GSPE attenuates CSE-induced lung inflammation and emphysema by regulating autophagy via the reactive oxygen species/TFEB signaling pathway.

Cigarette smoke can enhance reactive oxygen species (ROS) production in inflammatory and epithelial cells. Subsequently, ROS enhance autophagy-induced inflammation due to alveolar macrophages (AMs), the primary source of cytokines implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Therefore, we hypothesized that grape seed proanthocyanidin extract (GSPE), an effective antioxidant, could inhibit emphysema and airway inflammation by ameliorating cigarette smoke extract (CSE)-induced autophagy via suppressing oxidative stress in macrophages. We observed that GSPE significantly attenuated histological changes observed in CSE-induced emphysema and airway inflammation in the lungs of mice. Moreover, GSPE ameliorated lung inflammation by reducing the number of cells, macrophages, and neutrophils and the tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels measured in bronchioloalveolar lavage fluid. ROS levels increased after CSE instillation and significantly decreased with in vitro GSPE treatment. GSPE decreased transcription factor EB (TFEB) oxidation by reducing ROS, inhibiting TFEB nuclear translocation. Furthermore, GSPE inhibited ROS-induced autophagy in RAW 264.7 cells, bone marrow-derived macrophages, and AMs. Inhibiting autophagy through GSPE treatment diminishes CSE-induced lung inflammation by inhibiting the NLRP3 inflammasome. This study demonstrates that GSPE can ameliorate CSE-induced inflammation and emphysema via autophagy-induced NLRP3 inflammasome regulation through the ROS/TFEB signaling pathway in a COPD mouse model.

Impact of Postural Changes on Sinonasal Pressure After Endoscopic Endonasal Surgery.

BACKGROUND: Postoperative management following endoscopic endonasal surgery (EES) is important to prevent cerebrospinal fluid leak and preserve the integrity of the nasoseptal flap. No consensus regarding an optimal posture in the postoperative period has been established. We hypothesized that sinonasal pressure (SNP) can represent intracranial pressure affecting the sellar floor in the absence of the sellar bone after surgery. This study provides evidence for the effect of postural changes and recommends optimal posture to reduce SNP following EES. METHODS: The authors conducted a retrospective analysis of 50 patients who underwent reconstruction for skull base defects with nasoseptal flap after EES for resection of suprasellar tumor between March 2020 and August 2020. The Spiegelberg intracranial pressure probe was placed through the nostril over the nasoseptal flap. SNPs were measured in Fowler' (45° tilt) and supine positions, respectively, daily for the first 3 days immediately after EES. RESULTS: For the first 3 days after surgery, the mean SNP in Fowler' position (24.82 mmHg; standard deviation, 12.23 mmHg) was lower than that in the supine position (28.42 mmHg; standard deviation, 12.33 mmHg) (P < 0.01). There were no significant differences in mean SNP for age, sex, tumor size, presence of hydrocephalus, and body mass index. CONCLUSIONS: There was a significant correlation between Fowler' position and a decrease in SNP measurements. Placing a patient in Fowler' position after surgery can decrease the SNP. We recommend that patients should be placed in a Fowler' position as an optimal position after surgery.

Efficient brazzein production in yeast (Kluyveromyces lactis) using a chemically defined medium.

The sweet-tasting protein brazzein offers considerable potential as a functional sweetener with antioxidant, anti-inflammatory, and anti-allergic properties. Here, we optimized a chemically defined medium to produce secretory recombinant brazzein in Kluyveromyces lactis, with applications in mass production. Compositions of defined media were investigated for two phases of fermentation: the first phase for cell growth, and the second for maximum brazzein secretory production. Secretory brazzein expressed in the optimized defined medium exhibited higher purity than in the complex medium; purification was by ultrafiltration using a molecular weight cutoff, yielding approximately 107 mg L-1. Moreover, the total media cost in this defined medium system was approximately 11% of that in the optimized complex medium to generate equal amounts of brazzein. Therefore, the K. lactis expression system is useful for mass-producing recombinant brazzein with high purity and yield at low production cost and indicates a promising potential for applications in the food industry.

