Functional identification of protein phosphatase 1-binding consensus residues in NBCe1-B

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, ⁹²²FMDRLK⁹²⁷ , in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922–927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO₃⁻ transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO₃⁻ secretion in several types of epithelia.

m-Nisodipine inhibited 5-HT-induced proliferation of rat PASMCs through Rho/ROCK signal pathway / 药学学报

This paper is to report the exploration of the activation of Rho/ROCK signal pathway in 5-HT-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and the inhibitory effect of m-Nis on this pathway. PASMCs were cultured with the explant technique. MTT assay was used to explore the proliferation of PASMCs after 5-HT treated for different time and the intervening effect of m-Nis. RT-PCR and Western blot were used respectively to explore the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 in 5-HT-treated PASMCs and intervening effect of m-Nis. The results of MTT assay suggested that 5-HT (1 µmol · L(-1)) treatment for 12-72 h significantly induced the proliferation of rat PASMCs (P<0.05 or P < 0.01), which were inhibited by m-Nis (1 x 10(-5), 1 x 10(-6), l x 10(-7), 1 x10(-8) mol · L(-1)) in dose-dependent manners (P < 0.05 or P < 0.01). Similarly, the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 were also inhibited by m-Nis in different degrees (P < 0.05 or P < 0.01). Thus, the results of this study suggested that Rho/ROCK pathway played an important role in 5-HT-induced proliferation of rat PASMCs, m-Nis inhibited 5-HT-induced proliferation obviously, which may be related to the blockage of Rho/ROCK signal pathway.

Phytotoxicity associated to microcystins: a review / Fitotocixidade associada às microcistinas: uma revisão

Microcystins (MC) are the most studied toxins of cyanobacteria since they are widely distributed and account for several cases of human and animal poisoning, being potent inhibitors of the serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A). The phosphatases PP1 and PP2A are also present in plants, which may also suffer adverse effects due to the inhibition of these enzymes. In aquatic plants, biomass reduction is usually observed after absorption of cyanotoxins, which can bioaccumulate in its tissues. In terrestrial plants, the effects caused by microcystins vary from inhibition to stimulation as the individuals develop from seedling to adult, and include reduction of protein phosphatases 1 and 2A, oxidative stress, decreased photosynthetic activity and even cell apoptosis, as well as bioaccumulation in plant tissues. Thus, the irrigation of crop plants by water contaminated with microcystins is not only an economic problem but becomes a public health issue because of the possibility of food contamination, and this route of exposure requires careful monitoring by the responsible authorities.

Microcistinas (MC) são as toxinas de cianobactérias mais estudadas, uma vez que são amplamente distribuídas e responsáveis por vários casos de intoxicação humana e animal. São potentes inibidoras das proteínas fosfatases serina/treonina 1 (PP1) e 2A (PP2A). As fosfatases PP1 e PP2A também estão presentes em plantas, as quais podem sofrer efeitos adversos devido à inibição dessas enzimas. Em plantas aquáticas, a redução da biomassa é geralmente observada após absorção de cianotoxinas que podem bioacumular em seus tecidos. Em plantas terrestres, os efeitos causados pelas microcistinas variam de inibição ao estímulo, como no desenvolvimento de plântulas ao estádio adulto, e incluem a redução de proteínas fosfatases 1 e 2A, estresse oxidativo, diminuição da atividade fotossintética e até mesmo apoptose celular, bem como a bioacumulação em tecidos de plantas. Assim, a irrigação de plantas cultivadas com água contaminada com microcistina não é apenas um problema econômico, mas torna-se um problema de saúde pública, devido à possibilidade de contaminação dos alimento, sendo uma via de exposição que requer um monitoramento cuidadoso por parte das autoridades responsáveis.

Arsenite Acutely Decreases Nitric Oxide Production via the ROS-Protein Phosphatase 1-Endothelial Nitric Oxide Synthase-Thr497 Signaling Cascade

Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 (eNOS-Ser1179 in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of eNOS-Thr497, but not of eNOS-Ser116 or eNOS-Ser1179, which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on eNOS-Thr497 phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr497 phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr497 phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr497 phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing eNOS-Thr497 phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.

