins of 42- and 37-kDa in infected cells and 42-kDa protein in transfected cells. Here, we demonstrate that 22K protein localizes to the nucleus of BAdV-3 infected or transfected cells. Analysis of mutant 22K proteins suggested that amino acids 231–250 of non-conserved C-terminus of 22K are required for nuclear localization. The nuclear import of 22K appears to utilize multiple importin (α-5 and α-7) of importin α/β nuclear import pathway. Mutational analysis of 22K identified four basic residues 238RRRK241, which apparently are essential for the nuclear localization of 22K. Our results suggest that the nuclear localization of 22K appear essential for virus replication and production of progeny BAdV-3. Furthermore, we demonstrate that N-terminus amino acid 35–65 conserved in 22K and 33K interact with 52K protein in BAdV-3 infected cells.

Epitope mapping and identification of amino acids critical for mouse IgG-binding to linear epitopes on Gly m Bd 28K.

Gly m Bd 28K is one of the major allergens in soybeans, but there is limited information on its IgG-binding epitopes. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28K were synthesized, and 3 monoclonal antibodies against Gly m Bd 28K were utilized to identify the IgG-binding regions of Gly m Bd 28K. Three dominant peptides corresponding to (28)GDKKSPKSLFLMSNS(42)(G28-S42), (56)LKSHGGRIFYRHMHI(70)(L56-I70), and (154)ETFQSFYIGGGANSH(168)(E154-H168) were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of monoclonal antibody to Gly m Bd 28K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope. Two bioinformatics tools, ABCpred and BepiPred, were used to predict the epitopes of Gly m Bd 28K, and the predictions were compared with the epitopes that we had located by monoclonal antibodies.

Development of an Indirect Competitive ELISA Kit for the Detection of Soybean Allergenic Protein Gly m Bd 28K

The complementary DNA (cDNA) sequence of Gly m Bd 28K was expressed in Escherichia coli BL21 (DE3) as an inclusion body under the induction of 1.0 mmol/L of isopropyl β-D-thiogalactoside (IPTG). The inclusion bodies were solubilized in 6 M guanidine hydrochloride and refolded by rapid dilution. After renaturation, the purity of the recovered recombinant protein was up to 90 %, and its molecular weight was 29.1 kDa. A monoclonal antibody that was specific for recombinant Gly m Bd 28K protein was produced and characterized. Based on the mAb, an indirect competitive enzyme-linked immunosorbent kit (icELISA kit) for the specific detection of recombinant Gly m Bd 28K protein was developed, which showed that the IC50 was 3.19 μg/L and the sensitivity was 0.235 μg/L. The average relative standard deviation (RSD) was 10.28 %, and the average recovery was 96.73 %, which indicated that the kit was highly sensitive and accurate. The icELISA kit remained usable for more than 6 months when stored at 4 °C. Therefore, the icELISA is a valuable tool for the rapid and sensitive detection of Gly m Bd 28K in food and feed products from soybeans.

Low-Level Expression of the E1B 20-Kilodalton Protein by Adenovirus 14p1 Enhances Viral Immunopathogenesis.

Adenovirus 14p1 (Ad14p1) is an emergent variant of Ad serotype 14 (Ad14) that has caused increased severity of respiratory illnesses during globally distributed outbreaks, including cases of acute respiratory distress syndrome and death. We found that human cell infection with Ad14p1 results in markedly decreased expression of the E1B 20-kilodalton (20K) protein compared to that with infection with wild-type (wt) Ad14. This reduced Ad14p1 E1B 20K expression caused a loss-of-function phenotype of Ad-infected cell corpses that, in contrast to cells infected with wt Ad14, either failed to repress or increased NF-κB-dependent, proinflammatory cytokine responses of responder human alveolar macrophages. A small-animal model of Ad14-induced lung infection was used to test the translational relevance of these in vitro observations. Intratracheal infection of Syrian hamsters with Ad14p1 caused a marked, patchy bronchopneumonia, whereas hamster infection with wt Ad14 caused minimal peribronchial inflammation. These results suggest that this difference in E1B 20K gene expression during Ad14p1 infection and its modulating effect on the interactions between Ad14-infected cells and the host innate immune response could explain the increased immunopathogenic potential and associated increase in clinical illness in some people infected with the Ad14p1 outbreak strain.IMPORTANCE We previously reported that Ad-infected human cells exhibit E1B 19K-dependent repression of virally induced, NF-κB-dependent macrophage cytokine responses (J. R. Radke, F. Grigera, D. S. Ucker, and J. L. Cook, J Virol 88:2658-2669, 2014, http://dx.doi.org/10.1128/JVI.02372-13). The more virulent, emergent strain of Ad14, Ad14p1, causes increased cytopathology in vitro, which suggested a possible E1B 20K defect. Whether there is a linkage between these observations was unknown. We show that there is markedly reduced expression of E1B 20K in Ad14p1-infected human cells and that this causes an increased proinflammatory cytokine response of human alveolar macrophages and more severe inflammatory lung disease in infected hamsters. This is the first evidence of a clinical relevance of differential expression of the small Ad E1B gene product. The results suggest that there is a low, critical threshold of E1B 19/20K expression that is needed for viral replication and infection transmission but that a higher level of E1B 19/20K expression is required for the usual repression and control of the Ad-triggered host innate immune response.

