Search

Inflammatory bowel disease (IBD), which mainly includes ulcerative colitis (UC) and Crohn's disease (CD), is a group of chronic bowel diseases that are characterized by abdominal pain, diarrhea, and bloody stools. IBD is strongly associated with depression, and its patients have a higher incidence of depression than the general population. Depression also adversely affects the quality of life and disease prognosis of patients with IBD. The tryptophan-kynurenine metabolic pathway degrades more than 90% of tryptophan (TRP) throughout the body, with indoleamine 2,3-dioxygenase (IDO), the key metabolic enzyme, being activated in the inflammatory environment. A series of metabolites of the pathway are neurologically active, among which kynerunic acid (KYNA) and quinolinic acid (QUIN) are molecules of great interest in recent studies on the mechanisms of inflammation-induced depression. In this review, the relationship between depression in IBD and the tryptophan-kynurenine metabolic pathway is overviewed in the light of recent publications.

Indigo Naturalis has a long history of medicinal use with particularity and complexity in its processing. Before the Ming dynasty,Indigo Naturalis was extracted from the top layer of zymotic fluid,calledquot; purified Indigo Naturalisquot;. In modern processing,the precipitatequot; crude Indigo Naturalisquot; is dried to produce Indigo Naturalis after impurity removal. The form of Indigo Naturalis slices has undergone significant changes in ancient and modern times. In view of this,the quality comparison between crude Indigo Naturalis and purified Indigo Naturalis was conducted in this study with modern analytical techniques. Firstly,chemical composition was analyzed with UPLC-Q-TOF-MS,and the chemical composition of scent with HS-SPME/GC-MS/MS. The content of indigo,indirubin,total ash,and water-soluble extract was determined as well as the inorganic composition in crude Indigo Naturalis and purified Indigo Naturalis. Then,their microscopic morphology was observed and the surface element composition was investigated. Finally,the antipyretic activities of crude Indigo Naturalis and purified Indigo Naturalis were compared in the fever rat model induced by lipopolysaccharide and 2,4-dinitrophenol. The results demonstrated that the purified Indigo Naturalis had a faster and more lasting antipyretic effect,while the crude Indigo Naturalis had almost no antipyretic effect. This study is of great significance to the research on processing technology of Indigo Naturalis and provides reference for the formulation of its quality standards,production specifications and calibration procedures.

Escape is an evolutionarily conserved and essential avoidance response. Considered to be innate, most studies on escape responses focused on hard-wired circuits. We report here that peptidergic signaling is an integral and necessary component of the Caenorhabditis elegans escape circuit. Combining genetic screening, electrophysiology and calcium imaging, we reveal that a neuropeptide NLP-18 and its cholecystokinin receptor CKR-1 enable the escape circuit to execute a full omega (Ω) turn, the last motor step where the animal robustly steers away from its original trajectory. We demonstrate in vivo and in vitro that CKR-1 is a Gαq protein coupled receptor for NLP-18. in vivo, NLP-18 is mainly secreted by the gustatory sensory neuron (ASI) to activate CKR-1 in the head motor neuron (SMD) and the turn-initiating interneuron (AIB). Removal of NLP-18, removal of CKR-1, or specific knockdown of CKR-1 in SMD or AIB neurons lead to shallower turns hence less robust escape steering. Consistently, elevation of head motor neuron (SMD)’s Ca2+ transients during escape steering is attenuated upon the removal of NLP-18 or CKR-1. in vitro, synthetic NLP-18 directly evokes CKR-1-dependent currents in oocytes and CKR-1-dependent Ca2+ transients in SMD. Thus, cholecystokinin signaling modulates an escape circuit to generate robust escape steering.

FAM3C is a member of the family with sequence similarity 3 (FAM3) gene family, and this study determined its role and mechanism in regulation of hepatic glucose/lipid metabolism. In obese diabetic mice, FAM3C expression was reduced in the liver, and hepatic FAM3C restoration improved insulin resistance, hyperglycemia, and fatty liver. FAM3C overexpression increased the expression of heat shock factor 1 (HSF1), calmodulin (CaM), and phosphorylated protein kinase B (Akt) and reduced that of gluconeogenic and lipogenic genes in diabetic mouse livers with the suppression of gluconeogenesis and lipid deposition. In cultured hepatocytes, FAM3C overexpression upregulated HSF1 expression, which elevated CaM protein level by inducing CALM1 transcription to activate Akt in a Ca2+- and insulin-independent manner. Furthermore, FAM3C overexpression promoted nuclear exclusion of FOXO1 and repressed gluconeogenic gene expression and gluconeogenesis in a CaM-dependent manner in hepatocytes. Hepatic HSF1 overexpression activated the CaM-Akt pathway to repress gluconeogenic and lipogenic gene expression and improve hyperglycemia and fatty liver in obese diabetic mice. In conclusion, the FAM3C-HSF1-CaM-Akt pathway plays important roles in regulating glucose and lipid metabolism in hepatocytes independent of insulin and calcium. Restoring hepatic FAM3C expression is beneficial for the management of type 2 diabetes and fatty liver.

The IP3 receptor binding protein released with inositol 1,4,5-trisphosphate (IRBIT) plays important roles in the regulation of intracellular Ca2+ signaling and intracellular pH. The mammals express two IRBIT paralogs, i.e., IRBIT1 (encoded by AHCYL1) and IRBIT2 (encoded by AHCYL2). The clawed frog Xenopus laevis oocyte is widely used for biophysical studies on ion channels and transporters. It remains unknown whether endogenous IRBIT is expressed in Xenopus oocytes. Here, we cloned from frog oocyte irbit2.L and irbit2.S, orthologs of mammalian IRBIT2. When over-expressed, the frog IRBITs powerfully stimulate the electrogenic Na+/HCO3- cotransporter NBCe1-B as mouse IRBIT2-V2 does. Expression of an isolated Nt fragment of NBCe1-B containing the IRBIT-binding domain greatly decreases NBCe1-B activity in oocytes, suggesting that the basal activity of NBCe1-B contains a large component derived from the stimulation by endogenous frog IRBIT. The frog IRBITs are highly homologous to the mammalian ones in the carboxyl-terminal region, but varies greatly in the amino-terminal (Nt) appendage. Interestingly, truncation study showed that the Nt appendage of IRBIT1 and the long Nt appendage of IRBIT2-V2 modestly enhance, whereas the short Nt appendage of IRBIT2-V4 greatly inhibits the functional interaction between IRBIT and NBCe1-B. Finally, Ala-substitution of Ser68, a key phosphorylation site in the PEST domain of IRBIT, causes distinct functional consequences depending on the structural context of the Nt appendage in different IRBIT isoforms. We conclude that the Nt appendage of IRBITs is not necessary for, but plays an important regulatory role in the functional interaction between IRBIT and NBCe1-B.

To identify the disease-causing gene of a four-generation Chinese family with congenital cataract.To screen the disease-causing gene of the family, six disease genes of congenital cataract are screened by direct DNA sequencing, the cDNA of wild-type (WT) MIP gene, and P191R mutant MIP gene (MT) were constructed into pEGFP-C1 vector and pGH19 vector. The recombinant plasmids of pEGFP-C1, WT, and mutant MIP were transfected into Hela cell to check the localization and HEK293T cells to detect expression level of protein. The cRNA of WT and MT MIP gene were injected into Xenopus oocytes to measure the swelling rate.A novel missense mutation c.572CG（p.P191R）at exon 3 of the MIP gene was identified and co-segregated with disease in the Chinese family. The same amount of pEGFP-WT MIP and pEGFP- P191R MIP plasmids were transfected in Hela cells. Confocal microscopy imaging showed that WT MIP protein predominantly localized on the plasma membrane, the mutant protein was rich in the cytoplasm in Hela cells. Western blot results show that the expression level of P191R mutant MIP was significantly lower than WT MIPincell membrane enriched lysates in HEK293T cells. Xenopus oocytes swelling assay showed that the P191R mutation reduces the swelling rate of Xenopus oocytes.The novel missense mutation c.572CG（p.P191R）at exon 3 of the MIP gene was identified in a Chinese family of congenital cataract. The mutation affects the traffic of MIP protein in the cells and reduces the expression level of MIP protein in the cell membrane. The mutation of MIP gene reduces the swelling ratio of Xenopus oocytes.