Angiotensin IV upregulates the activity of protein phosphatase 1α in Neura-2A cells

The peptide angiotensin IV (Ang IV) is a derivative of angiotensin II. While insulin regulated amino peptidase (IRAP) has been proposed as a potential receptor for Ang IV, the signalling pathways of Ang IV through IRAP remain elusive. We applied high-resolution mass spectrometry to perform a systemic quantitative phosphoproteome of Neura-2A (N2A) cells treated with and without Ang IV using sta ble-isotope labeling by amino acids in cell culture (SILAC), and identified a reduction in the phosphorylation of a major Ser/Thr protein phosphorylase 1 (PP1) upon Ang IV treatment. In addition, spinophilin (spn), a PP1 regulatory protein that plays important functions in the neural system, was expressed at higher levels. Immunoblotting revealed decreased phosphorylation of p70S6 kinase (p70(S6K)) and the major cell cycle modulator retinoblastoma protein (pRB). These changes are consistent with an observed decrease in cell proliferation. Taken together, our study suggests that Ang IV functions via regulating the activity of PP1.

Fasudil reverses monocrotaline-induced pulmonary hypertension in rats / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe the effects and related mechanisms of fasudil on monocrotaline-induced pulmonary arterial hypertension (PAH) in rats.</p><p><b>METHODS</b>A total of 56 healthy male Sprague-Dawley rats were randomly divided into 5 groups: 4 weeks control group (N4), 4 weeks PAH group (M4), 8 weeks control group (N8), 8 weeks PAH group (M8), 8 weeks PAH and fasudil group (F8). PAH was induced by subcutaneous injection of monocrotaline (50 mg/kg). Animals in F8 group received intraperitoneal injection of fasudil hydrochloride (15 mg×kg(-1)×d(-1)) from the end of the 4th week to the end of the 8th week. Rats in control groups and PAH groups received equal volume saline injection. Polyethylene catheters were inserted into the RV through the jugular vein for right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) measurements after various treatment protocols. RV hypertrophy index [RV/(LV+S)] was also measured. Arteries of 50 to 150 µm were evaluated for the median wall thickness and wall area by HE staining as follows: percent wall thickness (WT%) = [(medial thickness×2/external diameter)]×100 and percent wall area (WA%) = (wall area/total area)×100%. The mRNA expression of ROCK-1 in lung tissue was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of ROCK-1 and MYPT-1 in lung tissue were analyzed by Western blot and MYPT-1 phosphorylation, respectively.</p><p><b>RESULTS</b>Forty-one rats survived and mortality rate was zero in N4, N8 and M4 groups. Survival rate was significantly higher in F8 group compared to M8 group (75.00% vs. 31.25%, P < 0.05). At the end of the 4th week, RVSP [(62.25 ± 3.24) vs. (31.33 ± 2.35) mm Hg(1 mm Hg = 0.133 kPa)], mPAP [(36.38 ± 2.31) vs.(20.32 ± 1.81) mm Hg], [RV/(LV+S)] (0.5648 ± 0.0580 vs. 0.3458 ± 0.0455), WT% [(25.63 ± 5.35)% vs.(13.38 ± 3.45)%], WA% [(60.36 ± 2.51)% vs. (38.42 ± 2.84)%] were all significantly higher in M4 group than in N4 group (all P < 0.01). RVSP [(54.64 ± 4.11) vs. (67.37 ± 4.68) mm Hg], mPAP [(26.25 ± 2.32) vs. (39.83 ± 1.83) mm Hg], and markedly relieve [RV/(LV+S)] (0.3985 ± 0.0210 vs. 0.7600 ± 0.0341), WT% [(15.64 ± 2.81)% vs. (28.26 ± 4.38)%], WA% [(40.35 ± 2.82)% vs. (68.83 ± 1.63)%] were all significantly lower in F8 group than in M8 group (all P < 0.05) while the expression of ROCK-1 mRNA (1.2139 ± 0.1778 vs. 1.6839 ± 0.3251, P < 0.01), and the protein expression of ROCK-1 and MYPT-1 as well as the extent of MYPT-1 phosphorylation were all downregualted in F8 group compared to M8 group (all P < 0.01).</p><p><b>CONCLUSIONS</b>Fasudil can effectively reverse the MCT-induced PAH in rats via downregulating ROCK-1 and MYPT-1.</p>

PPM1B and P-IKKβ expression levels correlated inversely with rat gastrocnemius atrophy after denervation

Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.

