OCTN2-mediated transport of carnitine in isolated Sertoli cells

Carnitine is extensively accumulated in epididymis. Carnitine is also accumulated in testis at higher concentration than in the plasma and is used in spite of the presence of the blood–testis barrier. In this study, we examined the characteristics of carnitine transport in primary-cultured rat Sertoli cells, which constitute a part of the blood–testis barrier. Uptake of [3H]carnitine (11.4 nM) from the basal side of Sertoli cells was Na+-dependent and was significantly decreased in the presence of 10 μM (48.0 ± 7.4% of control) or 100 μM unlabeled carnitine (14.6 ± 5.7% of control). Furthermore, the uptake was significantly inhibited in the presence of 100 μM acetyl-L-carnitine, 100 μM gamma-butyrobetaine or 500 μM quinidine. In RT-PCR analysis, the high-affinity carnitine transporter OCTN2 was detected in rat whole testis tissue and primary-cultured Sertoli cells. In contrast, the low-affinity carnitine transporter ATB0,+was detected in rat whole testis tissue, but not in primary cultured Sertoli cells. These results demonstrate that OCTN2 mediates carnitine supply to Sertoli cells from the circulation.

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l-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2

AJP Cell Physiology ◽

10.1152/ajpcell.00333.2003 ◽

2004 ◽

Vol 287 (2) ◽

pp. C263-C269 ◽

Cited By ~ 53

Author(s):

Karim Lahjouji ◽

Ihsan Elimrani ◽

Julie Lafond ◽

Line Leduc ◽

Ijaz A. Qureshi ◽

...

Keyword(s):

Brush Border ◽

Membrane Vesicles ◽

Organic Cation Transporter ◽

Plasma Membrane Vesicles ◽

Carnitine Transporter ◽

High Affinity ◽

Basal Plasma ◽

Carnitine Transport ◽

Ph Range ◽

Carnitine Uptake

Maternofetal transport of l-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that l-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent Km = 11.09 ± 1.32 μM; Vmax = 41.75 ± 0.94 pmol·mg protein−1·min−1), and was unchanged over the pH range from 5.5 to 8.5. l-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of l-carnitine, including d-carnitine, acetyl-d,l-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-l-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na+-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for l-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans.

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Expression of glypican-1, syndecan-1 and syndecan-4 mRNAs protein kinase C-regulated in rat immature Sertoli cells by semi-quantitative RT-PCR analysis

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/s0304-4165(00)00006-4 ◽

2000 ◽

Vol 1474 (1) ◽

pp. 31-40 ◽

Cited By ~ 11

Author(s):

S Brucato

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Sertoli Cells ◽

Pcr Analysis ◽

Rt Pcr ◽

Kinase C

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Coxsackievirus and Adenovirus Receptor Is Up-Regulated in Migratory Germ Cells during Passage of the Blood-Testis Barrier

Endocrinology ◽

10.1210/en.2007-0359 ◽

2007 ◽

Vol 148 (11) ◽

pp. 5459-5469 ◽

Cited By ~ 19

Author(s):

Momina Mirza ◽

Cecilia Petersen ◽

Katarina Nordqvist ◽

Kerstin Sollerbrant

Keyword(s):

Tight Junctions ◽

Adhesion Molecule ◽

Germ Cells ◽

Sertoli Cells ◽

Rt Pcr ◽

Adult Testis ◽

Functional Complex ◽

Coxsackievirus And Adenovirus Receptor ◽

Adenovirus Receptor ◽

Blood Testis Barrier

The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule expressed in epithelial tight junctions and other cell-cell contacts. Using indirect immunofluorescence, quantitative RT-PCR, and Western blots, the expression and distribution of CAR in developing and adult testis are examined. CAR is highly expressed in both Sertoli and germ cells during perinatal and postnatal development, followed by a rapid down-regulation of both mRNA and protein levels. Interestingly, we find that CAR is a previously unknown downstream target for FSH because CAR mRNA levels were induced in primary cultures of FSH-stimulated Sertoli cells. In contrast to other epithelia, CAR is not a general component of tight junctions in the seminiferous epithelium, and Sertoli cells in the adult testis do not express CAR. Instead, CAR expression is stage dependent and specifically found in migratory germ cells. RT-PCR also demonstrated the presence of junctional adhesion molecule-like (JAML) in the testis. JAML was previously reported by others to form a functional complex with CAR regulating transepithelial migration of leukocytes. The expression of JAML in the testis suggests that a similar functional complex might be present during germ cell migration across the blood-testis barrier. Finally, an intermediate compartment occupied by CAR-positive, migrating germ cells and flanked by two occludin-containing junctions is identified. Together, these results implicate a function for CAR in testis morphogenesis and in migration of germ cells across the blood-testis barrier during spermatogenesis.

