The Role of Ergosterol and Sphingolipids in the Localization and Activity of Candida albicans' Multidrug Transporter Cdr1p and Plasma Membrane ATPase Pma1p.

Opportunistic pathogen Candida albicans causes systemic infections named candidiasis. Due to the increasing number of multi-drug resistant clinical isolates of Candida sp., currently employed antifungals (e.g., azoles) are insufficient for combating fungal infection. One of the resistance mechanisms toward azoles is increased expression of plasma membrane (PM) transporters (e.g., Cdr1p), and such an effect was observed in C. albicans clinical isolates. At the same time, it has been proven that a decrease in PMs sphingolipids (SLs) content correlates with altered sensitivity to azoles and diminished Cdr1p levels. This indicates an important role for SL in maintaining the properties of PM and gaining resistance to antifungal agents. Here, we prove using a novel spot variation fluorescence correlation spectroscopy (svFCS) technique that CaCdr1p localizes in detergent resistant microdomains (DRMs). Immunoblot analysis confirmed the localization of CaCdr1p in DRMs fraction in both the C. albicans WT and erg11Δ/Δ strains after 14 and 24 h of culture. We also show that the C. albicanserg11Δ/Δ strain is more sensitive to the inhibitor of SLs synthesis; aureobasidin A (AbA). AbA treatment leads to a diminished amount of SLs in C. albicans WT and erg11Δ/Δ PM, while, for C. albicanserg11Δ/Δ, the general levels of mannose-inositol-P-ceramide and inositol-P-ceramide are significantly lower than for the C. albicans WT strain. Simultaneously, the level of ergosterol in the C. albicans WT strain after adding of AbA remains unchanged, compared to the control conditions. Analysis of PM permeabilization revealed that treatment with AbA correlates with the disruption of PM integrity in C. albicanserg11Δ/Δ but not in the C. albicans WT strain. Additionally, in the C. albicans WT strain, we observed lower activity of H+-ATPase, correlated with the delocalization of both CaCdr1p and CaPma1p.

Organ-specific expression of genes involved in iron homeostasis in wheat mutant lines with increased grain iron and zinc content.

Background: Iron deficiency is a well-known nutritional disorder, and the imbalance of trace-elements, specifically iron, is the most common nutrient deficiency of foods across the world, including in Kazakhstan. Wheat has significant nutritional relevance, especially in the provision of iron, however many bread wheat varieties have low iron despite the need for human nourishment. In this study, the expression profiles of wheat homologous genes related to iron homeostasis were investigated. The work resulted in the development of two new M5 mutant lines of spring bread wheat through gamma-irradiation (200 Gy) with higher grain iron and zinc content, lower phytic acid content, and enhanced iron bioavailability compared to the parent variety. Mutant lines were also characterized by higher means of yield associated traits such as grain number per main spike, grain weight per main spike, grain weight per plant, and thousand-grain weight. Methods: The homologous genes of bread wheat from several groups were selected for gene expression studies exploring the tight control of iron uptake, translocation rate and accumulation in leaves and roots, and comprised the following: (1) S-adenosylmethionine synthase (SAMS), nicotianamine synthase (NAS1), nicotianamine aminotransferase (NAAT), deoxymugineic acid synthetase (DMAS), involved in the synthesis and release of phytosiderophores; (2) transcription factor basic helix-loop-helix (bHLH); (3) transporters of mugineic acid (TOM), involved in long-distance iron transport; (4) yellow stripe-like (YSlA), and the vacuolar transporter (VIT2), involved in intracellular iron transport and storage; and lastly (5) natural resistance-associated macrophage protein (NRAMP) and ferritin (Fer1A). Results: The wheat homologous genes TaSAMS, TaNAS1, and TaDMAS, were significantly up-regulated in the roots of both mutant lines by 2.1-4.7-fold compared to the parent variety. The combined over-expression of TaYSlA and TaVIT2 was also revealed in the roots of mutant lines by 1.3-2.7-fold. In one of the mutant lines, genes encoding intracellular iron transport and storage genes TaNRAMP and TaFer1A-D showed significant up-regulation in roots and leaves (by 1.4- and 3.5-fold, respectively). The highest expression was recorded in the transcription factor TabHLH, which was expressed 13.1- and 30.2-fold in the roots of mutant lines. Our research revealed that genotype-dependent and organ-specific gene expression profiles can provide new insights into iron uptake, translocation rate, storage, and regulation in wheat which aid the prioritization of gene targets for iron biofortification and bioavailability.