Stable perovskite solar cells with efficiency exceeding 24.8% and 0.3-V voltage loss.

Further improvement and stabilization of perovskite solar cell (PSC) performance are essential to achieve the commercial viability of next-generation photovoltaics. Considering the benefits of fluorination to conjugated materials for energy levels, hydrophobicity, and noncovalent interactions, two fluorinated isomeric analogs of the well-known hole-transporting material (HTM) Spiro-OMeTAD are developed and used as HTMs in PSCs. The structure-property relationship induced by constitutional isomerism is investigated through experimental, atomistic, and theoretical analyses, and the fabricated PSCs feature high efficiency up to 24.82% (certified at 24.64% with 0.3-volt voltage loss), along with long-term stability in wet conditions without encapsulation (87% efficiency retention after 500 hours). We also achieve an efficiency of 22.31% in the large-area cell.

Histidine residues at the copper-binding site in human tyrosinase are essential for its catalytic activities.

Tyrosinase is a copper-binding enzyme involved in melanin biosynthesis. However, the detailed structure of human tyrosinase has not yet been solved, along with the identification of the key sites responsible for its catalytic activity. We used site-directed mutagenesis to identify the residues critical for the copper binding of human tyrosinase. Seven histidine mutants in the two copper-binding sites were generated, and catalytic activities were characterised. The tyrosine hydroxylase activities of the CuA site mutants were approximately 50% lower than those of the wild-type tyrosinase, while the dopa oxidation activities of the mutants were not significantly different from that of wild-type tyrosinase. By contrast, mutations at CuB significantly decreased both tyrosine hydroxylation and dopa oxidation activities, confirming that the catalytic sites for these two activities are at least partially distinct. These findings provide a useful resource for further structural determination and development of tyrosinase inhibitors in the cosmetic and pharmaceutical industries.

Quantitative image analysis using chest computed tomography in the evaluation of lymph node involvement in pulmonary sarcoidosis and tuberculosis.

PURPOSE: To evaluate the feasibility of quantitative analysis of chest computed tomography (CT) scans for the assessment of lymph node (LN) involvement in patients with pulmonary tuberculosis and sarcoidosis. METHODS: In 47 patients with tuberculosis (n = 26) or sarcoidosis (n = 21), 115 lymph nodes (tuberculous, 55; sarcoid, 60) were visually analyzed on chest CT scans according to their size, location, attenuation and shape. Each node was manually segmented using image analysis tool, which was quantitatively analyzed using the following variables: Feret's diameter, perimeter, area, circularity, mean grey value (Mean), standard deviation (SD) of grey value, minimum grey value (Min), maximum grey value (Max), median grey value (Median), skewness, kurtosis, and net enhancement. We statistically analyzed the visual and quantitative CT features of tuberculous and sarcoid LNs. RESULTS: In visual CT analysis, the mean node size in sarcoidosis was significantly greater than that in tuberculosis. There were no statistical differences between tuberculous and sarcoid LNs in terms of location and shape. Central low attenuation and peripheral rim enhancement were more frequently observed in tuberculous LNs than in the sarcoid ones. In quantitative CT analysis, there were significant differences in the values of the Feret's diameter, perimeter, area, circularity, mean grey value, SD, median, skewness, and kurtosis between tuberculous and sarcoid LNs. CONCLUSIONS: Quantitative CT analysis using CT parameters with pixel-by-pixel measurements can help to differentiate of tuberculous and sarcoid LNs.

Abnormal Human Sperm Parameters Contribute to Sperm DNA Fragmentation in Men with Varicocele.

