Amino acid supply in brain is regulated by the activity of the large neutral amino acid transporter (LAT) at the brain capillary endothelial cell, which forms the blood-brain barrier (BBB) in vivo. Bovine BBB poly(A) ؉ RNA was isolated from 2.0 kg of fresh bovine brain and size fractionated on a sucrose density gradient, and a size-fractionated bovine BBB cDNA library in the pSPORT vector was prepared. The full-length cDNA encoding the bovine BBB LAT was isolated from this library, and the predicted amino acid sequence was 89 -92% identical to the LAT1 isoform. The bovine BBB LAT1 mRNA produced a 10-fold enhancement in tryptophan transport into frog oocytes coinjected with bovine BBB LAT1 mRNA and the mRNA for 4F2hc, which encodes the heavy chain of the heterodimer. Tryptophan transport into the mRNA-injected oocytes was sodium independent and was specifically inhibited by other large neutral amino acids, and the Km of tryptophan transport was 31.5 ؎ 5.5 M. Northern blotting with the bovine BBB LAT1 cDNA showed that the LAT1 mRNA is 100-fold higher in isolated bovine brain capillaries compared with C6 rat glioma cells or rat brain, and the LAT1 mRNA was not detected in rat liver, heart, lung, or kidney. These studies show that the LAT1 transcript is selectively expressed at the BBB compared with other tissues, and the abundance of the LAT1 mRNA at the BBB is manyfold higher than that of transcripts such as the 4F2hc antigen, actin, or the Glut1 glucose transporter.biological transport ͉ endothelium ͉ gene expression A mino acid availability in brain plays an important role in the regulation of several pathways of brain amino acid metabolism, including neurotransmitter synthesis, S-adenosylmethionine production, and protein synthesis (1). The transport of essential amino acids from blood to brain intracellular space involves movement of amino acids through two biological membranes in series: the blood-brain barrier (BBB) and the plasma membrane of brain cells (neurons, glia). The brain capillary endothelial plasma membranes form the BBB in vivo. Because the surface area of the brain cell membrane is orders of magnitude greater than the surface area of the BBB (2), transport across the BBB is the rate-limiting step in amino acid movement from blood to brain intracellular spaces.The transport of large neutral amino acids across the BBB is mediated by a large neutral amino acid transporter (1), analogous to the leucine (L)-preferring system in peripheral tissues, and now designated LAT for large neutral amino acid transporter (3). However, the L-system at the BBB has a much higher affinity (lower K m ) for amino acids as compared with L-systems in peripheral tissues (1). Whereas the K m of the L-system in peripheral tissues is in the 1-10 mM range, the K m of large neutral amino acid transport by the BBB L-system is on the order of 10-100 M (4). The selective expression of a low-K m LAT at the BBB underlies the selective vulnerability of the brain to the pathologic effects of hyperaminoacidemias (1).Kanai and cowork...

The blood-brain barrier (BBB) is formed by the brain microvascular endothelium, and the unique transport properties of the BBB are derived from tissue-specific gene expression within this cell. The current studies developed a gene microarray approach specific for the BBB by purifying the initial mRNA from isolated rat brain capillaries to generate tester cDNA. A polymerase chain reaction-based subtraction cloning method, suppression subtractive hybridization (SSH), was used, and the BBB cDNA was subtracted with driver cDNA produced from mRNA isolated from rat liver and kidney. Screening 5% of the subtracted tester cDNA resulted in identification of 50 gene products and more than 80% of those were selectively expressed at the BBB; these included novel gene sequences not found in existing databases, ESTs, and known genes that were not known to be selectively expressed at the BBB. Genes in the latter category include tissue plasminogen activator, insulin-like growth factor-2, PC-3 gene product, myelin basic protein, regulator of G protein signaling 5, utrophin, IkappaB, connexin-45, the class I major histocompatibility complex, the rat homologue of the transcription factors hbrm or EZH1, and organic anion transporting polypeptide type 2. Knowledge of tissue-specific gene expression at the BBB could lead to new targets for brain drug delivery and could elucidate mechanisms of brain pathology at the microvascular level.

Loss of the histone H3.3‐specific chaperone component ATRX or its partner DAXX frequently occurs in human cancers that employ alternative lengthening of telomeres (ALT) for chromosomal end protection, yet the underlying mechanism remains unclear. Here, we report that ATRX/DAXX does not serve as an immediate repressive switch for ALT. Instead, ATRX or DAXX depletion gradually induces telomere DNA replication dysfunction that activates not only homology‐directed DNA repair responses but also cell cycle checkpoint control. Mechanistically, we demonstrate that this process is contingent on ATRX/DAXX histone chaperone function, independently of telomere length. Combined ATAC‐seq and telomere chromatin immunoprecipitation studies reveal that ATRX loss provokes progressive telomere decondensation that culminates in the inception of persistent telomere replication dysfunction. We further show that endogenous telomerase activity cannot overcome telomere dysfunction induced by ATRX loss, leaving telomere repair‐based ALT as the only viable mechanism for telomere maintenance during immortalization. Together, these findings implicate ALT activation as an adaptive response to ATRX/DAXX loss‐induced telomere replication dysfunction.