New lymphatic cell formation is associated with damaged brain tissue clearance after penetrating traumatic brain injury.

We characterized the tissue repair response after penetrating traumatic brain injury (pTBI) in this study. Seventy specific pathogen-free Kunming mice were randomly divided into the following groups: normal control, 1, 3, 7, 15, 21, and 30 days after pTBI. Hematoxylin and eosin (H&E) staining, immunohistochemistry, and immunofluorescence were performed to examine and monitor brain tissue morphology, and the distribution and expression of lymphatic-specific markers lymphatic vessel endothelial receptor-1 (LYVE-1), hematopoietic precursor cluster of differentiation 34 (CD34) antigen, and Prospero-related homeobox-1 (PROX1) protein. H&E staining revealed that damaged and necrotic tissues observed on day 1 at and around the injury site disappeared on day 7, and there was gradual shrinkage and disappearance of the lesion on day 30, suggesting a clearance mechanism. We explored the possibility of lymphangiogenesis causing this clearance as part of the post-injury response. Notably, expression of lymphangiogenesis markers LYVE-1, CD34, and PROX1 was detected in damaged mouse brain tissue but not in normal tissue. Moreover, new lymphatic cells and colocalization of LYVE-1/CD34 and LYVE-1/PROX1 were also observed. Our findings of the formation of new lymphatic cells following pTBI provide preliminary insights into a post-injury clearance mechanism in the brain. Although we showed that lymphatic cells are implicated in brain tissue repair, further research is required to clarify the origin of these cells.

Quantitative protein expression of blood-brain barrier transporters in the vasculature of brain metastases of patients with lung and breast cancer.

This study determined absolute transporter protein abundances in isolated microvessels of human noncancerous cerebral cortex as well as brain metastases of patients with lung and breast cancer, using a validated targeted proteomics approach. As compared with those in microvessels of noncancerous cerebral cortex, the median protein abundances of glucose transporter 1 (a brain endothelial marker) and sodium-potassium pump (Na/K ATPase, a plasma membrane marker) were decreased by ~ 80% in brain metastasis microvessels. The major efflux transporters (ABCB1 and ABCG2) fell to undetectable in microvessels of more than 80% of brain metastasis specimens. Monocarboxylate transporter 1 was undetectable in microvessels of more than 80% of brain metastases, whereas large neutral amino acid transporter 1 levels remained unchanged. In conclusion, the integrity of the physical and biochemical barrier with respect to transporter protein expression is largely disrupted in brain metastasis tumor vasculatures. Differential transporter protein abundances at the blood-brain barrier and blood-brain tumor barrier provided mechanistic and quantitative basis for prediction of heterogeneous drug penetration into human brain and brain tumors, which is critical not only to the understanding of the success or failure of systemic chemotherapy in the treatment of brain tumors but also to the development of more effective therapeutic strategies.

SLC14A1 prevents oncometabolite accumulation and recruits HDAC1 to transrepress oncometabolite genes in urothelial carcinoma.