A reduction in insulin-stimulated glucose uptake in skeletal muscles is a characteristic of insulin resistance and type 2 diabetes mellitus (T2DM). The glucagon-like peptide (GLP)-1 agonist liraglutide can reduce blood glucose levels in individuals with T2DM. However, its effect on insulin-induced glucose metabolism in the skeletal muscle of insulin resistance is unknown. We investigated the effects and action mechanisms of liraglutide on insulin resistance (IR) in the skeletal muscle cells treatment with palmitic acid (PA). The cell-surface GLUT4myc levels were determined by an antibody-coupled colorimetric assay. The phosphorylation levels of Akt, PI3K(p85α), AS160, IRS1, IKK, and JNK were determined by western blotting. The quantifications of mRNA levels of TNFα, IL-1β, and IL-6 were determined by real-time PCR. Analysis of variance was used for data analysis. PA elevated not only phosphorylation of JNK, IRS1 serines, and IKKα/β, but also the expression of IL-6, TNFα and IL-1β in C2C12–GLUT4myc cells. PA can reduce phosphorylation of IRS1 tyrosine. These effects of PA were reversed by liraglutide. In addition, liraglutide can reverse PA-decreased insulin-stimulated cell-surface GLUT4 levels, Akt, PI3K(p85α), and AS160 phosphorylation. Liraglutide can enhance insulin-induced GLUT4 translocation by inhibiting IRS1 serine phosphorylation in PA-treated muscle cells.

The kidney maintains systemic acid-base balance by reclaiming from the renal tubule lumen virtually all HCO3− filtered in glomeruli and by secreting additional H+ to titrate luminal buffers. For proximal tubules, which are responsible for about 80% of this activity, it is believed that HCO3− reclamation depends solely on H+ secretion, mediated by the apical Na+/H+ exchanger NHE3 and the vacuolar proton pump. However, NHE3 and the proton pump cannot account for all HCO3− reclamation. Here, we investigated the potential contribution of two variants of the electroneutral Na+/HCO3– cotransporter NBCn2, the amino termini of which start with the amino acids MCDL (MCDL-NBCn2) and MEIK (MEIK-NBCn2). Western blot analysis and immunocytochemistry revealed that MEIK-NBCn2 predominantly localizes at the basolateral membrane of medullary thick ascending limbs in the rat kidney, whereas MCDL-NBCn2 localizes at the apical membrane of proximal tubules. Notably, NH4Cl-induced systemic metabolic acidosis or hypokalemic alkalosis downregulated the abundance of MCDL-NBCn2 and reciprocally upregulated NHE3. Conversely, NaHCO3-induced metabolic alkalosis upregulated MCDL-NBCn2 and reciprocally downregulated NHE3. We propose that the apical membrane of the proximal tubules has two distinct strategies for HCO3− reclamation: the conventional indirect pathway, in which NHE3 and the proton pump secrete H+ to titrate luminal HCO3−, and the novel direct pathway, in which NBCn2 removes HCO3− from the lumen. The reciprocal regulation of NBCn2 and NHE3 under different physiologic conditions is consistent with our mathematical simulations, which suggest that HCO3− uptake and H+ secretion have reciprocal efficiencies for HCO3− reclamation versus titration of luminal buffers.

One of the neuropathological hallmarks of Alzheimer's disease (AD) is the occurrence of neurofibrillary tangles (NFTs) that are composed of abnormally hyperphosphorylated microtubule-associated protein tau. Abnormal tau hyperphosphorylation is mainly induced due to the imbalance between protein kinases and phosphatases. In the tanglerich subregions of the hippocampus and parietal cortex in the brain of AD patients, the levels of the phosphorylationdependent protein peptidyl-prolyl cis-trans isomerase (Pin1) were found to be low. Although Pin1 can regulate tau phosphorylation, it is not clear whether the inhibition of glycogen synthase kinase 3 (GSK-3), the primary mediator of tau phosphorylation in AD, could reverse tau hyperphosphorylation induced due to the down-regulation of Pin1. We found that while suppression of Pin1, either by using its inhibitor Juglone or a shRNA plasmid against Pin1, induces tau hyperphosphorylation and GSK-3β activation both in vivo and in vitro, inhibition of GSK-3β by SB216763 or LiCl reverses tau hyperphosphorylation. Our data suggest that GSK-3β activation plays an important role in tau hyperphosphorylation induced by the down-regulation of Pin1, and the inhibition of GSK-3β might be a potential therapeutic approach for AD pathology.

Hepatic FAM3A expression is repressed under obese conditions, but the underlying mechanism remains unknown. This study determined the role and mechanism of miR-423-5p in hepatic glucose and lipid metabolism by repressing FAM3A expression. miR-423-5p expression was increased in the livers of obese diabetic mice and in patients with nonalcoholic fatty liver disease (NAFLD) with decreased FAM3A expression. miR-423-5p directly targeted FAM3A mRNA to repress its expression and the FAM3A-ATP-Akt pathway in cultured hepatocytes. Hepatic miR-423-5p inhibition suppressed gluconeogenesis and improved insulin resistance, hyperglycemia, and fatty liver in obese diabetic mice. In contrast, hepatic miR-423-5p overexpression promoted gluconeogenesis and hyperglycemia and increased lipid deposition in normal mice. miR-423-5p inhibition activated the FAM3A-ATP-Akt pathway and repressed gluconeogenic and lipogenic gene expression in diabetic mouse livers. The miR-423 precursor gene was further shown to be a target gene of NFE2, which induced miR-423-5p expression to repress the FAM3A-ATP-Akt pathway in cultured hepatocytes. Hepatic NFE2 overexpression upregulated miR-423-5p to repress the FAM3A-ATP-Akt pathway, promoting gluconeogenesis and lipid deposition and causing hyperglycemia in normal mice. In conclusion, under the obese condition, activation of the hepatic NFE2/miR-423-5p axis plays important roles in the progression of type 2 diabetes and NAFLD by repressing the FAM3A-ATP-Akt signaling pathway.

The SLC4A10 gene, which is highly expressed in the mammalian brain, contains two known alternative splicing units, inserts A and B, and is theoretically capable of producing four NBCn2 splice variants: NBCn2-A, -B, -C, and -D. By immunoprecipitation and western blotting, a previous study showed the putative NBCn2-D to be expressed predominantly in the subcortex (SCX) and medulla (MD) of mouse brain. However, no evidence has been provided, in any species, for the existence of a full-length transcript encoding NBCn2-D. In the present study, we report for the first time the cloning of the full-length cDNAs encoding NBCn2-D from mouse SCX and MD. Based on the frequency of bacterial colonies obtained after PCR, we conclude that in SCX, the NBCn2-A transcript is dominant, whereas in MD, NBCn2-B is dominant. NBCn2-D is the least abundant transcript in each of these two brain regions. An analysis based upon the present PCR data as well as the previous immunoprecipitation/western-blot data suggests the following prevalence of NBCn2 variants in total mouse brain: NBCn2-A (~ 83%), NBCn2-B (~ 10%), NBCn2-C (~ 5%), and NBCn2-D (~ 2%). We also estimate the prevalence of each variant in each of the five brain regions (i.e., cerebral cortex, SCX, cerebellum, hippocampus, and MD). We hypothesize that the expression of different NBCn2 splice variants is characteristic of specific tissue/cells.