A Major Fimbrilin Variant of Mfa1 Fimbriae in Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is known to express 2 distinct types of fimbriae: FimA and Mfa1 fimbriae. However, we previously reported that fimbria-like structures were found in a P. gingivalis strain in which neither FimA nor Mfa1 fimbriae were detected. In this study, we identified a major protein in the bacterial lysates of the strain, which has been reported as the 53-kDa major outer membrane protein of P. gingivalis (53K protein) and subsequently reported as a major fimbrilin of a novel-type fimbria. Sequencing of the chromosomal DNA of the strain showed that the 53k gene (encoding the 53K protein) was located at a locus corresponding to the mfa1 gene (encoding the Mfa1 protein, which is a major fimbrilin of Mfa1 fimbriae) of the ATCC 33277 type strain. However, the 53K and Mfa1 proteins showed a low amino acid sequence homology and different antigenicity. The 53K protein was detected in 34 of 84 (41%) P. gingivalis strains, while the Mfa1 protein was detected in 44% of the strains. No strain expressed both 53K and Mfa1 proteins. Additionally, fimbriae were normally expressed in mutants in which the 53k and mfa1 genes were interchanged. These results indicate that the 53K protein is another major fimbrilin of Mfa1 fimbriae in P. gingivalis.

Detection of the Soybean Allergenic Protein Gly m Bd 28K by an Indirect Enzyme-Linked Immunosorbent Assay

The full-length cDNA sequence of Gly m Bd 28K was chemically synthesized and expressed in Escherichia coli (E. coli) BL21 (DE3) as an inclusion body under the induction of 0.2 mmol/L of isopropyl β-D-1-thiogalactopyranoside (IPTG). The purity of the recombinant protein was over 90% following Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and its molecular weight was 29.71 kDa. The polyclonal antibody (pAB) against Gly m Bd 28K was prepared and referred to as pAB-28K, and it exhibited high specificity for the protein in soybean meal. We established an indirect enzyme-linked immunosorbent assay (iELISA) using the pAB-28K and the recombinant Gly m Bd 28K protein to determine the Gly m Bd 28K content in soybean products. The R(2) value of the standard curve was 0.9910, the average relative standard deviation (RSD) was 16.93%, and the average recovery was 95.50%, which indicated that the iELISA was highly reproducible and accurate. Therefore, the pAB-28K and the iELISA provide valuable tools for the rapid and sensitive detection of Gly m Bd 28K in food and feed products from soybeans. This protocol meets the technical requirements for quality control and food safety as related to soybean.

Mapping of nuclear import signal and importin α3 binding regions of 52K protein of bovine adenovirus-3

The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues (105RKR107) of the identified domain (amino acids 102GMPRKRVLT110) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin α/β-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin α3. Although deletion of amino acid 102–110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90–133 are required for interaction with importin-α3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin α3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

EFFECTS OF DIETARY CARBOHYDRATES ON MINERAL METABOLISM IN LACTATING DAMS AND THEIR PUPS

Dramatic increases in fructose (F) consumption in the last 40 y have been tightly linked to obesity, insulin resistance and other metabolic abnormalities in adults. However, there is no information about the effects of F on maternal and fetal health. To evaluate the specific effects of excessive F and glucose consumption during gestation and lactation on dams and their litters, 3 groups of female rats were fed isocaloric, isolipidic 60% glucose, F or starch diets for 40 d, from day 2 of gestation to end of lactation. Compared to dams fed glucose and starch, dams fed F had lower rates of intestinal transepithelial Ca transport as well as lower levels of intestinal Ca and calbindin 9K protein, plasma Ca and plasma 1,25‐vitamin‐D. Dams fed glucose or F exhibited lower rates of transepithelial Pi transport, levels of plasma Pi, bone mineral content and bone mineral density (BMD). These marked perturbations in mineral homeostasis resulted in modest decreases in BMD as well as growth rates of pups from F‐fed dams. Our novel findings clearly show that over‐consumption of F and perhaps glucose during pregnancy and lactation impairs bone health of the dams because of decreases in active intestinal Ca and Pi transport caused by impaired production of 1,25‐vitamin D. Dams may mobilize bone Ca and Pi stores to dampen perturbations in mineral homeostasis transmitted to the offspring. (NIH RDK075617, NSF 722365)