PURPOSE: This study was performed to evaluate and compare threshold sperm parameters and sperm DNA fragmentation index (DFI), and further analyzed whether sperm DFI could be predicted from sperm parameters in men with varicocele. MATERIALS AND METHODS: A total of 157 semen samples underwent both semen analysis and sperm DNA fragmentation (SDF) testing in men with varicocele. Sperm parameters were assessed using the World Health Organization guidelines. SDF testing was performed using the Halosperm kit. Sperm parameters and sperm DFI results were compared. RESULTS: The overall sperm parameter results and sperm DFI showed normal values; however, the morphology value was at the lower limit of normal. High sperm DFI was associated with significantly lower motility and viability (p<0.001, respectively). Sperm motility and morphology were significantly higher in the higher sperm count group compared to the lower sperm count group (p<0.05), while sperm DFI was higher in the lower sperm count group (p<0.05). Sperm count and viability and sperm DFI were significantly associated with the quality of sperm motility (p<0.001). Sperm motility and sperm DFI were significantly different (p<0.001) between normal and abnormal sperm viability groups. Between normal and abnormal sperm morphology groups, sperm count, motility, and sperm DFI showed significant differences (p<0.001). CONCLUSIONS: In this study, a correlation between SDF and sperm parameters was confirmed in men with varicocele. SDF may be contributing factors to sperm motility, viability, and morphology. Abnormal sperm count, motility, and viability showed high sperm DFI. Therefore, lower sperm parameters were indicative of increasing SDF in men with varicocele.

Early Prediction of the Severity of Acute Pancreatitis Using Radiologic and Clinical Scoring Systems With Classification Tree Analysis.

OBJECTIVE: The objective of our study was to develop a decision tree model for the early prediction of the severity of acute pancreatitis (AP) using clinical and radiologic scoring systems. MATERIALS AND METHODS: For this retrospective study, 192 patients with AP who underwent CT 72 hours or less after symptom onset were divided into two cohorts: a training cohort (n = 115) and a validation cohort (n = 77). Univariate analysis was performed to identify significant parameters for the prediction of severe AP in the training cohort. For early prediction of disease severity, a classification tree analysis (CTA) model was constructed using significant scoring systems shown by univariate analysis. To assess the diagnostic performance of the model, we compared the area under the ROC curve (AUC) with each selected single parameter. We also evaluated the diagnostic performance in the validation cohort. RESULTS: The Acute Physiology and Chronic Health Evaluation (APACHE)-II score, bedside index for severity in acute pancreatitis (BISAP) score, extrapancreatic inflammation on CT (EPIC) score, and Balthazar grade were included in the CTA model. In the training cohort, our CTA model showed a trend of a higher AUC (0.853) than the AUC of each single parameter (APACHE-II score, 0.835; BISAP score, 0.842; EPIC score, 0.739; Balthazar grade, 0.700) (all, p > 0.0125) while achieving specificity (100%) higher than and accuracy (94.8%) comparable to each single parameter (both, p < 0.0125). In the validation cohort, the CTA model achieved diagnostic performance similar to the training cohort with an AUC of 0.833. CONCLUSION: Our CTA model consisted of clinical (i.e., APACHE-II and BISAP scores) and radiologic (i.e., Balthazar grade and EPIC score) scoring systems and may be useful for the early prediction of the severity of AP and identification of high-risk patients who require close surveillance.

Radiologic Findings in Extrapancreatic Solid Pseudopapillary Tumor with Aggressive Behavior: a Case Report.

Solid pseudopapillary tumor (SPT) is a low grade malignant tumor in the pancreas, and extrapancreatic SPT is extremely rare. We report a case of a 61-year-old woman who complained abdominal pain with diffuse tenderness. She was diagnosed with extrapancreatic SPT with extensive peritoneal dissemination and hepatic metastases. Although a few cases have reported imaging findings of extrapancreatic SPT, there have been no reports of extrapancreatic SPT with aggressive tumor behavior and dismal prognosis. Although imaging features closely resembled those of classical pancreatic SPTs, malignant transformation of extrapancreatic SPT should be considered when focal discontinuity of the tumor capsule with ill-defined margin and invasion of adjacent structures were identified.