BACKGROUND AND OBJECTIVE: Pyrazolone derivatives were reported to have a potent cytotoxicity against some tumor cells. In the present study, we evaluated the cytotoxic activity of a series of pyrazolone derivatives against four human tumor cell lines including HepG2, OVCAR3, KB, and multidrug resistance (MDR) KBv200 cell lines in vitro and in vivo. Additionally, the structure-activity relationships of these compounds were discussed. METHODS: To analyze the antiproliferative potential of the synthesized compounds against several human tumor cell lines, the 50% inhibitory concentration (IC50) values were determined by MTT assay. Besides, the KBv200 cell xenograft experimental model was established and the sensitivity to the pyrazolone compounds was compared between drug-sensitive parental KB cells and MDR KBv200 cells. RESULTS: Of 13 compounds screened, compound 9 presented remarkable anticancer effects, of which IC50 values were (3.24 ± 0.28), (2.58 ± 0.61), (3.81 ± 0.02), and (3.45 ± 0.03) μg/mL in HepG2, OVCAR3, KB and MDR KBv200 cells, respectively (P > 0.05). Furthermore, compound 9 effectively inhibited tumor growth of KBv200 cell xenografts in vivo, the inhibition ratio was 25.37%, 38.43%, and 47.50% for 1.5 mg/kg, 3 mg/kg, and 6 mg/kg of compound 9 groups, respectively. CONCLUSION: Compound 9 was the most promising antitumor agent in this study.

Evidence shows that women have lower tumour necrosis factor-alpha (TNF-alpha) levels and lower incidences of heart dysfunction and sepsis-related morbidity and mortality. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen and estrogen receptors (ERs), we investigated the effects of 17beta-estradiol (E(2)) and estrogen receptor alpha (ERalpha) on LPS-induced apoptosis by analyzing the activation of survival and death signalling pathways in doxycycline (Dox)-inducible Tet-On/ERalpha H9c2 myocardial cells and ERalpha-transfected primary cardiomyocytes overexpressing ERalpha. We found that LPS challenge activated JNK1/2, and then induced IkappaB degradation, NFkappaB activation, TNF-alpha up-regulation and subsequent myocardial apoptotic responses. In addition, treatments involving E(2), membrane-impermeable BSA-E(2) and/or Dox, which induces ERalpha overexpression, significantly inhibited LPS-induced apoptosis by suppressing LPS-up-regulated JNK1/2 activity, IkappaB degradation, NFkappaB activation and pro-apoptotic proteins (e.g. TNF-alpha, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardial cells. However, the cardioprotective properties of E(2), BSA-E(2) and ERalpha overexpression to inhibit LPS-induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that E(2), BSA-E(2) and ERalpha expression exert their cardioprotective effects by inhibiting JNK1/2-mediated LPS-induced TNF-alpha expression and cardiomyocyte apoptosis through activation of Akt.

Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2) transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters, although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1- or ABCG2-overexpressing cells and inhibited the transport of methotrexate and E217βG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [125I]iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic. [Cancer Res 2008;68(19):7905–14]

In the mammalian CNS, hypoxia causes a wide range of physiological effects, and these effects often depend on the stage of development. Among the effects are alterations in pH homeostasis. Na+-coupled HCO3−transporters can play critical roles in intracellular pH regulation and several, such as NCBE and NBCn1, are expressed abundantly in the central nervous system. In the present study, we examined the effect of chronic continuous hypoxia on the expression of two electroneutral Na-coupled HCO3−transporters, SLC4a7 (NBCn1) and SLC4a10 (NCBE), in mouse brain, the first such study on any acid-base transporter. We placed the mice in normobaric chambers and either maintained normoxia (21% inspired O2) or imposed continuous chronic hypoxia (11% O2) for a duration of either 14 days or 28 days, starting from ages of either postnatal age 2 days (P2) or P90. We assessed protein abundance by Western blot analysis, loading equal amounts of total protein for each condition. In most cases, hypoxia reduced NBCn1 levels by 20–50%, and NCBE levels by 15–40% in cerebral cortex, subcortex, cerebellum, and hippocampus, both after 14 and 28 days, and in both pups and adults. We hypothesize that these decreases, which are out of proportion to the expected overall decreases in brain protein levels, may especially be important for reducing energy consumption.

Aim: FG020326, a novel imidazole derivative, is a potent multidrug-resistance (MDR) modulator in vitro and in vivo . However, FG020326 is insoluble. PEDLLA-FG020326 is a FG020326-loaded nanoparticle formed with diblock copolymers of poly (ethylene glycol)-block-poly ( D,L -lactic acid) (PEG:PDLLA, PEDLLA) that can solubilize FG020326. This work was intended to evaluate the pharmacodynamics of PEDLLA-FG020326 on reversing MDR in vitro and in vivo . Methods: Cytotoxicity was determined by tetrazolium assay. The intracellular accumulation and efflux of doxorubicin (Dox) were detected by fluorescence spectrophotometry. The function of P-glycoprotein was examined by Rhodamine 123 (Rh123) accumulation detected by flow cytometry. The KBv200 cell xenograft model was established to investigate the effect of PEDLLA-FG020326 on reversing MDR in vivo . Results: PEDLLA-FG020326 and FG020326 exhibited 56.4- and 35.9-fold activity in reversing KBv200 cells to vincristine (VCR) resistance, respectively and 14.98-and 7.64-fold to Dox resistance, respectively. PEDLLA-FG020326 was much stronger than FG020326, resulting in the increase of Dox and Rh123 accumulation and the decrease of intracellular Dox extrusion in KBv200 cells. Importantly, PEDLLA-FG020326 exhibited more powerful activity than FG020326 in enhancing the effect of VCR against KBv200 cell xenografts in nude mice, but did not appear more toxic. Conclusion: The pharmacodynamics of FG020326 was improved by incorporating it into a micellar nanoparticle formed with PEG-block-PDLLA copolymers.

Pyrazolon derivatives were reported to have cytotoxicity to some tumour cells. In the present study, we investigated the effect of Lgf-YL-9 on cytotoxicity and cell apoptosis in human epidermoid carcinoma drug-sensitive parental KB cells and multidrug resistant (MDR) KBv200 cells. Lgf-YL-9 exhibited potent cytotoxicity not only to KB cells but also to KBv200 cells, and the IC(50) were 3.81 and 3.45 microg/mL in KB cells and KBv200 cells, respectively. Importantly, Lgf-YL-9 effectively inhibited tumour growth of KB cell xenografts in nude mice. Lgf-YL-9-induced cell apoptosis was confirmed by chromatin condensation, DNA fragmentation, Annexin-V and propidium iodide (PI) double-staining assay and poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, Lgf-YL-9-mediated apoptosis in KB cells and KBv200 cells was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)), the release of cytochrome c, and the activation of caspases-3, -7, and -9, but not by intercalating to DNA. Although Lgf-YL-9-induced apoptosis was associated with the decrease of DeltaPsi(m), reactive oxygen species (ROS) reduction was interestingly observed in both cell lines. The data suggest that Lgf-YL-9 has similar cytotoxicity to drug-sensitive parental KB cells and MDR KBv200 cells. Lgf-YL-9-induced apoptosis is involved in a new ROS-independent mitochondrial dysfunction pathway, but not in intercalating to DNA.

Tetrandrine (Tet), a multidrug resistant (MDR) modulator, was a potential candidate for use in cancer therapy and exhibited potent biological activity in vitro and in vivo when combined with anticancer agents such as doxorubicin, paclitaxel. Our aims were to determine whether serum concentration of Tet, which was capable of blocking P-gp in vitro, could be safely achieved in mice and whether Tet induced pharmacokinetic alterations in serum doxorubicin disposition in mice.Tet of 30 mg/kg dose used to reverse MDR was administrated intraperitoneally in mice. Plasma Tet and serum doxorubicin concentration were analyzed by HPLC. CYP 3A4 activity was examined by HPLC with the substrate of nifedipine.More than 1 micromol/L of Tet could at least tenfold reverse MDR in vitro. The plasma peak concentration of Tet was about 2 micromol/L and not less than 1 micromol/L until 18 h following Tet administration (i.p.) at 30 mg/kg. These suggested that the concentrations of Tet that were sufficient to inhibit P-gp might be achieved in mice receiving 30 mg/kg of Tet. Importantly, no significant difference was demonstrated between the doxorubicin pharmacokinetic parameters obtained in mice received doxorubicin only and doxorubicin plus Tet. This implied that Tet of 30 mg/kg did not alter the profiles of pharmacokinetics of doxorubicin including the clearance and AUC of doxorubicin. Furthermore, Tet did not significantly affect on CYP 3A4 activity in human liver microsomes until more than 25 micromol/L.Tet at the tested dose of combination treatment could achieve plasma concentrations that reversed MDR in experimental models and it had no apparent effect on doxorubicin pharmacokinetics in mice and CYP 3A4 activity in human liver microsomes.