Effects of Thiazide on the Expression of TRPV5, Calbindin-D28K, and Sodium Transporters in Hypercalciuric Rats

TRPV5 is believed to play an important role in the regulation of urinary calcium excretion. We assessed the effects of hydrochlorothiazide (HCTZ) on the expression of TRPV5, calbindin-D28K, and several sodium transporters in hypercalciuric rats. Sprague-Dawley rats were divided into 4 groups; control, HCTZ, high salt, and high salt with HCTZ group in experiment 1; control, HCTZ, high calcium (Ca), and high Ca with HCTZ group in experiment 2. To quantitate the expression of TRPV5, calbindin-D28K, and sodium transporters, western blotting was performed. In both experiments, HCTZ significantly decreased urinary calcium excretion. TRPV5 protein abundance decreased in all hypercalciuric rats, and restored by HCTZ in both high salt with HCTZ and high Ca with HCTZ group. Calbindin-D28K protein abundance increased in the high salt and high salt with HCTZ groups, but did not differ among groups in experiment 2. Protein abundance of NHE3 and NKCC2 decreased in all hypercalciuric rats, and were restored by HCTZ in only high Ca-induced hypercalciuric rats. In summary, protein abundance of TRPV5, NHE3, and NKCC2 decreased in all hypercalciuric rats. The hypocalciuric effect of HCTZ is associated with increased protein abundance of TRPV5 in high salt or calcium diet-induced hypercalciuric rats.

Structure and Function of the Pre-mRNA Splicing Machine

Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory pre-mRNA splicing event is fundamental in development and cancer. It occurs within a mega-Dalton multicomponent machine composed of RNA and proteins, which undergoes dynamic changes in RNA-RNA, RNA-protein, and protein-protein interactions during the splicing reaction. Recent years have seen progress in functional and structural analyses of the splicing machine and its subcomponents, and this review is focused on structural aspects of the pre-mRNA splicing machine and their mechanistic implications on the splicing of multi-intronic pre-mRNAs. It brings together, in a comparative manner, structural information on spliceosomes and their intermediates in the stepwise assembly process in vitro, and on the preformed supraspliceosomes, which are isolated from living cell nuclei, with a view of portraying a consistent picture.

The highly conserved orthopoxvirus 68k ankyrin-like protein is part of a cellular SCF ubiquitin ligase complex

The 68k ankyrin-like protein (68k-ank) of unknown function is highly conserved among orthopoxviruses and contains ankyrin repeats and an F-box-like domain. We performed a yeast-two-hybrid screen with 68k-ank to find interacting proteins. From a human and a murine cDNA library, 99% of the interaction partners were S-phase kinase-associated protein 1a (Skp1a), a part of the SCF ubiquitin ligase complex. 68k-ank co-immunoprecipitated with components of the endogenous, mammalian SCF ubiquitin ligase. This interaction was F-box domain dependent and could also be observed in infected cells, indicating that SCF complex formation might be important for the viral life cycle.

Murine TNFΔARE Crohnʼs disease model displays diminished expression of intestinal Ca2+ transporters