Natural course of the nodular bronchiectatic form of Mycobacterium Avium complex lung disease: Long-term radiologic change without treatment.

BACKGROUND/PURPOSE: Although the incidence of Mycobacterium avium complex (MAC) lung disease is increasing, the long-term natural course of the nodular bronchiectatic form of MAC lung disease is not well described. The objective of our study is to evaluate long-term radiologic changes in untreated MAC lung disease by analyzing serial chest computed tomography (CT) scan findings. METHODS: Of 104 patients with MAC lung disease, we selected 40 untreated nodular bronchiectatic MAC patients who underwent serial chest CTs without treatment for at least four years (mean = 6.23 years). Majority of patients have minimal symptoms. Two chest radiologists retrospectively reviewed initial and final chest CT scans. Each chest CT scan was scored for presence and extent of bronchiectasis, cellular bronchiolitis, consolidation, cavity, and nodule (maximum score: 30). RESULTS: Of 40 patients, 39 (97.5%) experienced a significant increase in overall CT score (overall difference = 4.89, p<0.001). On repeated measure analysis of variance analysis, cavity yielded the largest increase compared with cellular bronchiolitis (p = 0.013), nodule (p<0.001), and consolidation (p = 0.004). However, there was no significant difference in mean score change between cavity and bronchiectasis (p = 0.073). In analysis between radiologic parameters and the absolute number of involved segments, bronchiectasis showed most significant change compared with nodule (p<0.001) and consolidation (p<0.001). CONCLUSIONS: Most untreated nodular bronchiectatic MAC lung disease cases showed radiologic deterioration over long-term observation periods when we compared serial chest CT scans. Careful monitoring of MAC lung disease with serial chest CT scan can be beneficial in these untreated patients.

Pulmonary cryptococcosis manifesting as diffuse air-space consolidations in an immunocompetent patient.

Pulmonary cryptococcosis is an opportunity infection commonly occurred in the immunocompromised patients. However pulmonary cryptococcosis in the immunocompetent was reported up to 35% and these cases tend to show confined and localized radiologic findings than in immunocompromised patients. To our knowledge, extensive air-space consolidations have not frequently occurred in the immunocompetent patient. Therefore, in this case, we report a rare case of a 73-year-old woman who was diagnosed with pulmonary cryptococcosis, manifesting as diffuse air-space consolidations even though normal immune status. Thus, the possibility of pulmonary cryptococcosis should be considered when a patient with a normal immune status presents without respiratory symptoms are accompanied by consolidation on imaging.

Effects of maternal age on embryo quality and pregnancy outcomes using testicular sperm with intracytoplasmic sperm injection.

OBJECTIVE: The aim of this study was to evaluate the influence of maternal age on fertilization, embryo quality, and clinical pregnancy in patients undergoing intracytoplasmic sperm injection (ICSI) using testicular sperm from partners with azoospermia. METHODS: A total of 416 ICSI cycles using testicular spermatozoa from partners with obstructive azoospermia (OA, n=301) and non-obstructive azoospermia (NOA, n=115) were analyzed. Female patients were divided into the following age groups: 27 to 31 years, 32 to 36 years, and 37 to 41 years. The rates of fertilization, high-quality embryos, clinical pregnancy, and delivery were compared across maternal age groups between the OA and NOA groups. RESULTS: The rates of fertilization and high-quality embryos were not significantly different among the maternal age groups. Similarly, the clinical pregnancy and delivery rates were not significantly different. The fertilization rate was significantly higher in the OA group than in the NOA group (p<0.05). Age-group analysis revealed that the fertilization and high-quality embryo rates were significantly different between the OA and NOA groups in patients aged 27 to 31 years old, but not for the other age groups. Although the clinical pregnancy and delivery rates differed between the OA and NOA groups across all age groups, significant differences were not observed. CONCLUSION: In couples using testicular sperm from male partners with azoospermia, pregnancy and delivery outcomes were not affected by maternal age. However, women older than 37 years using testicular sperm from partners with azoospermia should be advised of the increased incidence of pregnancy failure.