The SLC4 genes are all capable of producing multiple variants by alternative splicing or using alternative promoters. The physiological consequences of such diversity are of great interest to investigators. Here, we identified two novel variants of the electroneutral Na+/"Equation missing" cotransporter NBCn1, one full-length starting with “MIPL” and the other Nt-truncated starting with “MDEL”. Moreover, we identified a new promoter of Slc4a10 encoding NBCn2 and a novel type of Nt-truncated NBCn2 starting with “MHAN”. When heterologously expressed, the new NBCn1 variants were well localized to the plasma membrane and exhibited characteristic NBCn1 activity. However, MHAN-NBCn2 was poorly localized on the plasma membrane. By deletion mutations, we identified the Nt regions important for the surface localization of NBCn2. Interestingly, coexpressing the full-length NBCn2 greatly enhances the surface abundance of the Nt-truncated NBCn2. Co-immunoprecipitation and bimolecular fluorescence complementation studies showed that the full-length and Nt-truncated NBCn2 interact with each other to form heterodimers in neuro-2A cells. Finally, we showed that the isolated Nt domain interacts with and enhances the surface abundance of the Nt-truncated NBCn2. The present study expands our knowledge of the NBCn1 and NBCn2 transcriptome and provides insights into how the Nt domain could affect transporter function by regulating its membrane trafficking.

Berberine is the major active constituent of Rhizoma coptidis. The present study was carried out to investigate the effect of berberine on diabetes in rats and its possible mechanisms. Diabetes was induced by tail vein injection with alloxan in Wistar rats. The amount of alloxan administered was 55 mg/kg. Diabetic rats were fed with a high-cholesterol diet. The fasting blood glucose, total cholesterol (TC), triglyceride (TG) and low density lipoprotein-cholesterol (LDL-c), high density lipoprotein-cholesterol (HDL-c), nitric oxide (NO) levels in serum and malondialdehyde (MDA),superoxide dismutase (SOD),glutathione peroxidase (GSH-px) contents in heart tissue were assayed by spectrophotometry. Pancreas samples collected after 3 weeks of alloxan treatment were stained with hematoxylin-eosin (HE) and examined under a light microscope, and scored. Intragastric administration of berberine (100 and 200 mg/kg) significantly decreased fasting blood glucose levels, serum content of TC, TG, LDL-c, and effectively increased HDL-c, NO level in diabetic rats. Furthermore, berberine treatment significantly blocked the increase of MDA, increased SOD and GSH-px levels in diabetic rats. Histopathological scores showed that berberine had restored the damage of pancreas tissues in rats with diabetes mellitus. The results showed berberine significantly inhibited the progression of diabetes induced by alloxan, and the inhibitory effect of berberine on diabetes might be associated with its hypoglycemic effect, modulating lipids metabolic effects and its ability to scavenge free radical.

Background and Objective: Little is known about the pathogenesis of apoptosis caused in cardiac tissues by periodontitis pathogens. The purpose of this study was to determine the related effect of periodontal pathogen Porphyromonas gingivalis on cardiac cell apoptosis. Methods: DNA fragmentation, nuclear condensation and activated apoptotic caspases were measured by agarose gel electrophoresis, nuclear DAPI (4′,6-diamidine-2-phenylindole dihydrochloride) stain and western blotting analysis following the surrounding medium of P. gingivalis and/or pre-administration of SB203580 (p38 inhibitor), U0126 [mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor], LY294002 [phosphoinositide 3-kinase (PI3K) inhibitor], cyclosporine A (CsA: calcineurin inhibitor), and Sp600125 [c-Jun N-terminal kinase (JNK) inhibitor] in cultured cardiac H9c2 cells. Results: The surrounding medium of periodontal pathogen P. gingivalis increased DNA fragmentation, nuclear condensation and the activated apoptotic caspase-3, -8, and -9 proteins in H9c2 cells. DNA fragmentation and nuclear condensation of H9c2 cells treated with P. gingivalis medium were completely blocked by SB203580 plus U0126 and were decreased after pre-administration of SB203580 only, U0126 only, LY294002, CsA, but were increased by Sp600125. Conclusion: Our findings suggest that the development of cardiac cell apoptosis can be directly induced by P. gingivalis medium. Porphyromonas gingivalis-related H9c2 cell apoptosis was mainly co-activated by p38 and ERK pathways and may be involved in death receptor-dependent (caspase 8) and mitochondria (caspase 9)-dependent apoptotic pathways. Porphyromonas gingivalis-related cardiac cell apoptosis was also partially mediated by PI3K or calcineurin signaling pathways, whereas the JNK pathway might play a protective role in P. gingivalis-related cardiac cell apoptosis.

Overexpression of P-glycoprotein (P-gp) encoded by MDR1 gene in cancer cells results in multidrug resistance (MDR) to structurally and mechanistically different chemotherapeutic drugs, which is a major cause for cancer chemotherapy failures to cancer patients. Recently, there were several reports showing that expression of siRNAs targeting MDR1 gene is able to reverse the P-gp mediated MDR, however, the in vivo reversal effects for MDR have still not been identified. We developed a novel MDR reversal system using RNA interference technique in human epidermoid carcinoma KBv200 cells. The stably expressing MDR1 shRNA cells (KBv200/MDR1sh) were established with transfection of vector pEGFPC2-H1-MDR1shDNA containing MDR1-V siRNA expression cassette, and we found that more than 90% of MDR1 mRNA and P-gp were reduced. KBv200/MDR1sh cells simultaneously showed stably expressing EGFP and kept low MDR1 expression beyond ten passages. Compared KBv200/MDR1sh cells with KBv200 cells, resistance to vincristine and doxorubicin decreased from 62.4-fold to 10.5-fold and from 74.5-fold to 9.5-fold respectively, and intracellular doxorubicin accumulation enhanced from 0.30 +/- 0.08 nmoles/10(6) cells to 0.86 +/- 0.16 nmoles/10(6) cells, and the fluorescence intensity of intracellular Rhodamine 123 accumulation increased from 3.58 +/- 1.63/10(6) cells to 13.96 +/- 3.07/10(6) cells. In the nude mice xenografts, vincristine (0.2 mg/kg of body weight) inhibited the growth of KBv200/MDR1sh solid tumors by 42.0%, but the same dose of vincristine didn't inhibit the growth of KBv200 solid tumors significantly. These results suggest that administration of RNAi targeted MDR1 gene can effectively reverse MDR both in vitro and in vivo models.

Overexpression of P-glycoprotein (P-gp) by tumours results in multidrug resistance (MDR) to structurally and functionally unrelated chemotherapeutic drugs. Combined therapy with MDR-related cytotoxins and MDR modulators is a promising strategy to overcome clinical MDR. This study was performed to explore the MDR reversal activity of a novel compound 2-[4-(2-pyridin-2-yl-vinyl) phenyl]-4,5-bis-(4-N,N-diethylaminophenyl)-1(H)-imidazole (FG020318) in-vitro and in-vivo. Tetrazolium (MTT) assay was used to evaluate the ability of FG020318 to reverse drug resistance in two P-gp-expressing tumour cell lines, KBv200 and MCF-7/adr. Intracellular doxorubicin accumulation was determined by fluorescence spectrophotometry in MCF-7/adr cell line. The effect of FG020318 on P-gp function was demonstrated by rhodamine 123 (Rh123) accumulation in KBv200 cells. KBv200 cell xenograft models were established to study the in-vivo effect of FG020318 on reversing MDR. FG020318 was not cytotoxic by itself against P-gp expressing KBv200 cells and MCF-7/adr cells and their parental drug-sensitive KB cells and MCF-7 cells. FG020318 could significantly increase the sensitivity of MDR cells to antitumour drugs including doxorubicin and vincristine in MCF-7/adr cells and KBv200 cells, respectively. It was much stronger than the positive control verapamil in reversal of MDR. FG020318 also increased the intracellular accumulation of doxorubicin in a concentration-dependent manner in MCF-7/adr cells, but did not affect the accumulation of doxorubicin in drug-sensitive MCF-7 cells. The Rh123 accumulation in resistant KBv200 cells was also increased by the addition of FG020318, but Rh123 accumulation was not affected by FG020318 in drug-sensitive KB cells. FG020318 potentiated the antitumour activity of vincristine to KBv200 xenografts and was an efficacious modulator in-vivo. Our results suggested that FG020318 was a highly potent, efficacious MDR modulator not only in-vitro but also in-vivo. The reversal of drug resistance by FG020318 was probably related to the increased anticancer drug accumulation and its inhibition of P-gp function of MDR tumour cells.