Urothelial carcinoma (UC), including upper tract urothelial carcinoma (UTUC) and urinary bladder urothelial carcinoma (UBUC), is a common malignant disease in developed countries. Oncogenic metabolic lesions have been associated with UC development. Methods: Using data mining, a series of studies were performed to study the involvement of SLC14A1 in UC specimens, animal models and UC-derived cell lines. Results: In two cohorts of UTUC (n = 340) and UBUC (n = 295), the SLC14A1 protein level was an independent prognostic factor. Epigenetic silencing contributed to SLC14A1 downregulation in UCs. Total and membranous SLC14A1 played tumor suppressive roles through the inhibition of cell proliferation and metastasis in distinct UC-derived cells and animal models. Functional SLC14A1 prevented the accumulation of arginine and urea, enhanced mitochondrial fusion and aerobic respiration, inhibited glycolysis by altering the expression levels of several related proteins and sensitized arginine-deprivation treatment in ASS1-deficient UC-derived cells. In vitro and in vivo, SLC14A1 inhibited the mTOR signaling pathway and subsequently tumorigenesis, supported by reduced arginine concentrations in vitro. Nuclear SLC14A1 transrepressed HK2 and DEGS1 genes via recruitment of HDAC1 and/or SIN3A to maintain metabolic homeostasis and thereafter impeded tumorigenesis. Conclusion: Clinical associations, animal models and in vitro indications provide solid evidence that the SLC14A1 gene is a novel tumor suppressor in UCs. Total and membranous SLC14A1 prevents urea and arginine accumulation via the mTOR signaling pathway. Nuclear SLC14A1 recruits HDAC1 to transrepress oncometabolite genes.

Prognostic impact of HERC2 protein and pink-eyed dilution protein in uveal melanoma.

Biological understanding of pigmentation and its association with clinicopathological implications in uveal melanoma (UM) risk is still unexplored. HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase 2 (HERC2) and Pink-eyed dilution protein (P-protein) are the important markers that regulate pigmentation in eye. Therefore, our aim of the study was to investigate the expression of HERC2 and P-protein in the UM patients and correlate with patient outcome. Fifty-two formalin-fixed paraffin-embedded UM tissue samples were included to detect the expression of HERC2 and P-protein by immunohistochemistry and validated by western blot. Cox proportional hazard model and log-rank test were used to determine the prognostic potential of these proteins. High pigmentation was seen in 67% of the UM cases. The expression of HERC2 and P-protein was present in 44% and 71% cases, respectively. On statistical analysis, increased pigmentation, epithelioid cell type, and ciliary body invasion were significant with the protein expressions (p < 0.05). Metastasis-free survival was reduced in UM cases which expressed HERC2 and P-protein. On multivariate analysis, P-protein expression was found to be an independent prognostic factor. Our findings suggest that HERC2 and P-protein could be used as novel predictors of high pigmentation in UM cases which have high metastatic potential.

Gestational Age-Dependent Abundance of Human Placental Transporters as Determined by Quantitative Targeted Proteomics.

Some women take medication during pregnancy to address a variety of clinical conditions. Because of ethical and logistical concerns, it is impossible to determine fetal drug exposure, and therefore fetal risk, during pregnancy. Hence, alternative approaches need to be developed to predict maternal-fetal drug exposure throughout pregnancy. To do so, we previously developed and verified a maternal-fetal physiologically based pharmacokinetic model, which can predict fetal exposure to drugs that passively cross the placenta. However, many drugs are actively transported by the placenta (e.g., human immunodeficiency virus protease inhibitors). To extend our maternal-fetal physiologically based pharmacokinetic model to these actively transported drugs, we determined the gestational age-dependent changes in the protein abundance of placental transporters. Total cellular membrane fractions from first trimester (T1; n = 15), second trimester (T2; n = 19), and term (n = 15) human placentae obtained from uncomplicated pregnancies were isolated by ultracentrifugation. Transporter protein abundance was determined by targeted quantitative proteomics using liquid chromatography tandem mass specrometry. We observed that breast cancer resistance protein and P-glycoprotein abundance significantly decreased from T1 to term by 55% and 69%, respectively (per gram of tissue). Organic anion-transporting polypeptide (OATP) 2B1 abundance significantly decreased from T1 to T2 by 32%. In contrast, organic cation transporter (OCT) 3 and organic anion transporter 4 abundance significantly increased with gestational age (2-fold from T1 to term, 1.6-fold from T2 to term). Serotonin transporter and norepinephrine transporter did not change with gestational age. The abundance of bile salt export pump, multidrug resistance-associated protein 1-5, Na+-taurocholate cotransporting polypeptide, OATP1B1, OATP1B3, OCTN1-2, concentrative nucleoside transporter 1-3, equilibrative nucleoside transporter 2, and multidrug and toxin extrusion 1 could not be quantified. These data can be incorporated into our maternal-fetal physiologically based pharmacokinetic model to predict fetal exposure to drugs that are actively transported across the placenta. SIGNIFICANCE STATEMENT: We quantified the protein abundance of key placental uptake and efflux transporters [organic cation transporter (OCT) 3, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP)] across gestational ages (first trimester, second trimester, and term) using quantitative targeted proteomics. We observed that the protein abundance of P-gp and BCRP decreased, whereas that of OCT3 increased with gestational age. Incorporating the protein abundance determined in this study into maternal-fetal physiologically based pharmacokinetic model can help us better predict fetal drug exposure to substrates of these transporters.