Patients suffering from Crohn's disease (CD) show increased incidence of low bone mineral density. Investigating this complication is difficult because the exact etiology of CD remains elusive. Mice carrying a deletion in the tumor necrosis factor (TNF) AU-rich elements (ARE) are reported as a model for human CD and are characterized by elevated TNF-alpha levels and inflammations in the terminal ileum. To evaluate whether these mice have a Ca(2+) handling problem, this study analyzed the Ca(2+) homeostasis in heterozygous TNF(DeltaARE) mice (TNF(DeltaARE/+)) in comparison to wildtype littermates. Beside serum Ca(2+) and vitamin D levels, the expression of Ca(2+) transporters was analyzed in intestine, kidney and bone using quantitative real-time PCR, Western blot and immunohistochemistry. Bone scans were performed to measure bone parameters. Ca(2+) transporters in duodenum (TRPV6, calbindin-D(9K), PMCA1b) and kidney (TRPV5, calbindin-D(28K), NCX1) showed significantly reduced mRNA expression levels in TNP(DeltaARE/+) mice, except for renal TRPV5. In bone, only calbindin-D(9K) mRNA displayed a significant down-regulation. These findings were supported by declined duodenal calbindin-D(9K) and renal calbindin-D(28K) protein values. Likely, this down-regulation of Ca(2+) transporters in TNP(DeltaARE/+) mice is mediated by the 58 +/- 9% reduction in serum 1,25(OH)(2)D(3) levels. Diminished expression of Ca(2+) transporters combined with unchanged serum Ca(2+) levels assumes Ca(2+) loss from bone to compensate for the body's overall Ca(2+) shortage. Indeed, microcomputed tomography scanning demonstrated reduced trabecular and corticol bone thickness and volume in TNF(DeltaARE/+) mice. This finding is further supported by increased total deoxypyridinoline in serum. Our results imply that TNF(DeltaARE/+) mice have a disturbed Ca(2+) homeostasis characterized by reduced duodenal and renal Ca(2+) transporters, diminished 1,25(OH)(2)D(3) levels, and increased bone resorption associated with profound bone abnormalities.

Anti-progestogenic effect of flutamide on uterine expression of calbindin-D 9k mRNA and protein in immature mice

A calcium binding protein, calbindin-D 9k (CaBP-9k), is a cytosolic protein and regulated by steroid hormones in the reproductive tissues. Mouse CaBP-9k gene was predominantly regulated by progesterone (P4), whereas rat CaBP-9k was mainly regulated by 17beta-estradiol (E2) in the uterus. The induction of CaBP-9k can be employed as a biomarker for steroidal substrates as endocrine disruptors (EDs). Flutamide (FLU) is a non-steroidal anti-androgen or pro-drug that is rapidly metabolized to hydroxyflutamide, which may have both an anti-androgenic and anti-progestogenic activities. Thus, in the present study, we employed immature mice (14-day-old) subcutaneously injected with P4 (20 mg/kg/day) and/or FLU (5 mg/kg/day) for 3 consecutive days in the presence or absence of RU486, a pure PR antagonist (30 mg/kg/day), to analyze uterine CaBP-9k expression in this model. When immature mice were treated with P4, the expression levels of CaBP-9k mRNA and protein were significantly increased by P4. P4-induced expression levels of CaBP-9k mRNA and protein were abolished by FLU, in part, suggesting that FLU is a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mice. In addition, P4-induced CaBP-9k expression was completely reversed by RU486. Increased expression levels of CaBP-9k mRNA and protein were maintained for 24 h after final injection with P4 in a time-dependent manner. However, CaBP-9k mRNA rapidly disappeared after 48 h and its protein level is similar with its mRNA. Treatment with FLU suppressed partially P4-induced CaBP-9k mRNA and protein until 24 h. Taken together, these results indicate that FLU has an anti-progestogenic activity and plays a role as a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mouse model.

Helicobacter Genus DNA Fragments Are Commonly Detectable in Bile from Patients with Extrahepatic Biliary Diseases and Associated with Their Pathogenesis

Several Helicobacter species are known to colonize the biliary tract in animals and have been identified in the gallbladder bile of a high proportion of Chilean patients with gallbladder cancer. In this study, we tried to examine the presence of Helicobacter species in the bile to know their participation in the development of extrahepatic biliary diseases. DNA was extracted from 57 bile samples from 30 patients with benign biliary diseases (cholecystolithiasis and choledochocystolithiasis), 6 malignant biliary diseases (gallbladder cancer and common bile duct cancer), and 21 nonbiliary diseases. The presence of Helicobacter genus-, H. pylori-, H. hepaticus-, and H. bilis-specific 16S rRNA genes, the H. pylori urease A gene, and the H. pylori 26K protein gene in the bile was determined by PCR and sequencing analysis. Helicobacter genus DNA (shorter amplicons, 400 bp) was statistically frequently detected in biles from 53% (16/30) and 86% (5/6) of benign and malignant biliary diseases, compared with 9% (2/21) of nonbiliary diseases, but longer amplicons (1200 bp) were not detectable in any samples. The H. pylori urease A gene (nested amplicon) was also frequently found in bile, whether benign, malignant, or control, though neither H. pylori 16S rRNA nor the 26K protein gene was detectable in any bile samples. H. bilis-16S rRNA genes were detectable in only two cases. H. hepaticus was not detectable in any samples. DNA fragments of Helicobacter species other than H. pylori, H. hepaticus, and H. bilis are commonly detectable in the bile of patients with extrahepatic biliary diseases, whether benign or malignant, implying that the Helicobacter genus may be directly or indirectly involved in the pathogenesis of these diseases.

