pH stat studies on bicarbonate secretion in the isolated mouse ileum

Bicarbonate secretion occurs in almost all segments of the gastrointestinal tract. This study examined HCO(3)(-) secretion in the ileum, since it is less understood than HCO(3)(-) secretion in other intestinal segments. Mouse ileal mucosa was mounted in vitro in Ussing chambers, and the mucosal alkalinization rate (J(OH)) was determined by pH stat titration, while the mucosal side was bathed with a buffer-free solution (100% O(2)) and the serosal side with a HCO(3)(-)/CO(2)-buffered solution. The transmural potential difference (PD) was recorded. The mucosal alkalinization rate (J(OH)) was higher in the presence of mucosal Cl(-) than in its absence. Forskolin, an activator of adenylate cyclase, enhanced J(OH) and PD in both the presence and absence of mucosal Cl(-). Mucosal SO(4)(2-) also caused an increase in J(OH), although the magnitude was smaller than that induced by Cl(-). Mucosal Cl(-)-dependent J(OH) was partially inhibited by acetazolamide, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tenidap and probably also by niflumic acid, but not by glibenclamide, DIDS or bumetanide. The forskolin-induced J(OH) value and PD were both inhibited by NPPB and probably also by tenidap. It is concluded that HCO(3)(-)- secretion in the ileum follows a mucosal Cl(-)-dependent pathway and a cAMP-activated pathway, each being distinct from each other. The Cl(-)-dependent pathway is probably mediated by the slc26a6 anion exchanger, and possibly also by the slc26a3 anion exchanger. The cAMP-activated HCO(3)(-) secretion is probably mediated by the cystic fibrosis transmembrane conductance regulator.     

Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier

Purpose

To investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB).

Methods

EverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively.

Results

The signal of EFV was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker^® Red, a lysosomotropic agent, and was not altered by acute treatment with NH₄Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH₄Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH₄Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC₅₀ of 98.0 μM.

Conclusions

Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB.

     

Sucutinirane-diterpene derivatives induce apoptosis via oxidative stress in HL-60 cells

Previously, we reported the isolation of cassane-type diterpenes, sucutiniranes A–F, from the seeds of Bowdichia nitida. In this study, a series of sucutinirane derivatives was prepared, and their in vitro toxicity in the HL-60 cell line was evaluated. Then the action mechanism of a representative compound that induces cell death was investigated. Whereas C-6 or C-7 diol esters and ether decreased the activity against the HL-60 cell line, furan-oxidized derivatives 12 and 13 showed improvement or retention of the activity compared with those of the natural products sucutinirane A (11), E (1), and F (2). Treatment with sucutinirane derivative 13 elevated caspase 3/7 activity and also decreased expression of Bcl-2 family proteins, Mcl-1, and Bid. Derivative 13 generated reactive oxygen species in HL-60 cells, whose apoptotic effects were attenuated by the addition of an antioxidant, N-acetyl-l-cysteine. These results suggest that cassane butenolide 13 induces apoptosis in HL-60 via its oxidative effects.     

In vivo supramolecular templating enhances the activity of multivalent ligands: a potential therapeutic against the Escherichia coli O157 AB5 toxins

We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.     

Characterization of complement C3, C4, and factor B molecules in human bile

We performed molecular analysis of complement components (C3, C4, and factor B) in human bile by sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Complement C3 was detected as a molecule composed of a 115-kDa α-chain linked to a 70-kDa β-chain by disulfide bonds, and C3 levels ranged from 45 to 650μg/ml (n=15). C4 was detected as a triple chain (98-kDa α-chain, 73-kDa β-chain, and 33-kDa γ-chain) molecule linked by disulfide bonds, and C4 levels ranged from 2.5 to 60μg/ml. Factor B, a component of the alternative pathway, was also detected, as an intact form. Factor B levels ranged from 0.3 to 8.0μg/ml. The sizes and subunit structures of complement components in human bile were compatible with those reported in human serum. The results of a hemolytic assay indicated that complement molecules in human bile were functionally active. These molecules may participate in local immune and inflammatory responses in the biliary tract.     

A novel splice variant of the nuclear coactivator p120 functions strongly for androgen receptor: characteristic expression in prostate disease

We cloned a novel splicing variant for nuclear coactivator p120(alpha), designated as p120beta and studied its function and expression in several human prostate diseases. Transfection assays demonstrated that p120beta functions as a strong coactivator for androgen receptor (AR), but weakly for other nuclear receptors. GST-pull down assay showed that a glutamine-rich region of the p120 bound to the ligand-binding domain of AR. Interestingly, p120beta mRNAs were expressed predominantly in the normal prostate, androgen-responsive prostate cancers and an androgen-sensitive prostate cancer cell line, LNCaP, but weakly in recurrent cancers and the androgen-insensitive prostate cancer cell lines PC3 and DU145. Furthermore, knockdown of p120alpha by siRNA abolished coactivator activity on thyroid hormone receptors (TR) and PPARgamma, but did not affect that of ARs in PC3 cells. In addition, competitive assay with other nuclear receptors demonstrated that TR and PPARgamma did not inhibit p120beta-induced stimulation. These findings suggested that while p120alpha was essential for ligand-dependent stimulation of TRs and PPARgamma, p120beta acted as a coactivating protein predominantly for AR.     

Ex utero intrapartum treatment for an infant with cerebro‐costo‐mandibular syndrome

Cerebro‐costo‐mandibular syndrome (CCMS) is a rare disorder characterized by multiple rib abnormalities, micrognathia described as Pierre–Robin sequence, and cerebral involvement. Appropriate management of respiratory distress immediately after birth is crucial to rescue these patients. A boy, having a mother with Pierre–Robin sequence and a sister with CCMS, was diagnosed prenatally with CCMS and successfully treated with ex utero intrapartum treatment (EXIT) at 36 weeks 6 days of gestation. EXIT would be an effective option for rescuing patients with prenatally diagnosed CCMS and preventing neonatal hypoxia.