Effects of intrahippocampal administration of the phosphatase inhibitor okadaic acid: dual effects on memory formation / Efeitos da administração intra-hipocampal do inibidor de fosfatases ácido okadaico: efeito duplo sobre a formação de memória

Protein phosphorylation mediated by serine-threonine kinases in the hippocampus is crucial to the synaptic modifications believed to underlie memory formation. The role of phosphatases has been the focus of comparatively little study. Objectives: Here we evaluate the contribution of the serine-threonine protein phosphatases 1 and 2A (PP1, PP2A) on memory consolidation. Methods: We used immediate post-training bilateral hippocampal infusions of okadaic acid (OA, 0.01 and 10 pmol/side), a potent inhibitor of PP1 and PP2A, and measured short- [3 h] and long-term memory [24 h] (STM, LTM) of step-down inhibitory avoidance. Results: At the lower dose, OA inhibited both STM and LTM whereas at the higher dose it instead enhanced LTM. Pre-test infusion of these two doses of OA had no effect on retrieval. Conclusions: These two doses of OA are known to selectively inhibit PP1 and PP2A respectively. These findings point to the importance of these enzymes in memory formation and also suggest a deleterious influence of endogenous hippocampal PP2A on LTM formation.

A fosforilação de proteínas mediada por serina-treonina quinases no hipocampo é crucial para as modificações sinápticas que se acredita sejam necessárias para a formação de memórias. O papel das fosfatases tem sido comparativamente pouco estudado. Objetivos: Aqui avaliamos a contribuição das fosfatases serina-treonina 1 e 2 (PP1, PP2A) sobre a consolidação da memória. Métodos: Usamos infusões imediatamente após o treino de ácido okadaico (OA, 0.01 e 10 pmol/lado), um potente inibidor de PP1 e medimos memória de curta [3 h] e longa duração [24 h] (STM, LTM) de esquiva inibitória de evitar descer de uma plataforma. Resultados: Na dose menor, OA inibiu tanto STM como LTM. Na dose maior, produziu, em vez disso, uma melhora da LTM. A infusão pré-teste de qualquer uma das duas doses de OA não teve efeito sobre a evocação. Conclusões: Estas duas doses de OA são conhecidas por inibir seletivamente PP1 a PP2 respectivamente. Estes resultados apontam à importância das duas enzimas na formação de memória e sugerem, adicionalmente, uma influência deletérea da PP2A endógena sobre a formação de LTM.

Action of protein phosphatase-1 on Tat-dependent HIV-1 transcription and its related inhibitors / 药学学报

Host cell protein phosphatase-1 (PP1) is an important regulator of human immunodeficiency virus-1 (HIV-1) transcription. PP1 is involved in the regulation of HIV-1 transcription, and dephosphorylates RNA polymerase II C-terminal domain (RNAPII CTD) or CycT1-dependent kinase 9 (CDK9) to increase Tat-dependent HIV-1 transcription. In this review, we discuss the action of PP1 in Tat-induced HIV-1 transcription and related to PP1 inhibitors.

Preliminary structural and functional study on a novel gene HSPCSET / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To characterize the structural and the functional feature of a novel gene HSPCSET isolated from human CD34+ hematopoietic stem/progenitor cells (HS/PCs).</p><p><b>METHODS</b>Bioinformatic technology was used to identify the structural features of the HSPCSET protein and perform the multiple sequence alignment. Yeast-two-hybrid system was used to identify the proteins interacting with the HSPCSET protein. After sequencing, we selected out the positive clones which had clear functions, and carried out beta-gal experiment and GST pull down assay to confirm the results. The cellular location of the HSPCSET was checked by immunofluorescence assay.</p><p><b>RESULTS</b>The HSPCSET protein belongs to a SET domain family, which is evolutionarily conserved across species. It implied that HSPCSET may have biologically important function. Using yeast-two-hybrid system, we showed that the protein sequence with SET domain might bind to 13 proteins, which involved in signaling transduction, transcriptional regulation, apoptosis, tumorigenesis, development, etc. And 4 proteins (GADD34, SIVA, DNAJ and PHF1) were confirmed by one-on-one back of the hybrid experiment, beta-gal test and GST pull down assay. When GADD34 and HSPCSET were co-transfected, they co-localized in the nucleus, suggesting a strong interaction.</p><p><b>CONCLUSION</b>The novel gene HSPCSET is likely to have biologically important function. This study provides the basis for further studies of its function in hematopoiesis and tumorigenesis.</p>