The human U5 snRNP 52K protein (CD2BP2) interacts with U5-102K (hPrp6), a U4/U6.U5 tri-snRNP bridging protein, but dissociates upon tri-snRNP formation

The U5 snRNP plays an essential role in both U2- and U12-dependent splicing. Here, we have characterized a 52-kDa protein associated with the human U5 snRNP, designated U5-52K. Protein sequencing revealed that U5-52K is identical to the CD2BP2, which interacts with the cytoplasmic portion of the human T-cell surface protein CD2. Consistent with it associating with an snRNP, immunofluorescence studies demonstrated that the 52K protein is predominantly located in the nucleoplasm of HeLa cells, where it overlaps, at least in part, with splicing-factor compartments (or "speckles"). We further demonstrate that the 52K protein is a constituent of the 20S U5 snRNP, but is not found in U4/U6.U5 tri-snRNPs. Thus, it is the only 20S U5-specific protein that is not integrated into the tri-snRNP and resembles, in this respect, the U4/U6 di-snRNP assembly factor Prp24p/p110. Yeast two-hybrid screening and pulldown assays revealed that the 52K protein interacts with the U5-specific 102K and 15K proteins, suggesting that these interactions are responsible for its integration into the U5 particle. The N-terminal two-thirds of 52K interact with the 102K protein, whereas its C-terminal GYF-domain binds the 15K protein. As the latter lacks a proline-rich tract, our data indicate that a GYF-domain can also engage in specific protein-protein interactions in a polyproline-independent manner. Interestingly, the U5-102K protein has been shown previously to play an essential role in tri-snRNP formation, binding the U4/U6-61K protein. The interaction of 52K with a tri-snRNP bridging protein, coupled with its absence from the tri-snRNP, suggests it might function in tri-snRNP assembly.

Downregulation of Ca(2+) and Mg(2+) transport proteins in the kidney explains tacrolimus (FK506)-induced hypercalciuria and hypomagnesemia.

FK506 (tacrolimus) and dexamethasone are potent immunosuppressants known to induce significant side effects on mineral homeostasis, including hypercalciuria and hypomagnesemia. However, the underlying molecular mechanisms remain unknown. The present study investigated the effects of FK506 and dexamethasone on the expression of proteins involved in active Ca(2+) reabsorption: the epithelial Ca(2+) channel TRPV5 and the cytosolic Ca(2+)-binding protein calbindin-D(28K). In addition, the renal expression of the putative Mg(2+) channel TRPM6, suggested to be involved in transcellular Mg(2+) reabsorption, was determined. Administration of FK506 to rats by daily oral gavage during 7 d significantly enhanced the urinary excretion of Ca(2+) and Mg(2+) and induced a significant hypomagnesemia. FK506 significantly decreased the renal mRNA expression of TRPV5 (62 +/- 7% relative to controls), calbindin-D(28K) (9 +/- 1%), and TRPM6 (52 +/- 8%), as determined by real-time quantitative PCR analysis. Furthermore, semiquantitative immunohistochemistry showed reduced renal protein abundance of TRPV5 (24 +/- 5%) and calbindin-D(28K) (29 +/- 4%), altogether suggesting that downregulation of these transport proteins is responsible for the FK506-induced Ca(2+) and Mg(2+) wasting. In contrast, dexamethasone significantly enhanced renal TRPV5 (150 +/- 15%), calbindin-D(28K) (177 +/- 23%), and TRPM6 (156 +/- 20%) mRNA levels along with TRPV5 (211 +/- 8%) and calbindin-D(28K) (176 +/- 5%) protein abundance in the presence of significantly increased Ca(2+) and Mg(2+) excretion. This indicated that these proteins are directly or indirectly regulated by dexamethasone. In conclusion, FK506 and dexamethasone induce renal Ca(2+) and Mg(2+) wasting, albeit by different mechanisms. Downregulation of specific Ca(2+) and Mg(2+) transport proteins provides a molecular mechanism for FK506-induced hypercalciuria and hypomagnesemia, whereas dexamethasone positively regulates these proteins.