Molecular and cytogenetic studies of 101 infertile men with microdeletions of Y chromosome in 1,306 infertile Korean men.

OBJECTIVES: To determine the prevalence of Y chromosome microdeletions in infertile Korean men with abnormal sperm counts and to assess the clinical features and frequency of chromosomal abnormalities in Korean patients with microdeletions. METHODS: A total of 1,306 infertile men were screened for Y chromosome microdeletions, and 101 of them had microdeletions. These 101 men were then retrospectively studied for cytogenetic evaluation, testicular biopsy and outcomes of IVF and ICSI. RESULTS: The overall prevalence of Y chromosome microdeletions in infertile men was 7.7% (101/1,306). Most microdeletions were in the AZFc region (87.1%), including deletions of AZFbc (24.7%) and AZFabc (8.9%). All patients with AZFa, AZFbc and AZFabc deletions had azoospermia, whereas patients with an AZFc deletion usually had low levels of sperm in the ejaculate or in the testis tissues. Chromosomal studies were performed in 99 men with microdeletions, 36 (36.4%) of whom had chromosomal abnormalities. Among the infertile men with Y chromosome microdeletions in this study, the incidence of chromosomal abnormality was 48.6% in the azoospermic group and 3.7% in the oligozoospermic group. Among the 69 patients with microdeletions and available histological results, 100.0% of the azoospermic group and 85.7% of the oligozoospermic group had histological abnormalities. The frequency of both chromosomal abnormalities and histological abnormalities was higher in the azoospermic group compared to the oligozoospermic group. Thirty-four ICSI cycles with either testicular (n = 14) or ejaculated spermatozoa (n = 20) were performed in 23 couples with men with AZFc microdeletion. Thirteen clinical pregnancies (39.4%) were obtained, leading to the birth of 13 babies. CONCLUSIONS: The study results revealed a close relationship between microdeletions and spermatogenesis, although IVF outcome was not significantly affected by the presence of the AZFc microdeletion. Nevertheless, Y chromosome microdeletions have the potential risk of being transmitted from infertile fathers to their offspring by ICSI. Therefore, before using ICSI in infertile patients with severe spermatogenic defects, careful evaluations of chromosomal abnormalities and Y chromosome microdeletions screening should be performed and genetic counseling should be provided before IVF-ET.

In vitro development and gene expression of frozen-thawed 8-cell stage mouse embryos following slow freezing or vitrification.

OBJECTIVE: This study was performed to compare the efficiency of slow freezing and vitrification based on survival, development to blastocysts, and cell numbers of blastocysts. Changes in embryonic gene expression in fresh and frozen-thawed embryos were also examined. METHODS: Eight-cell stage embryos were collected from superovulated female BDF1 mice. The collected embryos were randomly divided into three groups. One group was maintained as fresh controls (n=42), one was frozen by slow freezing (n=43), and one was cooled by vitrification (n=43). After thawing or cooling, survival rates, development to blastocyst, and cell numbers and inner cell mass (ICM) cell numbers of blastocysts were compared with those of the control group. The expressions of eight genes (Rbm3, Birc5, Sod1, Sod2, Cirbp, Caspase3, Trp53, Hsp70.1) were examined by real time-quantitative polymerase chain reaction in the fresh and frozen-thawed embryos. RESULTS: There were no significant differences in the slow freezing and vitrification groups' survival rate after thawing (88.4% vs. 88.4%), development to blastocyst (100% vs. 97.4%), cell numbers (107.0±21.0 vs. 115.0±19.7), or ICM cell numbers of blastocysts (11.3±5.2 vs. 11.1±3.7). Cell numbers of blastocysts were significantly (p<0.05) lower in the frozen-thawed embryos than the fresh embryos. There were no significant differences in the slow freezing and the vitrification groups' expressions of the eight genes. The expressions of CirbP and Hsp70.1 were higher in the frozen-thawed embryos than in the fresh embryos but there were no significant differences. CONCLUSION: These results suggest that there were no significant differences between embryos that underwent slow freezing and vitrification.

