Rafiqul Islam Khan

Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction

Disruption of epithelial barrier function was identified as one of the pathologic mechanisms in inflammatory bowel diseases (IBD). Epithelial barrier consists of various intercellular junctions, in which the tight junction (TJ) is an important component. However, the regulatory mechanism of tight junction is still not clear. Here we examined the role of focal adhesion kinase (FAK) in the epithelial barrier function on Caco-2 monolayers using a specific FAK inhibitor, PF-573, 228 (PF-228). We found that the decrease of transepithelial resistance and the increase of paracellular permeability were accompanied with the inhibition of autophosphorylation of FAK by PF-228 treatment. In addition, PF-228 inhibited the FAK phosphorylation at Y576/577 on activation loop by Src, suggesting Src-dependent regulation of FAK in Caco-2 monolayers. In an ethanol-induced barrier injury model, PF-228 treatment also inhibited the recovery of transepithelial resistance as well as these phosphorylations of FAK. In a sucrose gradient ultracentrifugation, FAK co-localized with claudin-1, an element of the TJ complex, and they co-migrate after ethanol-induced barrier injury. Immunofluorescence imaging analysis revealed that PF-228 inhibited the FAK redistribution to the cell border and reassembly of TJ proteins in the recovery after ethanol-induced barrier injury. Finally, knockdown of FAK by siRNA resulted in the decrease of transepithelial resistance. These findings reveal that activation of FAK is necessary for maintaining and repairing epithelial barrier in Caco-2 cell monolayer via regulating TJ redistribution.

Activation of muscarinic receptors prevents TNF-α-mediated intestinal epithelial barrier disruption through p38 MAPK

Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gαq/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gαq/11 dependent intracellular Ca2+ mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gαq/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR.

Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Peroxo complexes of ZrIV, ThIV, MoVI and UVI ions containing some bidentate organic ligands

Abstract Several new organoperoxo complexes of Zr IV , Th IV , Mo IV and U VI have been synthesized and characterized. The complexes have the general formulae, [Zr(O 2 )C 6 H 4 (CO 2 ) 2 ·.2C 5 H 5 NO], [Th(O 2 ) 2 L], [Th(O 2 )C 6 H 4 (CO 2 ) 2 ·.2OPPh 3 ], [Th(O 2 )L′ 2 ], [MO(O 2 ) 2 L], [MO(O 2 )L′ 2 ] and [MO(O 2 )L″H 2 O] [L = bidentate, neutral; L′ = bidentate, uninegative and L″ = bidentate, dinegative ligands; M = Mo VI and U VI ], respectively. The complexes were found to oxidize PPh 3 and AsPh 3 to their oxides. The IR spectra of the complexes indicate that the frequency of the ν 1 mode of the M(O 2 ) grouping, which is essentially an OO stretch, decreases with the increase in the atomic number of metals in a particular group.

Crystal Structure of the 3.8-MDa Respiratory Supermolecule Hemocyanin at 3.0 Å Resolution.

Molluscan hemocyanin, a copper-containing oxygen transporter, is one of the largest known proteins. Although molluscan hemocyanins are currently applied as immunotherapeutic agents, their precise structure has not been determined because of their enormous size. Here, we have determined the first X-ray crystal structure of intact molluscan hemocyanin. The structure unveiled the architecture of the 3.8-MDa supermolecule composed of homologous functional units (FUs), wherein the dimers of FUs hierarchically associated to form the entire cylindrical decamer. Most of the specific inter-FU interactions were localized at narrow regions in the FU dimers, suggesting that rigid FU dimers formed by specific interactions assemble with flexibility. Furthermore, the roles of carbohydrates in assembly and allosteric effect, and conserved sulfur-containing residues in copper incorporation, were revealed. The precise structural information obtained in this study will accelerate our understanding of the molecular basis of hemocyanin and its future applications.

Activation of muscarinic cholinoceptor ameliorates tumor necrosis factor-α-induced barrier dysfunction in intestinal epithelial cells.

Impaired intestinal barrier function is one of the critical issues in inflammatory bowel diseases. The aim of this study is to investigate muscarinic cholinoceptor (mAChR)‐mediated signaling for the amelioration of cytokine‐induced barrier dysfunction in intestinal epithelium. Rat colon challenged with TNF‐α and interferon γ reduced transepithelial electrical resistance (TER). This barrier injury was attenuated by muscarinic stimulation. In HT‐29/B6 intestinal epithelial cells, muscarinic stimulation suppressed TNF‐α‐induced activation of NF‐κB signaling and barrier disruption. Finally, muscarinic stimulation promoted the shedding of TNFR1, which would be a mechanism for the attenuation of TNF‐α/NF‐κB signaling and barrier disruption via mAChR.

Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Muscarinic cholinoceptor-mediated activation of JNK negatively regulates intestinal secretion in mice.

Regulation of intestinal secretion is important for body fluid homeostasis. We investigated the role of three MAP kinases (MAPKs) as negative regulators in muscarinic cholinoceptor (mAChR)-mediated intestinal secretion in mice. Electrophysiological analyses revealed that mAChR stimulation enhanced intestinal chloride secretion, which was further augmented by the inhibition of JNK but not by that of ERK or p38 with specific inhibitors SP600125, U0126 or SB203580, respectively. Immunoblot analyses in colonic mucosa showed that mAChR stimulation increased MAPKs phosphorylation that was suppressed by the specific inhibitor for each MAPK. This suggests that JNK is a major negative regulator in mAChR-induced intestinal secretion.

Assessment of Cardiovascular Diseases & Drug Therapy in Northern and Southern Parts of Bangladesh

Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels. Bangladesh is currently facing the silent epidemic of CVD. Medications are a cornerstone of the prevention and management of CVD. Here we studied to assess the currently occurring CVD and their drug therapy in the northern and southern parts of Bangladesh through survey. In the prospective study, we assessed the condition of patients and frequently used drugs therapy with standard questionnaires by interviewing individuals (35 to 60 years old) with a history of coronary heart disease, admitted to some renowned governmental and non-governmental hospital in the northern and southern parts of Bangladesh. From the interview of 263 patients (177 male and 86 female) associated with diabetes were 63 and not associated with diabetes were 200, having asthma 92, smoking habit 97, high sodium intake tendency 93. The most frequently reported diseases were myocardial infarction (MI), unstable angina (UA), hypertension (HTN), and stroke. Sometimes left ventricular failure (LVF) and ischemic heart disease (IHD) was also reported. The most prescribed drugs were diuretics and β-blockers. This survey provides unique contemporary data on characteristics and management of patients with CVD in northern and southern parts of Bangladesh.