Verification of the Saccharina japonica Translocon Tic20 and its Localization in the Chloroplast Membrane in Diatoms.

Tic20 is an important translocon protein that plays a role in protein transport in the chloroplast. The sequence of Tic20 was determined in the lower brown alga Saccharina japonica. Structural analysis of SjTic20 revealed a noncanonical structure consisting of an N-terminal non-cyanobacterium-originated EF-hand domain (a helix-loop-helix structural domain) and a C-terminal cyanobacterium-originated Tic20 domain. Subcellular localization and transmembrane analysis indicated that SjTic20 featured an "M"-type Nin-Cin-terminal orientation, with four transmembrane domains in the innermost membrane of the chloroplast in the microalga Phaeodactylum tricornutum, and the EF-hand domain was entirely extruded into the chloroplast stroma. Our study provides information on the structure, localization, and topological features of SjTic20, and further functional analysis of SjTic20 in S. japonica is needed.

Anti-biofilm and antimicrobial effects of zerumbone against Bacteroides fragilis.

Enterotoxigenic Bacteroides fragilis (ETBF) can form biofilms in the colon mucosal membrane and may promote chronic infection and tumor formation. The objective of this study was to determine the effect of zerumbone extracted from Zingiber zerumbet (L.) Smith, on B. fragilis biofilm formation. Inhibitory effects of zerumbone on planktonic cell growth and biofilm formation were examined by crystal-violet biofilm assays and XTT metabolic assays. Results showed that zerumbone significantly inhibited biofilm formation and eradicated established biofilms. Furthermore, B. fragilis biofilms inhibited by zerumbone were observed by confocal laser scanning microscopy. qRT-PCR was used to support the phenotypic results and to investigate the expression levels of an efflux pump-related gene (bmeB). Specifically, among the efflux pump-related genes, zerumbone significantly suppressed the expression level of bmeB12. These results indicate that zerumbone might be a promising candidate as an anti-biofilm and antimicrobial agent to treat and prevent biofilm-related infections caused by B. fragilis.

Identification of 3q oncogene SEC62 as a marker for distant metastasis and poor clinical outcome in invasive ductal breast cancer.

PURPOSE: In previous studies, we have shown that SEC62 has an essential function in cell migration, epithelial-to-mesenchymal transition, and endoplasmic reticulum stress tolerance of cancer cells. SEC62 expression correlated with distant and lymph node metastasis and poor outcome in different cancer entities. In this initial study, we investigated SEC62 expression and its possible role as a prognostic and predictive biomarker in breast cancer (BC). METHODS: Formalin-fixed, paraffin-embedded tissue samples of 53 BC patients were analyzed by immunohistochemistry. The immunoreactive score (IRS) according to Remmele and Stegner was evaluated and correlated with clinico-pathological findings and overall survival (OS). RESULTS: We found increased SEC62 protein levels in tumor tissue compared to tumor-free tissue samples from the same patients. Tumors with high SEC62 expression (IRS > 8), or containing isolated cells with high SEC62 staining intensity, independent of the IRS, had more frequently distant metastases (48.4% vs. 18.2%; p = 0.024 and 47.4 vs. 6.7%; p = 0.005, respectively). Overall survival was significantly worse in BC patients with high SEC62 expression (SEC62 IRS > 8) (54.8% vs. 81.8%; p = 0.011) and in cases with isolated high-intensity SEC62 staining cells independently of SEC62 IRS (55.3% vs 93.3%; p = 0.024). CONCLUSIONS: We are the first to describe the SEC62 expression and its correlation to clinicopathological parameters in mammary carcinoma. Our results suggest that SEC62 expression may serve as a prognostic marker for patients with invasive ductal breast cancer.