Establishment and characterization of mammalian cell lines stably expressing human L-type amino acid transporters.

System L (SL), a basolateral amino acid transporter, transports large neutral amino acids (LNAAs) in a Na(+)-independent manner. Previously, we identified two isoforms of transporters: L-type amino acid transporter 1 (LAT1) and 2 (LAT2) and revealed their distinct substrate selectivity and transport properties. In this study, to establish more stable human LAT1 (hLAT1) and LAT2 (hLAT2) in vitro assay systems, we established mouse cell lines stably expressing hLAT1 (S2-LAT1) and hLAT2 (S2-LAT2). Real-time quantitative RT-PCR analysis revealed that S2-LAT1 and S2-LAT2 cells express hLAT1 and hLAT2 mRNAs at 20 - 1000-fold higher levels than those of endogenous mouse Lat1 and Lat2. S2-LAT1 and S2-LAT2 mediated [(14)C]L-leucine transport properties were measured and corresponded to results observed via Xenopus oocytes. Using these cells, the data demonstrate that hLAT1 and hLAT2 exhibit different characters in the acceptance of alpha-methyl amino acids and amino acid-related compounds with bulky side chains such as thyroid hormones and melphalan. S2-LAT1 and S2-LAT2 cells are expected to facilitate hLAT1 and hLAT2 substrate recognition research and contribute to drug development by providing an efficient assay system to screen for chemical compounds that interact with hLAT1 and hLAT2.

Preimplantation genetic diagnosis for ornithine transcarbamylase deficiency by simultaneous analysis of duplex-nested PCR and fluorescence in situ hybridization: a case report.

Ornithine transcarbamylase (OTC) deficiency is an X-linked co-dominant disorder. A couple, with a previous history of a neonatal death and a therapeutical termination due to OTC deficiency, was referred to our center for preimplantation genetic diagnosis (PGD). The female partner has a nonsense mutation in the exon 9 of the OTC gene (R320X). We carried out nested polymerase chain reaction (PCR) for R320X mutation and fluorescence in situ hybridization (FISH) for aneuploidy screening. Among a total of 11 embryos, two blastomeres per embryo from 9 embryos were biopsied and analyzed by duplex-nested PCR and FISH, and one blastomere per embryo from 2 embryos by only duplex-nested PCR. As a result of PCR and restriction fragment length polymorphism analysis, four embryos were diagnosed as unaffected embryos having the normal OTC gene. Among these embryos, only one embryo was confirmed as euploidy for chromosome X, Y and 18 by FISH analysis. A single normal embryo was transferred to the mother, yielding an unaffected pregnancy and birth of a healthy boy. Based on our results, PCR for mutation loci and FISH for aneuploidy screening with two blastomeres from an embryo could provide higher accuracy for the selection of genetically and chromosomally normal embryos in the PGD for single gene defects.

Identification of a novel single nucleotide polymorphism of HADHA gene at a referred primer-binding site during pre-diagnostic tests for preimplantation genetic diagnosis.