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Carnitine/organic cation transporter OCTN2-mediated transport of carnitine in primary-cultured epididymal epithelial cells

Reproduction ◽

10.1530/rep.1.00737 ◽

2005 ◽

Vol 130 (6) ◽

pp. 931-937 ◽

Cited By ~ 12

Author(s):

Daisuke Kobayashi ◽

Masanori Irokawa ◽

Tomoji Maeda ◽

Akira Tsuji ◽

Ikumi Tamai

Keyword(s):

Epithelial Cells ◽

Organic Cation ◽

Basolateral Membrane ◽

Organic Cation Transporter ◽

Systemic Circulation ◽

Transport Systems ◽

Pcr Analysis ◽

Carnitine Transporter ◽

Carnitine Transport ◽

Cationic Compounds

Carnitine is essential for the acquisition of motility and maturation of spermatozoa in the epididymis, and is accumulated in epididymal fluid. In this study, carnitine transport into primary-cultured rat epididymal epithelial cells was characterized to clarify the nature of the transporter molecules involved. Uptake of carnitine by primary-cultured epididymal epithelial cells was time, Na+and concentration dependent. Kinetic analysis of carnitine uptake by the cells revealed the involvement of high- and low-affinity transport systems withKm values of 21 μM and 2.2 mM respectively. The uptake of carnitine by the cells was significantly reduced by inhibitors of carnitine/organic cation transporter (OCTN2), such as carnitine analogues and cationic compounds. In RT-PCR analysis, OCTN2 expression was detected. These results demonstrated that the high-affinity carnitine transporter OCTN2, which is localized at the basolateral membrane of epididymal epithelial cells, mediates carnitine supply into those cells from the systemic circulation as the first step of permeation from blood to spermatozoa.

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Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Applied Sciences ◽

10.3390/app11135776 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5776

Author(s):

Varvara G. Blinova ◽

Natalia S. Novachly ◽

Sofya N. Gippius ◽

Abdullah Hilal ◽

Yulia A. Gladilina ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Inflammatory Reactions ◽

Effector Cells ◽

Cycle Regulation ◽

Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption

Dentistry Journal ◽

10.3390/dj9010007 ◽

2021 ◽

Vol 9 (1) ◽

pp. 7

Author(s):

Yusuke Makino ◽

Kaoru Fujikawa ◽

Miwako Matsuki-Fukushima ◽

Satoshi Inoue ◽

Masanori Nakamura

Keyword(s):

Bacterial Infections ◽

Tooth Movement ◽

Tooth Eruption ◽

Intercellular Adhesion Molecule ◽

Enamel Organ ◽

Pcr Analysis ◽

Maturation Stage ◽

Oral Epithelium ◽

Rt Pcr ◽

Inflammatory Reactions

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1β, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

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RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)

Journal of General Virology ◽

10.1099/vir.0.81401-0 ◽

2005 ◽

Vol 86 (12) ◽

pp. 3419-3424 ◽

Cited By ~ 210

Author(s):

Constanze Yue ◽

Elke Genersch

Keyword(s):

Varroa Destructor ◽

Pcr Analysis ◽

Body Parts ◽

Rt Pcr ◽

Larval Food ◽

Viral Pathogen ◽

Deformed Wing Virus ◽

Significant Difference ◽

Almost All ◽

Viral Sequences

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

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Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) – ITI target genes in mouse ovary identified by microarray analysis

Journal of Endocrinology ◽

10.1677/joe.1.05803 ◽

2004 ◽

Vol 183 (1) ◽

pp. 29-38 ◽

Cited By ~ 14

Author(s):

Mika Suzuki ◽

Hiroshi Kobayashi ◽

Yoshiko Tanaka ◽

Naohiro Kanayama ◽

Toshihiko Terao

Keyword(s):

Real Time ◽

Trypsin Inhibitor ◽

Target Genes ◽

Reproductive Function ◽

Pcr Analysis ◽

Rt Pcr ◽

Reproductive Process ◽

Female Mice ◽

Retinoid Metabolism ◽

Regulatory Effects

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik−/− female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

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