Effects of valsartan on sarcoplasmic reticulum calcium adenosine triphosphatase, protein kinase A and protein phosphatase 1 alpha in a rabbit model of heart failure / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of valsartan on expressions of myocardial sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2), protein kinase A (PKA) and protein phosphatase 1 alpha (PP1alpha) in a rabbit model of heart failure.</p><p><b>METHODS</b>Rabbits were divided into sham-operated group, heart failure group (volume overload by aortic valve destruction induced aortic insufficiency plus pressure overload induced by abdominal aortic banding) and heart failure plus valsartan (20 mgxkg(-1)xd(-1), n = 6 each). Seven weeks later, echocardiography examination was performed and SERCA2, PKA, PP1alpha protein and mRNA expressions were detected by Western blot and RT-PCR.</p><p><b>RESULTS</b>Compared with the with sham operated rabbits, LVMI and LVEDP in heart failure rabbits were significantly increased while left ventricular shorten fraction (LVFS) and ejection fraction (EF) were significantly decreased (all P < 0.05), these changes could be significantly attenuated by valsartan treatment (all P < 0.05). SERCA2, PKA expressions at protein and mRNA levels were significantly downregulated and PP1alpha expressions significantly upregulated in heart failure rabbits than sham operated rabbits (all P < 0.05) and these changes could be significantly attenuated by valsartan (all P < 0.05).</p><p><b>CONCLUSION</b>Valsartan improved cardiac function in volume and pressure overload induced heart failure rabbits possibly by upregulating expressions of myocardial SERCA2, PKA and downregulating expression of myocardial PP1alpha.</p>

Mutations of t-complex testis expressed gene 5 transcripts in the testis of sterile t-haplotype mutant mouse / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To determine the possible roles of the t-complex testis expressed gene 5 (Tctex5) on sperm functions, the full-length sequence of mRNA was studied and compared in the testis between the normal wild-type and the sterile t-haplotype mutant mice.</p><p><b>METHODS</b>We applied rapid amplification of cDNA ends, Northern blot and reverse transcription polymerase chain reaction to analyze the full length of Tctex5 mRNAs isolated from testes of the wild-type and the t-haplotype mice. Reverse transcription polymerase chain reaction was used to semi-quantitatively compare expression of Tctex5 transcripts in the 16 tissues and 9.5 day stage embryos in the wild-type mice. E-translation was applied to estimate the amino acid sequences.</p><p><b>RESULTS</b>One long and one short transcript of Tctex5 mRNA were discovered in mouse testis of wild-type (Tctex5(long-+) and Tctex5(short-+)) and t-haplotype (Tctex5(long-t) and Tctex5(short-t)) mice, respectively. Being enhanced only in the testis, Tctex5(long-t) had 17 point mutations and one 15-bp-deletion in the exon 1 region, comparing with the Tctex5(long-+), whereas the Tctex5(short-t) was similar to the Tctex5(short-+). The short isoforms of Tctex5 mRNAs in the two models encoded exactly the same peptides, but the long isoforms did not. The estimated peptide encoded by Tctex5(long-t) had significant mutations on putative sites of phosphorylation and PP1 binding.</p><p><b>CONCLUSION</b>We established that mutations that occur in the Tctex5 long transcript of the t-haplotype mice are important for normal sperm function, whereas the short transcript of Tctex5 might have a conserved function among different tissues.</p>