The pre-diagnostic test for preimplantation genetic diagnosis (PGD) of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency was performed by polymerase chain reaction (PCR) and direct sequencing for hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (HADHA) gene. We obtained unexpected genotyping results of HADHA gene by allele drop-out in the analysis of patients' genomic DNA samples with a referred PCR primer set. Upon further analysis with a re-designed primer set, we found a novel single nucleotide polymorphism (SNP) at the referred primer-binding site in the normal allele of HADHA gene (NT_022184, 5233296 a>t). We found that the frequency of this novel SNP was 0.064 in Korean population. Pre-diagnostic test using single lymphocytes and clinical PGD were successfully performed with the re-designed primer set. Nineteen embryos (95.0%) among 20 were successfully diagnosed to 5 homozygous mutated, 8 heterozygous carrier and 6 wild type. Among 6 normal embryos, well developed and selected 4 embryos were transferred into the mother's uterus, but a pregnancy was not achieved. We proposed that an unknown SNP at primer-binding sites would be a major cause of allele drop-out in the PGD for single gene dis-order.

Optimization of real time RT-PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos.

This study was carried out to optimize conditions for using real time RT-PCR as an efficient and precise quantitative method for estimating the transcript levels of genes expressed in samples containing miniscule amounts of RNA, such as single mammalian oocytes and embryos. First, using mouse eggs and blastocysts, we tested three kinds of RNA isolation or collection methods: TRIZOL reagent, oligo-dT conjugated beads, or three freeze/thaw cycles with the reverse transcription buffer. There were no significant differences among three groups in mRNA quantity as assayed by real time RT-PCR analysis. Second, we compared the efficacy of real time analysis between TaqMan fluorescent probes and the SYBR-green dye system. The two systems presented similar real time RT-PCR profiles for the 16s ribosomal protein gene from oocytes to blastocysts. Third, RNA from mouse embryos at defined stages of preimplantation development were isolated and the levels of transcripts encoded by several housekeeping genes (GAPDH, beta-actin, ribosomal protein L7, 16s ribosomal protein, histone H2A.Z) were quantitatively analyzed by real time RT-PCR. The histone H2A.Z and 16s ribosomal protein slightly increased from the egg to blastocyst stages by approximately 10- and 30-fold, respectively. However, other transcripts increased more than 300-fold as a function of developmental stage from eggs to blastocysts. Our results suggest that the simple freezing/thawing method for RNA collection, the economic SYBR-green dye system, and histone H2A.Z gene as an internal control should be useful for the real time RT-PCR analysis of single mouse eggs and preimplantation embryos.

Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells.

System L is a major nutrient transport system responsible for the Na(+)-independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L-type amino acid transporter 1 (LAT1) is up-regulated to support tumor cell growth. LAT1 is also essential for the permeation of amino acids and amino acid-related drugs through the blood-brain barrier. To search for in vitro assay systems to examine the interaction of chemical compounds with LAT1, we have investigated the expression of system L transporters and the properties of [14C]L-leucine transport in T24 human bladder carcinoma cells. Northern blot, real-time quantitative PCR and immunofluorescence analyses have reveled that T24 cells express LAT1 in the plasma membrane together with its associating protein 4F2hc, whereas T24 cells do not express the other system L isoform LAT2. The uptake of [14C]L-leucine by T24 cells is Na(+)-independent and almost completely inhibited by system L selective inhibitor BCH. The profiles of the inhibition of [14C]L-leucine uptake by amino acids and amino acid-related compounds in T24 cells are comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [14C]L-leucine uptake is, therefore, mediated by LAT1 in T24 cells. Consistent with LAT1 in Xenopus oocytes, the efflux of preloaded [14C]L-leucine is induced by extracellularly applied substrates of LAT1 in T24 cells. This efflux measurement has been proven to be more sensitive than that in Xenopus oocytes, because triiodothyronine, thyroxine and melphalan were able to induce the efflux of preloaded [14C]L-leucine in T24 cells, which was not detected for Xenopus oocyte expression system. T24 cell is, therefore, proposed to be an excellent tool to examine the interaction of chemical compounds with LAT1.

Identification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of a heterodimeric amino acid transporter family associated with unknown heavy chains.

We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.