Multidrug resistance (MDR) is one of the main obstacles in tumor chemotherapy. A promising approach to solving this problem is to utilize a nontoxic and potent modulator able to reverse MDR, which in combination with anticancer drugs increases the anticancer effect. Experiments were carried out to examine the potential of tetrandrine (Tet) as a MDR-reversing agent. Survival of cells incubated with Tet at 2.5 micromol/l for 72 h was over 90%. Tet at 2.5 micromol/l almost completely reversed resistance to vincristine (VCR) in KBv200 cells. Tet at a concentration as low as 0.625 micromol/l produced a 7.6-fold reversal of MDR, but showed no effect on the sensitivity of drug-sensitive KB cells in vitro. In the KBv200 cell xenograft model in nude mice, neither Tet nor VCR inhibited tumor growth. However, VCR and Tet combined inhibited tumor growth by 45.7%, 61.2% and 55.7% in three independent experimental settings. In the KB cell xenograft model in nude mice, Tet did not inhibit tumor growth, but VCR and the combination of VCR and Tet inhibited tumor growth by 40.6% and 41.6%, respectively. Mechanism studies showed that Tet inhibited [(3)H]azidopine photoaffinity labeling of P-gp and increased accumulation of VCR in MDR KBv200 cells in a concentration-dependent manner. The results suggest that Tet is a potent MDR-reversing agent in vitro and in vivo. Its mechanism of action is via directly binding to P-gp and increasing intracellular VCR accumulation.

To investigate the role of C-peptide in the folding of insulin precursor, a series of C-peptide mutant proinsulin genes were constructed, overexpressed in Escherichia coli and the proteins purified. Correct disulfide linkages of these proteins were confirmed by both tryptic peptide mapping and insulin receptor binding analyses. In vitro refolding experiments were performed with the purified proteins and showed that mutations on the glycine-rich middle segment of C-peptide, GGGPGAG, and deletion of the C-terminal pentapeptide, EGSLQ, as well as mutations on the two pairs of dibasic residues at the two ends of C-peptide did not significantly affect the refolding yields. However, both alanine replacement mutation and deletion of three highly conserved acidic residues (EAED) at the N-terminus of the C-peptide resulted in serious aggregation during refolding. The results indicate that the highly conserved acidic N-terminal part of C-peptide is very important for insulin precursor folding, and that C-peptide may have some intramolecular chaperone-like function in the folding of insulin precursor.

HCO3(-) reabsorption in the renal tubules plays a critically important role in maintaining the global acid-base balance. Loss of HCO3(-) causes metabolic acidosis. Proximal renal tubule is the major site for HCO3(-) reabsorption, accounting for more than 80% of total HCO3(-) reabsorption along the nephron. Over the past more than half centuries, tremendous progresses have been made on understanding the molecular mechanisms underlying the HCO3(-) reabsorption in proximal tubules. The transepithelial movement of HCO3(-) involves the coordinated operation of machineries on both the apical and the basolateral membranes of the epithelial cells. On the apical domain, Na(+)-H(+) exchanger NHE3 and the vacuolar H(+)-ATPase are two major pathways mediating the apical uptake of HCO3(-)-related species. Taken together, NHE3 and H(+)-ATPase are responsible for about 80% of HCO3(-) reabsorption in the proximal tubule. The remaining 20% is likely mediated by pathways yet to be characterized. On the basolateral membrane, NBCe1 represents the only major known pathway mediating the extrusion of HCO3(-) coupled with Na(+) into the interstitial space. In the present article, we provide a historical view about the studies on the mechanisms of HCO3(-) reabsorption since 1940s. Moreover, we summarize the latest progresses emerging over the past decade in the physiological as well as pathological roles of acid-base transporters underlying the HCO3(-) reabsorption in proximal tubules.

AIM: To distinguish between the esophagus and adjacent organs using extraesophageal saline injection (ESI) in a canine model. METHODS: ESI was performed through the esophagus under the guidance of linear-array endoscopic ultrasonography (EUS). Approximately 15 mL of methylene blue saline (0.5%) was then injected through each of the extraesophageal puncture points using a 22 G needle. Radial EUS examinations were conducted before and after ESI. EUS images of the trachea, tracheal bifurcation, arcus aortae and thoracic aorta were recorded. Vital signs were monitored during the ESI procedure and EUS examination. The dogs were then sacrificed for exploratory thoracotomy. RESULTS: No obvious fluctuation in vital signs or serious adverse events occurred during the ESI procedure. On EUS imaging, an apparent hypoechoic area outside the esophagus, which separated the esophagus and adjacent organs, was visualized. The adventitious of the esophagus and adjacent organs were easily distinguished. The findings of subsequent exploratory thoracotomy confirmed the EUS findings: obvious accumulation of a blue liquid in the extraesophageal tissues, as well as in the esophageal-thoracic aorta space, esophageal-arcus aortae space and esophageal-tracheal space. CONCLUSION: The esophagus and adjacent organs were successfully separated by ESI, and extraesophageal saline acted as an effective ultrasonic contrast agent.

The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus – MEAD in rat only, MERF in human only – as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3− transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.

Na⁺/HCO₃⁻ cotransporter NBCe1 is an electrogenic member of the solute carrier 4 (SLC4) family and plays important roles in intracellular pH regulation as well as transepithelial HCO₃⁻ movement. The physiological and pathological significance of NBCe1 has been well established by genetic studies with humans as well as knock-out study with mouse. NBCe1 is expressed in diverse tissues in mammals. The transporter plays an essential role in the maintenance of acid-base homeostasis in our body, being responsible for more ~80% of HCO₃⁻ reabsorption in the proximal renal tubule. In humans, a number of SLC4A4 mutations have been associated with proximal renal tubule acidosis that is often accompanied with short stature, ocular abnormalities (including cataract, glaucoma, and band keratopathy), migraine, and/or defects in dental enamel development. In the present article, we review the molecular physiology, the structure/function relationship, the mechanisms underlying the functional regulation of NBCe1, as well as the physiological and pathological roles of the transporter.

HCO3− translocation across the plasma membrane via the electrogenic Na/HCO3− cotransporter NBCe1 plays an important role in intracellular pH regulation and transepithelial HCO3− transport. However, the structural determinants of transporter function remain largely unknown. A previous study showed that the putative fourth extracellular loop (EL4) plays an essential role in determining the electrogenicity of NBCe1. In the present study, we generated eight new chimeras of human NBCe1-A and NBCn1-A. All possess the putative NBCe1 EL4 and are electrogenic. Chimera O, in which the putative sixth transmembrane segment (TM6) and EL5 through the C terminus (Ct) of NBCe1 was replaced by corresponding NBCn1 sequence, produces the smallest hyperpolarization (1–2 mV) when CO2/HCO3− is added to the extracellular solution. Biotinylation experiments show that O has a very low abundance at the plasma membrane. However, chimeras in which we simultaneously replaced the putative TM6 and smaller subdomains of the EL5-Ct region for the NBCn1 sequence were strongly electrogenic except for chimera T, in which we replaced TM6 and TM12 of NBCe1 with the corresponding regions of NBCn1. T exhibited greatly reduced transporter surface expression compared to wild-type NBCe1-A, while retaining at least some electrogenic character. We hypothesize that putative TM6 and TM12 are part of a functional unit and that if the two TMs are replaced by those of the same transporter type, high surface expression would require that the surrounding TMs are also from the same transporter type.