Microarray Analysis of Gene Expression in Rat Glioma after Ethanol Treatment

Objetives: Identification of target genes for ethanol in neurons is important for understanding its molecular and cellular mechanism of action and the neuropathological changes seen in alcoholics. The purpose of this study is to identify of altered gene expression after acute treatmet of ethanol in rat gliom cells. METHODS: We used high density cDNA microarray chip to measure the expression patterns of multiple genes in cultured rat glioma cells. DNA microarrays allow for the simultaneous measurement of the expression of several hundreds of genes. RESULTS: After comparing hybridized signals between control and ethanol treated groups, we found that treatment with ethanol increased the expression of 15 genes and decreased the expression of 12 genes. Upregulated genes included Orthodenticle(Drosophila) homolog 1, procollagen type II, adenosine A2a receptor, GATA-bindning protein 2. Downregulated genes included diacylglycerol kinase beta, PRKC, Protein phosphatase 1, clathrin-associated protein 17, nucleoporin p58, proteasome. CONCLUSION: The gene changes noted were those related to the regulation of transcription, signal transduction, second messenger systems. modulation of ischemic brain injury, and neurodengeneration.Although some of the genes were previously known to be ethanol responsive, we have for the most part identified novel genes involved in the brain response to ethanol.

Down regulation of mitogen activated protein kinase phosphatase-1 mediate acquired multidrug resistance in pancreatic adenocarcinoma cell line SW1990/Fu / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the role of mitogen activated protein kinase phosphatase-1 (MKP-1) in mediating acquired multidrug resistance in pancreatic adenocarcinoma cell line SW1990/Fu.</p><p><b>METHODS</b>To detect MKP-1 mRNA expression, Northern blot analysis was carried out in well established drug resistant pancreatic adenocarcinoma cell line SW1990/Fu, SW1990 and MiaPaCa-2 cell lines. To further elucidate the exact role of MKP-1, Western blot hybridization was performed in these three cell lines.</p><p><b>RESULTS</b>Northern blot analysis of total RNA isolated from SW1990/Fu, SW1990 and MiaPaCa-2 cell lines revealed the presence of the 2400 bp MKP-1 transcript 7 at relatively high levels in pancreatic cancer cell lines SW1990 and MiaPaCa-2. In the SW1990/Fu, the MKP-1 transcript was detectable at very low level. Densitometric analysis with normalization to 7S indicated that MKP-1 mRNA expression level was significantly decreased in SW1990/Fu in comparison with the parental and MiaPaCa-2 cell lines. MKP-1 protein expression level in SW1990/Fu detected by Western blot was coincident with mRNA level.</p><p><b>CONCLUSIONS</b>MKP-1 may be involved in acquired multidrug resistance in pancreatic adenocarcinoma, and we could hypothesized that alterations of intra-cellular transduction signal system acts as an important role in multidrug resistance of tumor cells.</p>

Effects of Tautomycetin on the Proliferation and Fibronectin Secretion in Vascular Smooth Muscle Cells and Glomerular Mesangial Cells / 대한이식학회지

PURPOSE: Proliferation and extracellular matrix (ECM) accumulation in the vascular smooth muscle cell (VSMC) and glomerular mesangial cell (MC) play key roles in the development and the progression of transplant glomerulosclerosis and chronic allograft nephropathy. Tautomycetin (TMC), a newly developed immunosuppressive agent, induces T-lymphocyte apoptosis through the inhibition of tyrosine kinase and protein phosphatase 1. We examined the effects of TMC on platelet-derived growth factor (PDGF)-induced proliferation and ECM synthesis in cultured VSMCs and MCs of Sprague- Dawley rats, and investigated the molecular mechanisms that are involved. METHODS: Different concentrations of TMC were administered 1 hour before the addition of PDGF 10 ng/mL into the growth-arrested and synchronized cells. Cell proliferation was assessed by methylthiazoletetrazolium (MTT) assay. Caspase-3 cleavage, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK were assessed by Western blot analysis, respectively. RESULTS: PDGF 10 ng/ mL increased cell proliferation, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK in both VSMCs and MCs. In both cultured cells, TMC at above 1 microgram/mL significantly reduced basal MTT and increased cleavage caspase-3 in a dose-dependent manner. TMC at 100 ng/mL decreased the PDGF-induced VSMC and MC proliferation without cytotoxicity. However, fibronectin secretion and the activation of Akt, ERK, and p38 MAPK were not affected at this low concentration of TMC, respectively. CONCLUSION: The present data demonstrated that low-dose TMC reduced PDGF-induced VSMC and MC proliferation without affecting the fibronectin secretion and cellular kinase activation.

