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The mouse telomerase holoenzyme, which synthesizes telomeric DNA de novo, is a ribonucleoprotein complex that includes the mouse telomerase RNA component (mTERC), mouse telomerase-associated protein (mTEP1) and mouse telomerase reverse transcriptase (mTERT). To determine the role of telomerase in urethane-induced lung tumorigenesis in A/J mice we examined telomerase activity and the expression of each telomerase subunit in 20 tumor samples, harvested at 16, 28, 40 and 50 weeks after urethane treatment. The telomeric repeat amplification protocol assay showed that statistically significant telomerase activation occurred both early and late in tumorigenesis. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNA expression levels of mTEP1 and mTERT were up-regulated during tumor progression, while mTERC expression was not significantly different between tumors and normal lung. We further examined mTEP1 protein expression in normal lung tissue and lung tumors; western blot analysis showed preferential expression of mTEP1 protein in lung tumors compared with normal lung and immunohistochemistry revealed that a majority of the adenoma cells were positively stained in the nucleus, whereas only a few of the adjacent normal alveolar cells were immunoreactive. In addition, we investigated DNAs of the 20 tumor samples by single strand conformation polymorphism and sequencing analyses to examine whether alterations of the p53 gene in exons 5-8 were associated with telomerase activity. Although we found one nonsense, two missense, two silent and one simultaneous double mutation at different codons in six late stage tumors, there was no apparent correlation between telomerase activity and p53 mutations. Collectively, these results suggest that mTEP1 as well as mTERT may be involved in the regulation of telomerase activity and that telomerase activation may contribute to lung tumorigenesis in A/J mice independently of p53 gene alterations.

An inwardly rectifying potassium channel predominantly expressed in glial cells, Kir4.1/KAB-2, has a sequence of Ser-Asn-Val in its carboxyl-terminal end, suggesting a possible interaction with an anchoring protein of the PSD-95 family. We examined the effects of PSD-95 on the distribution and function of Kir4.1 in a mammalian cell line. When Kir4.1 was expressed alone, the channel immunoreactivity was distributed homogeneously. In contrast, when co-expressed with PSD-95, prominent clustering of Kir4.1 in the cell membrane occurred. Kir4.1 was co-immunoprecipitated with PSD-95 in the co-expressed cells. Glutathione S-transferase-fusion protein of COOH terminus of Kir4.1 bound to PSD-95. These interactions disappeared when the Ser-Asn-Val motif was deleted. The magnitude of whole-cell Kir4.1 current was increased by 2-fold in cells co-expressing Kir4.1 and PSD-95 compared with cells expressing Kir4. 1 alone. SAP97, another member of the PSD-95 family, showed similar effects on Kir4.1. Furthermore, we found that Kir4.1 as well as SAP97 distributed not diffusely but clustered in retinal glial cells. Therefore, PSD-95 family proteins may be a physiological regulator of the distribution and function of Kir4.1 in glial cells.

We have isolated a cDNA encoding a novel isoform of the sulfonylurea receptor from a mouse heart cDNA library. Coexpression of this isoform and BIR (Kir6.2) in a mammalian cell line elicited ATP-sensitive K+ (KATP) channel currents. The channel was effectively activated by both diazoxide and pinacidil, which is the feature of smooth muscle KATP channels. Sequence analysis indicated that this clone is a variant of cardiac type sulfonylurea receptor (SUR2). The 42 amino acid residues located in the carboxyl-terminal end of this novel sulfonylurea receptor is, however, divergent from that of SUR2 but highly homologous to that of the pancreatic one (SUR1). Therefore, this short part of the carboxyl terminus may be important for diazoxide activation of KATP channels. The reverse transcription-polymerase chain reaction analysis showed that mRNA of this clone was ubiquitously expressed in diverse tissues, including brain, heart, liver, urinary bladder, and skeletal muscle. These results suggest that this novel isoform of sulfonylurea receptor is a subunit reconstituting the smooth muscle KATP channel.

We have isolated a novel inward rectifier K+ channel predominantly expressed in glial cells of the central nervous system. Its amino acid sequence exhibited 53% identity with ROMK1 and approximately 40% identity with other inward rectifier K+ channels. Xenopus oocytes injected with cRNA derived from this clone expressed a K+ current, which showed classical inward rectifier K+ channel characteristics. Intracellular Mg.ATP was required to sustain channel activity in excised membrane patches, which is consistent with a Walker type-A ATP-binding domain on this clone. We designate this new clone as KAB-2 (the second type of inward rectifying K+ channel with an ATP-binding domain). In situ hybridization showed KAB-2 mRNA to be expressed predominantly in glial cells of the cerebellum and forebrain. This is the first description of the cloning of a glial cell inward rectifier potassium channel, which may be responsible for K+ buffering action of glial cells in the brain.

Dopa decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; aromatic-L-amino-acid carboxylase, EC 4.1.1.28) was purified from rat liver and its partial sequence was determined. Synthetic oligonucleotides were used to construct and screen rat liver cDNA libraries, and three clones were isolated and sequenced. The 2 kilobases of DDC cDNA cloned consisted of a 5'-noncoding segment of 78 nucleotides, a coding region of 1440 nucleotides, and a 3'-noncoding region of 438 nucleotides. The encoded protein of 480 amino acid residues had a molecular weight of 54,000. A special feature of the primary structure of rat DDC was a repeating structure consisting of 29 amino acid residues. A sequence of 58 amino acid residues, including this repeating structure of rat DDC, was found to show homologies with those of rat tyrosine hydroxylase, human dopamine beta-hydroxylase, and bovine phenylethanolamine N-methyltransferase, other mammalian enzymes that synthesize catecholamines. These results indicate that catecholamine biosynthetic enzymes are structurally related and suggest that their homologous domains are important for catechol-protein interactions.