SOX10, GATA3, GCDFP15, Androgen Receptor, and Mammaglobin for the Differential Diagnosis Between Triple-negative Breast Cancer and TTF1-negative Lung Adenocarcinoma.

Triple-negative breast cancer (TNBC) patients have an increased risk of developing visceral metastases and other primary nonbreast cancers, particularly lung cancer. The differential diagnosis of TNBC metastases and primary cancers from other organs can be difficult due to lack of a TNBC standard immunoprofile. We analyzed the diagnostic value of estrogen receptor, progesterone receptor, human epidermal growth factor receptor, thyroid transcription factor-1 (TTF1), Napsin A, mammaglobin, gross cystic disease fluid protein 15 (GCDFP15), Sry-related HMg-Box gene 10 (SOX10), GATA-binding protein 3 (GATA3), and androgen receptor in a series of 207 TNBC and 152 primary lung adenocarcinomas (LA). All tested TNBCs were TTF1 and Napsin A-negative. When comparing TNBC and TTF1-positive or negative LA, SOX10 had the best sensitivity (62.3%) and specificity (100%) as a marker in favor of TNBC compared with LA, irrespective of TTF1 status (P<0.0001). GATA3 had moderate sensitivity (30.4%) and excellent specificity (98.7%) and misclassified only 2/152 LA (1.3%). GCDFP15 had a moderate sensitivity (20.8%) and excellent specificity (98%) and misclassified only 3/152 (2%) LA. Mammaglobin and androgen receptor had moderate sensitivities (38.2% and 30%), good specificities (81.6% and 86%), and misclassified 28/152 and 21/152 LAs, respectively. In multivariate analysis, the best markers, enabling the distinction between SOX10-negative TNBC and TTF1 and Napsin A-negative LA were GATA3 (odds ratio=33.5; 95% confidence interval, 7.3-153.5; P<0.0001) and GCDFP15 (odds ratio=31.7; 95% confidence interval, 6.9-145.6; P<0.0001). Only 13/207 (6.3%) TNBC cases did not express any aforementioned marker. On the basis of our results, the best sequential immunohistochemical analysis to differentiate TNBC from TTF1-negative LA is first SOX10 followed by GATA3, and finally GCDFP15. This order is important in the diagnostic workup of small biopsies from lung nodules in women with a previous history of TNBC.

Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.

Tubulointerstitial inflammation plays a critical role in the progression of diabetic nephropathy (DN), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes contribute to renal interstitial inflammation in DN. Decreased expression of optineurin (OPTN) is also associated with the progression of DN. We investigated the role of OPTN in activation of the NLRP3 inflammasome in DN. We initially examined the renal biopsy tissues of 172 patients with type 2 DN and 32 nondiabetic patients with renal hamartoma. Expression of renal OPTN was significantly lower in patients with DN and negatively correlated with urinary levels of IL-1ß and IL-18. Confocal microscopy analysis of the biopsies indicated no NLRP3 or IL-1ß staining in OPTN-positive renal tubular epithelial cells (RTECs), indicating a negative correlation of OPTN expression with the activation of NLRP3 inflammasome. In vitro studies of murine RTECs indicated the levels of OPTN mRNA and protein decreased significantly after stimulation by a high glucose (HG) treatment. Relative to RTECs given HG, RTECs overexpressing OPTN showed significantly lower levels of NLRP3 expression, cleavage of caspase-1 and IL-1ß, and release of IL-1 ß and IL-18. Overexpression of OPTN in the presence of HG significantly increased the costaining of microtubule-associated protein 1A/1B-light chain 3-II and translocase of outer mitochondrial membrane 20 in RTECs, suggesting that OPTN enhances mitophagy. In addition, mitochondrial division inhibitor 1 blocked the inhibitory effect of OPTN overexpression on the activation of NLRP3 inflammasome in the presence of HG, indicating that OPTN overexpression inhibited NLRP3 inflammasome activation by enhancement of mitophagy. OPTN gene silencing significantly enhanced production of mitochondrial reactive oxygen species (mtROS) in the presence of HG. Compared with HG+OPTN small interfering RNA (siRNA)-treated RTECs, HG+OPTN siRNA+MitoTempo-treated RTECs attenuated NLRP3 inflammasome activation, suggesting that OPTN gene silencing activates the NLRP3 inflammasome by increasing mtROS in HG-treated RTECs. Taken together, our results demonstrate that OPTN inhibits the activation of NLRP3 inflammasome by enhancing mitophagy.-Chen, K., Feng, L., Hu, W., Chen, J., Wang, X., Wang, L., He, Y. Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.