The cDNA microarray study for the effect of FGF-5 administration on fibroblast

Study on the association of PPP1R3 gene polymorphism with type 2 diabetes in Han population of Anhui province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the association of muscle-specific glycogen-targeting regulatory subunit of the glucogen-bound protein phosphatase 1 (PPP1R3) gene codon 905 Asp/Tyr polymorphism with type 2 diabetes in Chinese Han population in Hefei region of Anhui province.</p><p><b>METHODS</b>PPP1R3 gene Asp905Tyr polymorphism was detected by polymerase chain reaction and appropriate restriction enzyme (PCR-RFLP) in 262 type 2 diabetic cases and 104 normal controls. Case and control groups were divided into subgroups by body mass index (BMI) 25 kg/m2.</p><p><b>RESULTS</b>When PPP1R3 gene Asp905Tyr polymorphism was not associated with type 2 diabetes mellitus. When subjects with BMI < 25 kg/m2 and Tyr/Tyr genotypes were used as reference. Subjects with Asp905 and BMI > or = 25 kg/m2 had a 3.69-fold increase of risk suffering from type 2 diabetes (OR = 3.69, 95% CI: 1.38-8.89, P=0.006).</p><p><b>CONCLUSIONS</b>PPP1R3 gene Asp905Tyr polymorphism did not seem to play a critical role in the development of type 2 diabetes mellitus in Han population of Chinese in Anhui province but interaction between the Asp905 and BMI cause the increase of risk of type 2 diabetes.</p>

Study on association of PPP1R3 gene 5 bp deletion/insertion within 3'-untranslated region polymorphism with type 2 diabetes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine whether the muscle-specific glycogen-targeting regulatory subunit of the glucogen bound protein phosphatase 1 (PPP1R3) gene 5 bp deletion/insertion(D/I) within 3'-untranslated region ( 3'-UTR) polymorphism is associated with type 2 diabetes in Chinese Han population in Hefei region of Anhui province.</p><p><b>METHODS</b>The PPP1R3 gene 3'-UTR 5 bp D/I polymorphism was detected by polymerase chain reaction in 268 patients with type 2 diabetes and 106 normal controls.</p><p><b>RESULTS</b>(1) The distributions of the frequency of three genotypes and two alleles of the PPP1R3 gene 5 bp D/I polymorphism showed no significant difference between the type 2 diabetic cases and the normal controls. (2) In both the cases and controls, there was no significant difference in age at onset, duration of disease, blood glucose, blood lipid profile, blood pressure, insulin sensitive index, body mass index, and waist hip ratio between the three genotypic groups(P 0.05). (3) The PPP1R3 gene 3'-UTR polymorphism in Chinese Han population in Hefei region of Anhui province was found to be similar to that in both Japanese population and Canadian population, and to be different from that in Piman Indians and the Caucasians in Sweden.</p><p><b>CONCLUSION</b>The PPP1R3 gene 5 bp D/I within 3'-UTR polymorphism taking on genetic variation among the different races of mankind may not play a critical role in the development of type 2 diabetes mellitus in Chinese Hans of Hefei region in Anhui province.</p>

Effects of low concentrations of okadaic acid in HeLa cells.