To evaluate the inhibitory effect of statins on glucose-stimulated insulin secretion (GSIS) of pancreatic islet in rat and to explore its mechanisms.According to the average volume, freshly isolated or 24-hour cultured pancreatic islets were randomly divided into control group (incubated with Kreb-Ringer bicarbonate buffer), the atorvastatin group (incubated with 100 µmol/L atorvastatin), the fluvastatin group (incubated with 100 µmol/L fluvastatin) and the pravastatin group (incubated with 100 µmol/L pravastatin). Stimulated by 2.8, 5.5, 11.1, 16.7 mmol/L and 25.0 mmol/L glucose respectively, the effect of 100 µmol/L statins on ATP content and GSIS was compared in the four groups. GSIS was performed by the 37°C bath incubation method and ATP content was measured by chemiluminescence method.Incubated with 100 µmol/L atorvastatin for 30 minutes, in the presence of 16.7 mmol/L glucose, the ATP content [(9.54 ± 1.64) pmol/islet vs (12.33 ± 1.89) pmol/islet] and GSIS (1.60 ± 0.21 vs 2.39 ± 0.30) were significantly reduced in comparison with the control group (P0.05). Cultured with 100 µmol/L fluvastatin for 24 hours, the ATP content [(10.24 ± 2.01) pmol/islet vs (12.31 ± 2.16) pmol/islet] and GSIS (3.12 ± 0.32 vs 4.17 ± 0.37) were all significantly decreased at the higher glucose concentration of 16.7 mmol/L (P0.05).Atorvastatin and fluvastatin may inhibit GSIS by decreasing ATP content in pancreatic islet and the inhibitory effect is related to the strength of its lipophilicity.

Neuroglobin (Ngb) is a recently identified member of hemoglobin family, distributed mainly in central and peripheral nervous systems. Recent studies suggest that Ngb can protect neural cells from β-amyloid-induced toxicity in Alzheimer disease (AD). Hyperphosphorylation of tau is another characterized pathological hallmark in the AD brains; however, it is not reported whether Ngb also affects tau phosphorylation. In this study, we found that the level of Ngb was significantly reduced in Tg2576 mice (a recognized mouse model of AD) and TgMAPt mice, and the level of Ngb was negatively correlated with tau phosphorylation. Over-expression of Ngb attenuates tau hyperphosphorylation at multiple AD-related sites induced by up-regulation of glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase. While Ngb activates Akt and thus inhibits GSK-3β, simultaneously inhibition of Akt abolishes the effects of Ngb on GSK-3β inhibition and tau hyperphosphorylation. Our data indicate that Ngb may attenuate tau hyperphosphorylation through activating Akt signaling pathway, implying a therapeutic target for AD.

The Na+–HCO3− cotransporters (NBCs) of the solute carrier 4 family (SLC4) are critical for regulating pH in cells as well as in fluids such as blood and cerebrospinal fluid. Moreover, mutations and gene disruptions in NBC are linked to a wide range of pathologies. NBCe1 (SLC4A4) is electrogenic because it has an apparent Na+:HCO3− stoichiometry of 1:2 or 1:3, whereas NBCn1 (SLC4A7) is electroneutral because it has an apparent stoichiometry of 1:1. Because stoichiometry influences the effect of transport on membrane potential and vice versa, a central question is what structural features underlie electrogenicity versus electroneutrality. A previous study on rat NBCe1/n1 chimeras demonstrated that the structural elements determining the electrogenicity of NBCe1-A are located within the transmembrane domain, excluding the large third extracellular loop. In the present study we generated a series of chimeras of human NBCe1-A and human NBCn1-A. We found that replacing merely the predicted fourth extracellular loop (EL4) – containing 32 amino acid residues that include 7 prolines – of human NBCe1-A with EL4 of NBCn1-A creates an electroneutral NBC. The opposite switch converts an electroneutral construct to one with electrogenic properties. The introduction of an N-glycosylation site into EL4 confirms that at least a part of it is exposed to the extracellular fluid. We hypothesize that putative EL4 either contributes to the substrate-binding vestibule or indirectly influences substrate binding by interacting with one or more transmembrane segments, thereby controlling the nature of transport.

The mammalian aquaporins AQP1, AQP4, and AQP5 have been shown to function not only as water channels but also as gas channels. Zebrafish have two genes encoding an AQP1 homologue, aqp1a and aqp1b. In the present study, we cloned the cDNA that encodes the zebrafish protein Aqp1a from the 72-h postfertilization (hpf) embryo of Danio rerio, as well as from the swim bladder of the adult. The deduced amino-acid sequence of aqp1a consists of 260 amino acids and is 59% identical to human AQP1. By analyzing the genomic DNA sequence, we identified four exons in the aqp1a gene. By in situ hybridization, aqp1a is expressed transiently in the developing vasculature and in erythrocytes from 16 to 48 h of development. Later, at 72 hpf, aqp1a is expressed in dermal ionocytes and in the swim bladder. Western blot analysis of adult tissues reveals that Aqp1a is most highly expressed in the eye and swim bladder. Xenopus oocytes expressing aqp1a have a channel-dependent (*) osmotic water permeability (Pf*) that is indistinguishable from that of human AQP1. On the basis of the magnitude of the transient change in surface pH (ΔpHS) that were recorded as the oocytes were exposed to either CO2or NH3, we conclude that zebrafish Aqp1a is permeable to both CO2and NH3. The ratio (ΔpHS*)CO2/Pf*is about half that of human AQP1, and the ratio (ΔpHS*)NH3/Pf*is about one-quarter that of human AQP1. Thus, compared with human AQP1, zebrafish Aqp1a has about twice the selectivity for CO2over NH3.

The five known Na-coupled HCO(3)(-) transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO(3)(-) transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO(3)(-) cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants-NBCn2-A, -B, and -C-as well as a potential variant-D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem.

Our previous studies have shown that tetrandrine (Tet) reverses the effect of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) both in vitro and in vivo. 5-Bromotetrandrine (Br-Tet) is a newly synthesized brominated derivative of Tet. In this study, we investigated the MDR reversal activity of Br-Tet in vitro and in vivo and the mechanism involved in this reversal.The ability of Br-Tet to reverse drug resistance in vitro was evaluated by MTT assay in human MDR KBv200 cells and the parental drug-sensitive KB cells. A KBv200 cell xenograft model was established to investigate the MDR reversal activity of Br-Tet in vivo. Doxorubicin (Dox) accumulation in KBv200 and KB cell lines was determined by flow cytometry and Dox accumulation in KBv200 xenografts tissue was examined by spectrofluorometer. The effect of Br-Tet on the expression of P-glycoprotein was detected by flow cytometry and Western blot, respectively.Br-Tet significantly enhanced the cytotoxicity of Dox, paclitaxel, taxotere, vincristine and epirubicin in KBv200 cells but not in KB cells. Co-administration of 10 mg/kg Br-Tet and 2 mg/kg epirubicin significantly enhanced the antitumor activity of epirubicin without increasing the toxicity. Br-Tet increased the Dox accumulation in the MDR KBv200 cell line and in KBv200 xenograft tissue in a time- and dose-dependent manner. However, it did not reduce the expression of P-gp in KBv200 cells.Br-Tet caused a significant reversal of P-gp-mediated MDR, not only in vitro but also in vivo. The MDR reversal activity of Br-Tet in vivo was associated with the enhancement of accumulation of chemotherapeutical drugs in tumor tissue.