Adipocytes fuel gastric cancer omental metastasis via PITPNC1-mediated fatty acid metabolic reprogramming.

Omental metastasis occurs frequently in gastric cancer (GC) and is considered one of the major causes of gastric cancer-related mortality. Recent research indicated that omental adipocytes might mediate this metastatic predilection. Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) was identified to have a crucial role in metastasis. However, whether PITPNC1 participates in the interaction between adipocytes and GC omental metastasis is unclear. Methods: We profiled and analyzed the expression of PITPNC1 through analysis of the TCGA database as well as immunohistochemistry staining using matched GC tissues, adjacent normal gastric mucosa tissues (ANTs), and omental metastatic tissues. The regulation of PITPNC1 by adipocytes was explored by co-culture systems. By using both PITPNC1 overexpression and silencing methods, the role of PITPNC1 in anoikis resistance and metastasis was determined through in vitro and in vivo experiments. Results: PITPNC1 was expressed at higher rates in GC tissues than in ANTs; notably, it was higher in omental metastatic lesions. Elevated expression of PITPNC1 predicted higher rates of omental metastasis and a poor prognosis. PITPNC1 promoted anoikis resistance through fatty acid metabolism by upregulating CD36 and CPT1B expression. Further, PITPNC1 was elevated by adipocytes and facilitated GC omental metastasis. Lastly, in vivo studies showed that PITPNC1 was a therapeutic indicator of fatty acid oxidation (FAO) inhibition. Conclusion: Elevated expression of PITPNC1 in GC is correlated with an advanced clinical stage and a poor prognosis. PITPNC1 promotes anoikis resistance through enhanced FAO, which is regulated by omental adipocytes and consequently facilitates GC omental metastasis. Targeting PITPNC1 might present a promising strategy to treat omental metastasis.

Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies.

Subcellular fractionation of tissue homogenate provides enriched in vitro models (e.g., microsomes, cytosol, or membranes), which are routinely used in the drug metabolism or transporter activity and protein abundance studies. However, batch-to-batch or interlaboratory variability in the recovery, enrichment, and purity of the subcellular fractions can affect performance of in vitro models leading to inaccurate in vitro to in vivo extrapolation (IVIVE) of drug clearance. To evaluate the quality of subcellular fractions, we developed a simple, targeted, and sensitive LC-MS/MS proteomics-based strategy, which relies on determination of protein markers of various cellular organelles, i.e., plasma membrane, cytosol, nuclei, mitochondria, endoplasmic reticulum (ER), lysosomes, peroxisomes, cytoskeleton, and exosomes. Application of the quantitative proteomics method confirmed a significant effect of processing variables (i.e., homogenization method and centrifugation speed) on the recovery, enrichment, and purity of isolated proteins in microsomes and cytosol. Particularly, markers of endoplasmic reticulum lumen and mitochondrial lumen were enriched in the cytosolic fractions as a result of their release during homogenization. Similarly, the relative recovery and composition of the total membrane fraction isolated from cell vs tissue samples was quantitatively different and should be considered in IVIVE. Further, analysis of exosomes isolated from sandwich-cultured hepatocyte media showed the effect of culture duration on compositions of purified exosomes. Therefore, the quantitative proteomics-based strategy developed here can be applied for efficient and simultaneous determination of multiple protein markers of various cellular organelles when compared to antibody- or activity-based assays and can be used for quality control of subcellular fractionation procedures including in vitro model development for drug metabolism and transport studies.