HeLa cells treated for prolonged period with okadaic acid (OA; 5-10nM) inhibiting protein phosphatase 2A (PP2A) and also protein phosphatase 1 (PP1) partially showed prolonged effects on mitotic progression. In the presence of OA cells progressed normally in mitosis almost upto 4 hr, then a progressive accumulation of mitotic cells could be noticed. Most of the mitotic cells seemed to be arrested at the metaphase-anaphase transition point. In arrested mitotic cells the chromosomes remained arranged at the equiatorial plate, but with prolonged treatment the chromosomes got either scattered or clumped. However, a slow release into anaphase could also be observed after 15 hr treatment. Immunofluorescence studies for microtubules and electron microscope investigations indicated the dearrangement of spindle fibres, and a prolonged treatment led to the formation of multipolarity. This was also confirmed by spread preparations of chromosomes and the formation of multinucleate cells in preparations released from the mitotic block. Chromosomes became highly condensed showing mostly nondisjunction, but separation of sister chromatids could be observed in many cells. Immunoblot assays indicated a degradation of cyclin A, but the cyclin B1 level was significantly higher in the arrested mitotic cells after 12 hr treatment. After 24 hr of treatment the cyclin B1 level was slightly lower in arrested cells. Possible roles of protein phosphatase 2A inhibition and a prolonged partial inhibition of PP1 on the mitotic progression and the cyclin degradation at the metaphase-anaphase transition have been discussed.

Molecular Mechanism of Fas-mediated Apoptosis in Colon Cancer Cell Line

BACKGROUND/AIMS: Physiologic cell death occurs primarily through an evolutionary conserved form of cellular suicide termed apoptosis. Recent evidence suggests that alterations in regulation of apoptosis contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, degenerative diseases and inflammatory diseases. Fas antigen(APO-1, CD95) is a cell surface receptor protein that is broadly expressed in normal and neoplastic cells and can mediate apoptosis in susceptible cells. Fas is involved in immune-related apoptosis including T-cell selection in thymus, down regulation of immune response and cytotoxic T-cell mediated cytotoxicity. In contrast to immune system, little is known about the function of Fas antigen expressed on epithelial cells. Recently, however, it has been shown that Fas is also important for the pathogenesis of liver disease and inflammatory skin disease. We have recently reported that although colon cancer cells HT-29 express Fas antigen on their surface, Fas ligation using IgM anti-Fas monoclonal antibody(CH-11) is not sufficient to induce apoptosis. In addition, cellular activation by IFN-gamma not only enhances Fas expression but also sensitizes HT-29 to apoptosis induced by Fas ligation as well as treatment with cycloheximide and actinomycin D. However, molecular mechanisms of Fas-mediated apoptosis are yet far from complete understanding. We, therefore, studied the functional role of Fas and apoptosis-related gene expression in apoptosis of colon cancer cell line HT-29 and signal transduction pathways including protein kinase C as well as protein phosphatase I and 2A. METHODS: Fas, Fas ligand and apoptosis related gene mRNA expression was measured by RT-PCR. Cytotoxicity and cell survival were assessed by LDH assay and MTT assay, respectively. Apoptosis was detected by confocal microscopic observation of chromatin condensation after DAPI stain and confirmed by demonstration of DNA fragmentation in agarose gel electrophoresis as well as TUNEL assay. DNA content was deteunined by flow cytometry after staining with propidium iodide and sub-G1 peak was considered as apoptotic cells. Results: Fas ligation by IgM anti-Fas monoclonal antibody(CH-11) tailed to induce poptosis in control HT-29. However, Fas ligation in IFN- gamma pretreated HT-29 induced apoptosis dose-dependently. HT-29 expressed very low level of bcl-2 mRNA, which was not changed by IFN-gamma pretreatment. IFN-gamma pretreatment did not alter the mRNA expression levels of bax, c-myc, p53, and caspases such as ICE, hich and cpp32. Protein kinase C inhibitor such as staurosporine and H7 did not inhibit Fas-mediated apoptosis of IFN-gamma pretreated HT-29. Fas-mediated apoptosis of IFN-gamma pretreated HT-29 was not suppressed as well by protein phosphatase 1 and 2A inhibitor calyculin A. Conclusions: Colon cancer cell line HT-29 expresses Fas antigen on the surface which is not sufficient to induce apoptosis. IFN-gamma pretreatment sensitizes HT-29 to Fas-mediated apoptosis, but dose not alter the expression of apoptosis-related genes including bcl-2, bax, p53 and caspases. Fas-mediated apoptotic signal in IFN-gamma pretreated HT-29 maybe independent with protein kinase C as well as with protein phosphatase 1 and 2A.