SUMMARYMammalian genomes contain 10 SLC4 genes that, between them, encode three Cl–HCO3 exchangers, five Na+-coupled HCO3 transporters (NCBTs), one reported borate transporter, and what is reported to be a fourth Cl–HCO3 exchanger. The NCBTs are expressed throughout the body and play important roles in maintaining intracellular and whole-body pH, as well as contributing to transepithelial transport processes. The importance of NCBTs is underscored by the genetic association of dysfunctional NCBT genes with blindness, deafness, epilepsy,hypertension and metal retardation. Key to understanding the action and regulation of NCBTs is an appreciation of the diversity of NCBT gene products. The transmembrane domains of human NCBT paralogs are 50–84% identical to each other at the amino acid level, and are capable of a diverse range of actions, including electrogenic Na/HCO3 cotransport (i.e. NBCe1 and NBCe2) and electroneutral Na/HCO3 cotransport (i.e. NBCn1 and NBCn2), as well as Na+-dependent Cl–HCO3 exchange(i.e. NDCBE). Furthermore, by the use of alternative promoters and alternative-splicing events, individual SLC4 genes have the potential to generate multiple splice variants (as many as 16 in the case of NBCn1), each of which could have unique temporal and spatial patterns of distribution, unitary transporter activity (i.e. flux mediated by one molecule), array of protein-binding partners, and complement of regulatory stimuli. In the first section of this review, we summarize our present knowledge of the function and distribution of mammalian NCBTs and their multiple variants. In the second section of this review we consider the molecular consequences of NCBT variation.

The water channel aquaporin 1 (AQP1) and certain Rh-family members are permeable to CO 2 and NH 3 . Here, we use changes in surface pH (pH S ) to assess relative CO 2 vs. NH 3 permeability of Xenopus oocytes expressing members of the AQP or Rh family. Exposed to CO 2 or NH 3 , AQP1 oocytes exhibit a greater maximal magnitude of pH S change (ΔpH S ) compared with day-matched controls injected with H 2 O or with RNA encoding SGLT1, NKCC2, or PepT1. With CO 2 , AQP1 oocytes also have faster time constants for pH S relaxation (τ pHs ). Thus, AQP1, but not the other proteins, conduct CO 2 and NH 3 . Oocytes expressing rat AQP4, rat AQP5, human RhAG, or the bacterial Rh homolog AmtB also exhibit greater ΔpH S (CO 2 ) and faster τ pHs compared with controls. Oocytes expressing AmtB and RhAG, but not AQP4 or AQP5, exhibit greater ΔpH S (NH 3 ) values. Only AQPs exhibited significant osmotic water permeability (P f ). We computed channel-dependent (*) ΔpH S or P f by subtracting values for H 2 O oocytes from those of channel-expressing oocytes. For the ratio ΔpH S (CO 2 )*/P f *, the sequence was AQP5 > AQP1 ≅ AQP4. For ΔpH S (CO 2 )*/ΔpH S (NH 3 )*, the sequence was AQP4 ≅ AQP5 > AQP1 > AmtB > RhAG. Thus, each channel exhibits a characteristic ratio for indices of CO 2 vs. NH 3 permeability, demonstrating that, like ion channels, gas channels can exhibit selectivity.

Others have shown that exposing oocytes to high levels of \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \)(10–20 mM) causes a paradoxical fall in intracellular pH (pHi), whereas low levels (e.g., 0.5 mM) cause little pHi change. Here we monitored pHi and extracellular surface pH (pHS) while exposing oocytes to 5 or 0.5 mM NH3/NH4+. We confirm that 5 mM \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \) causes a paradoxical pHi fall (−ΔpHi ≅ 0.2), but also observe an abrupt pHS fall (−ΔpHS ≅ 0.2)—indicative of NH3 influx—followed by a slow decay. Reducing [NH3/NH4+] to 0.5 mM minimizes pHi changes but maintains pHS changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpHS at both \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \) levels. During removal of 0.5 or 5 mM NH3/NH4+, failure of pHS to markedly overshoot bulk extracellular pH implies little NH3 efflux and, thus, little free cytosolic NH3/NH4+. A new analysis of the effects of NH3 vs. NH4+ fluxes on pHS and pHi indicates that (a) NH3 rather than NH4+ fluxes dominate pHi and pHS changes and (b) oocytes dispose of most incoming NH3. NMR studies of oocytes exposed to 15N-labeled \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \) show no significant formation of glutamine but substantial \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \) accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pHi and pHS demonstrate that NH3 flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

The effectiveness of chemotherapeutic treatment is usually limited by the overexpression of adenosine triphosphate binding cassette (ABC) transporters, which mediate multidrug resistance (MDR) by acting as efflux pumps to remove chemotherapeutic agents from MDR cancer cells. Thus, the inhibition of ABC transporters may represent a promising strategy to reverse MDR. This study was to characterize the in vitro and in vivo actions of FG020326, a newly synthesized triaryl-substituted imidazole derivative, to reverse MDR. FG020326 significantly potentiated the cytotoxicity of paclitaxel, doxorubicin, and vincristine in the ABCB1 (P-glycoprotein, P-gp) overexpressing cells KBv200 and MCF-7/adr, but not in the ABCB1 negative parental cell lines KB and MCF-7. However, FG020326 did not alter the cytotoxicity of the aforementioned drugs in ABCC1 (MRP1), ABCC4 (MRP4), ABCG2 (BCRP) and LRP overexpressing cell lines, KB-CV60, NIH3T3/MRP4-2, S1-M1-80 and SW1573/2R120, respectively. FG020326, following p.o. administration, was present in concentrations sufficient for reversal of MDR in mice. The co-administration of FG020326 with paclitaxel or vincristine significantly enhanced the antitumor activity of these drugs without significantly increasing toxicity in the mice bearing the KBv200 cell xenografts. In addition, FG020326, at concentrations that reversed MDR, did not significantly affect the activity of CYP 3A4 or alter the pharmacokinetic profile of paclitaxel after co-administration with paclitaxel. FG020326 produced a significant concentration-dependent displacement of [3H]-azidopine and inhibition of efflux of drug from cells. Furthermore, FG020326 was colocalized with ABCB1 in cell membranes. Hence, FG020326 is characterized as a third generation MDR modulator that holds great promise for the treatment of cancer patients with ABCB1 mediated MDR.

To investigate the inhibitory effect of Oridonin injection on heterotransplanted tumors of human gastric adenocarcinoma cell line BGC823 cells in nude mice and explore its mechanism.Heterotransplanted models of human gastric adenocarcinoma cell line BGC823 cells in nude mice were established. They were divided at random into three groups as control group, low-dose group and high-dose group. The Oridonin solution at concentration of 37.5 mg x kg(-1 x d(-1) and 75 mg x kg(-1) x d(-1) were injected to the mice in low-dose group and high-dose group, respectively, and 0.9% sodium chloride was injected to the mice of control group per day for 10 days sequentially. The mice of the three groups were sacrificed at 11th day after the first injection of Oridonin. The tumor weight of the sacrificed mice was measured. Morphological and ultrastructural examinations of the tumors were carried out by light and electron microscopy. The expression of bcl-2, Bax, Fas and FasL was detected by immunohistochemistry.Oridonin injection showed a suppressive effect on the growth of heterotransplanted tumors in the nude mice. The tumor growth inhibition rates were 48.5% and 70.7% in the low-dose and high-dose groups, respectively. The morphological study demonstrated that tumor cells displayed a typical appearance of apoptosis. The expression of bcl-2 was down-regulated, while Bax, Fas and FasL were up-regulated.Oridonin can markedly inhibit the growth of heterotransplanted human gastric adenocarcinoma in nude mice. It was due, at least in part, to the induction of apoptosis in cancer cells.

Na-coupled HCO3 transporters (NCBTs) play important roles in brain pH regulation. One NCBT, the Na-driven Cl-HCO3 exchanger (SLC4A8 or NDCBE), appears to be the major regulator of intracellular pH (pHi), at least in some hippocampal pyramidal neurons. NDCBE is widely expressed throughout the central nervous system in rodent brain. In a previous study, it has been demonstrated that CCH decreases the abundance of NBCn1 and NBCn2 proteins in four regions of the mouse brain: cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and hippocampus (HC). Here we report the effect of CCH (11% O2) on the expression of NDCBE protein in mouse brain. Neonates (beginning at age P2) or adult mice (beginning at P90) were subjected to either normoxia or CCH for durations of 14 or 28 days. Membrane-protein levels were assessed by western blotting using our polyclonal antibody directed against NDCBE. In neonates, CCH significantly decreased NDCBE expression in HC after 14 days and SCX after 28 days, but had no significant effect for other combinations of region/duration. In adults, however, CCH significantly decreased (by 20–50%) the expression of NDCBE in all four brain regions, both with 14 and 28 day duration. Thus, the mouse brain exhibits marked developmental differences in the response of NDCBE protein expression to CCH. We hypothesize that decreases in adult NDCBE protein levels, which are probably out of proportion to the decreases in other proteins, may be part of an adaptive response that reduces energy consumption and/or stabilizes brain pHi. The smaller or absent responses in the young animals could be related to neonatal hypoxia tolerance.