Alteration of transporter activities in the epididymides of infertile initial segment-specific Pten knockout mice.

A fully functional initial segment, the most proximal region of the epididymis, is important for male fertility. Our previous study generated a mouse model to investigate the importance of initial segment function in male fertility. In that model, phosphatase and tensin homolog (Pten) was conditionally removed from the initial segment epithelium, which resulted in epithelial de-differentiation. When spermatozoa progressed through the de-differentiated epithelial duct, they developed angled flagella, suggesting compromised sperm maturation, which eventually resulted in male infertility. To understand the molecular mechanisms, by which PTEN regulates epididymal sperm maturation, we compared the transcriptome profile of the initial segment between controls and initial segment-specific Pten knockouts and revealed that water, ion, and organic solute transporter activities were one of the top molecular and cellular functions altered following loss of Pten. Alteration in protein levels and localization of several transporters following loss of Pten were also observed by immunofluorescence analysis. Epithelial cells of the initial segment from knockouts were more permeable to fluorescein isothiocyanate-dextran (4000 Da) compared to controls. Interestingly, conditional deletion of Pten from other organs also resulted in changes in transporter activity, suggesting a common role of PTEN in regulation of transporter activity. Taken together, our data support the hypothesis that loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, which in turn altered the luminal fluid microenvironment that is so important for sperm maturation and male fertility.

Quantification of membrane transporter proteins in human lung and immortalized cell lines using targeted quantitative proteomic analysis by isotope dilution nanoLC-MS/MS.

Information is needed on the expression of transporters in lung to inform drug development and therapeutic decisions. Much of the information currently available is from semiquantitative gene expression or immunometric densitometry studies reported in the literature. NanoLC-MS/MS (MRM mode) isotope dilution targeted quantitative proteomics was used here to quantify twelve selected transporters in fresh human lung membrane fraction samples and in the membrane fraction of selected immortalized human lung epithelial cell line samples. Fractionation was undertaken by homogenization in crude membrane lysis buffer followed by differential centrifugation of the homogenate. In lung membranes we found OATPs to be the most highly expressed transporters of those measured, followed by PEPT2 and ABCs (P-gp & BCRP). SLC22A transporters (OCTs 2 & 3 and OCTN1) were also found to be expressed. OATP2A1, also known as the prostaglandin transporter, was the most highly expressed transporter, being low in two subjects who were at least occasional smokers. One subject, a non-smoker, had an OATP2A1 concentration that was 8.4 times higher than the next nearest concentration, which itself was higher than the concentration of any other transporter. OATP2A1 is known, from gene expression and animal functional studies, to be present in lung. These results inform the understanding of xenobiotic disposition in the lung and show the distinct profile of transporters in lung compared to other tissues.

Tobacco and Antiretrovirals Modulate Transporter, Metabolic Enzyme, and Antioxidant Enzyme Expression and Function in Polarized Macrophages.