To explore the effect and mechanism of compound Rhizoma Coptidis capsule (CRCC) on diabetic nephropathy in experimental rats.The rat model of early diabetic nephropathy was induced by injection of streptozotocin (STZ). The rats were divided into 6 groups: normal control group, model group, 3 CRCC treatment groups and XKW treatment group. The fasting blood glucose (FBG), blood urea nitrogen (BUN), creatinine (Cr), insulin (Ins) and urinary protein (Upro) were tested 30 days later. The expression of transforming growth factor-beta1 (TGF-beta1) and type IV collagen (IV-C) proteins and the pathological changes in renal tissue of diabetic rats with nephropathy were observed by optical micrography.CRCC could reduce the levels of FBG, BUN, Cr, Upro and the expression of TGF-beta1 and IV-C proteins, and alleviate pathological lesion in renal tissue of diabetic rats with nephropathy.CRCC may protect the renal function and slow down the progression of diabetic nephropathy in rats by suppressing the expression of TGF-beta1 and IV-C proteins in renal tissue.

The Na(+)-driven Cl-HCO(3) exchanger (NDCBE or SLC4A8) is a member of the solute carrier 4 (SLC4) family of HCO(3)(-) transporters, which includes products of 10 genes with similar sequences. Most SLC4 members play important roles in regulating intracellular pH (pH(i)). Physiological studies suggest that NDCBE is a major pH(i) regulator in at least hippocampal (HC) pyramidal neurons. We generated a polyclonal rabbit antibody directed against the first 18 residues of the cytoplasmic N terminus (Nt) of human NDCBE. By Western blotting, the antibody distinguishes NDCBE-as a purified Nt peptide or a full-length transporter (expressed in Xenopus oocytes)-from other Na(+)-coupled HCO(3)(-) transporters. By Western blotting, the antiserum recognizes an approximately 135-kDa band in several brain regions of adult mice: the cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and HC. In CX, PNGase F treatment reduces the molecular weight to approximately 116 kDa. By immunocytochemistry, affinity-purified (AP) NDCBE antibody stains the plasma membrane of neuron cell bodies and processes of rat HC neurons in primary culture as well as freshly dissociated mouse HC neurons. The AP antibody does not detect substantial NDCBE levels in freshly dissociated HC astrocytes, or astrocytes in HC or CB sections. By immunohistochemistry, the AP antibody recognizes high levels of NDCBE in neurons of CX, HC (including pyramidal neurons in Cornu Ammonis (CA)1-3 and dentate gyrus), substantial nigra, medulla, cerebellum (especially Purkinje and granular cells), and the basolateral membrane of fetal choroid plexus. Thus, NDCBE is in a position to contribute substantially to pH(i) regulation in multiple CNS neurons.

NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO(3)(-) transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting--both of purified peptides representing the initial approximately 120 aa of the transporters and of full-length transporters expressed in Xenopus oocytes--demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of embryonic day 18 (E18) fetal and adult choroid plexus. NCBE protein is present by Western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By Western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (approximately 150 kDa). PNGase F reduces the molecular weight (MW) of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression.

We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).

Maintaining genetic diversity is a major issue in conservation biology. In this study, we demonstrate the differences of genetic diversity levels between wild and captive individuals of Elliot’s Pheasant Syrmaticus ellioti. Wild individuals showed a higher genetic diversity level than that of the captive individuals. Nucleotide diversity and haplotype diversity of wild individuals were 0.00628 and 0.993, while those of captive individuals were 0.00150 and 0.584 respectively. Only 3 haplotypes of mtDNA control region sequence were identified among 36 captive individuals, while 16 unique haplotypes were identified among the 17 wild individuals in this study. One captive haplotype was shared by a wild individual from Anhui Province. It is concluded that a low number of founders was the likely reason for the lower level genetic diversity of the captive group. Careful genetic management is suggested for captive populations, particularly of such an endangered species, to maintain genetic variability levels.

Annonaceous acetogenin 89-2 was obtained from atemoya plant. To investigate the effect of 89-2 on experimental chemotherapy against xenografts derived from multidrug resistant KBv200 cells and parental drug-sensitive KB cells.Cytotoxicity was determined by tetrazolium (MTT) assay. The models of KB and KBv200 xenografts in nude mice were established to investigate the effect of 89-2 on experimental chemotherapy against cancer in vivo. Mechanistic experiments were conducted to examine the function of P-gp by Fura 2-AM assay.The compound 89-2 showed potent cytotoxicity in KBv200 and KB cells, and the mean IC50 of 89-2 to KBv200 and KB cells was 48.7 and 64.6 nmol.L-1, respectively. The IC50 of 89-2 to multidrug resistant (MDR) cells was similar to that to the parental drug-sensitive cells (P0.05). In the models of KBv200 and KB cell xenografts in nude mice, 89-2 (0.90 mg.kg-1, q2d x 6) exhibited 52.3% and 56.5% in inhibiting the growth of xenografts, respectively. The toxicity was endurable. The intracellular accumulation of Fura-2 in KBv200 cells increased to 1.66, 2.03, and 2.74-fold, respectively, by addition of 12.8, 64 and 320 nmol.L-1 of 89-2.Both MDR KBv200 cells and parental drug-sensitive KB cells were sensitive to the treatment of 89-2 in vitro and in vivo. The mechanism of overcoming MDR was associated with the decrease of P-gp function MDR cells.

Many studies showed that there is complementary effect between topoisomerase I inhibitor and topoisomerase II inhibitor. Combination of them showed synergistic action. This study was designed to investigate the experimental therapeutic effect of the combination of topoisomerase I inhibitor hydroxycamptothecin (HCPT) with topoisomerase II inhibitor etoposide (VP-16) on nasopharyngeal carcinoma (NPC), the effect of the combination of HCPT with VP-16 against NPC was studied in vitro and in vivo.Cytotoxicity of HCPT alone, VP-16 alone, and combination of HCPT and VP-16 on NPC cell strain CNE2 were measured by MTT assay. The models of nude mice xenografts were established for investigating the effect of the combination regimen against NPC in vivo.HCPT alone and VP-16 alone have potent cytotoxicity to CNE2 cells. The IC(50) values were 13.5+/-1.2 micromol/L and 13.5+/-1.0 micromol/L, respectively. The combination of HCPT with VP-16 (1:1) showed synergistic cytotoxicity to CNE2 cells in vitro (CI1). In vivo experiment showed that HCPT alone (1.5 mg/kg, i.p., q2d) exhibited 13.6% inhibition growth rate;VP-16 alone (10 mg/kg, i.p., q2d) exhibited 27.3% inhibition growth rate; the combination regimen exhibited 50% inhibition growth rate.The combination of HCPT with VP-16 had significant synergistic effect against NPC in vitro and in vivo. These results suggested that the combination of HCPT with VP-16 is a promising regimen to treat NPC in clinic.

Male copulation calls are found in a number of polygynous primate species, but their functions still represent a puzzle [1]. It has been proposed that among rhesus macaques, male copulation calls are sexually selected handicap signals that females can use to evaluate male quality on account of the cost to males of calling (specifically increased aggression shown to callers) [2]. Evidence in favor of this hypothesis is inconclusive, however, and an alternative explanation of their occurrence is that the calls simply reflect overall levels of male courtship intensity [3]. This report describes for the first time the acoustic structure and pattern of occurrence of male copulation calls among the little-studied Formosan macaques. This study also evaluates whether calling is related to male rank and leads to increased levels of aggressive harassment received by mating males, as predicted by the handicap signal hypothesis.