BACKGROUND: Cigarette smoking increases systemic oxidative stress, inflammation, and viral replication in individuals with HIV. Macrophages are infected during HIV infection and serve as an important reservoir throughout the process. Macrophages exist in two phenotypes, the classically activated M1 macrophage and alternatively activated M2 macrophage. The expression of drug efflux transporters and metabolic enzymes, which have direct effects on intracellular drug concentrations, differ between the pro-inflammatory M1 macrophage and the anti-inflammatory M2 macrophage. OBJECTIVE: To further explain the role of tobacco use in worsened outcomes in the HIV + population receiving antiretroviral therapy. METHODS: Western blotting was used to examine macrophage polarization and expression of drug efflux transporters, CYP enzymes, and antioxidant enzymes. The arginase assay was used to measure arginase activity. Cytokine production was measured using the human multiplex inflammatory cytokine assay kit. The 8-OHdG DNA Damage Quantification Direct Kit was used to quantify DNA damage. Viral replication under the influence of tobacco and antiretroviral drug use was measured by p24 Elisa. RESULTS: We observed phenotypic shifts from M1 to M2 with both individual and combination treatments with cigarette smoke condensate and the protease inhibitor antiretroviral drug lopinavir. These shifts lead to changes in cytokine production, the expression of CYP enzymes, anti-oxidant enzymes, and drug efflux transporters, as well as changes in viral replication. CONCLUSION: This data suggest a mechanism by which tobacco use impairs HIV antiretroviral therapy to increase intracellular drug concentrations in this important cellular reservoir.

Pendrin Expression in Preeclampsia: A Prospective Immunohistochemical Staining Study on Placental Bed Biopsies.

INTRODUCTION: To assess the pendrin expression density in placental bed biopsies from preeclampsia cases in comparison with healthy term controls. MATERIAL AND METHODS: A prospective case-control study with 106 placental bed biopsies obtained during cesarean deliveries. Pendrin expression was evaluated by immunohistochemical staining in different hypertensive disorders of pregnancy. RESULTS: Pendrin immunostaining frequency was higher in the hypertensive disorders group (p: 0.024), which was a result of the high frequency in the early-onset preeclampsia group. Uterine artery pulsatility indices were higher in pendrin positive patients than in the negatives in the case group. Gravidity was not found to affect the pendrin expression frequency in the placental bed. CONCLUSION: Placental ischemia seems to be an important determinant of pendrin expression in pregnant decidua. Increased pendrin density in early-onset preeclampsia could be a pathogenetic mechanism in or a part of the adaptational response to the development of the hypertension.

Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells.

Quantitative proteomics, using LC-MS/MS, is increasingly used to quantify drug transporters present in tissues and cells. Most of these investigations quantify total transporter expression in the cells by utilizing a total membrane fraction, not only the plasma membrane. Here, we report development and optimization of a biotinylation method to quantify protein expression of transporters in the plasma membrane of cells. The Pierce cell surface isolation protocol was optimized for plasma membrane isolation. Incubation of OATP1B1-expressing CHO cells with 0.78 mg/mL of membrane impermeable biotinylation reagent (sulfo-NHS-SS-biotin) at 37°C for 1 h resulted in optimum isolation of the plasma membrane. Subsequently, the expression of transporters in the plasma membrane as a percent of the total was determined by quantitative proteomics using LC-MS/MS. Mean (±SD) plasma membrane expression of OATP1B1 in plated OATP1B1-expressing CHO, MDCKII, and HEK293 cells was found to be 79.7% (±4.7%), 67.7% (±12.2%), and 65.3% (±6.8%) of total cell OATP1B1 expression. Mean (±SD) plasma membrane expression of OATP1B3 in plated OATP1B3-expressing HEK293 cells, OATP2B1 in plated OATP2B1-expressing MDCKII cells, and sodium/taurocholate co-transporting polypeptide (NTCP) in plated NTCP-expressing CHO cells was 63.2% (±1.6%), 37.1% (±15.7%), and 71.7% (±1.2%), respectively. This method of quantifying transporter protein expression in the plasma membrane will be useful in the future to predict transporter-mediated drug disposition.

Sorting receptor SORLA: cellular mechanisms and implications for disease.

Sorting-related receptor with A-type repeats (SORLA) is an intracellular sorting receptor that directs cargo proteins, such as kinases, phosphatases, and signaling receptors, to their correct location within the cell. The activity of SORLA assures proper function of cells and tissues, and receptor dysfunction is the underlying cause of common human malignancies, including Alzheimer's disease, atherosclerosis, and obesity. Here, we discuss the molecular mechanisms that govern sorting of SORLA and its cargo in multiple cell types, and why genetic defects in this receptor results in devastating diseases.