Several Amino Acids and Carnitine Transport Activities of the Epithelial Cells of Bovine Mammary Gland

We investigated several amino acids and carnitine transport activities of bovine mammary gland epithelial cells. Gly, Ala, Gln, Glu, Arg, Leu, cystine and carnitine transport activities at 1 μmol/L substrate concentration were 24.0 ± 3.97, 90.9 ± 13.4, 32.5 ± 9.0, 14.2 ± 5.1, 48.9 ± 11.4, 48.8 ± 5.1, 22.7 ± 6.8 and 2.56 ± 0.96 nmol/mg protein/min, respectively. Na-dependency of transport was observed in Gly, Ala and Gln, but not in Arg, Leu and carnitine. Glu and Cys transport activity without Na condition were reduced to 36%, 63% in Na-free condition, respectively. Carnitine transport activities were low but detectable with or without Na condition. There was no correlation between amino acid transport activities and their concentrations in milk. The data clarified in this paper will be basic data for metabolic analysis of bovine mammary gland

Identification of cytochrome P450 monooxygenase genes from the white-rot fungus Phlebia brevispora

Three cytochrome P450 monooxygenase (CYP) genes, designated pb-1, pb-2 and pb-3, were isolated from the white-rot fungus, Phlebia brevispora, using reverse transcription PCR with degenerate primers constructed based on the consensus amino acid sequence of eukaryotic CYPs in the O2-binding, meander and heme-binding regions. Individual full-length CYP cDNAs were cloned and sequenced, and the relative nucleotide sequence similarity of pb-1 (1788 bp), pb-2 (1881 bp) and pb-3 (1791 bp) was more than 58%. Alignment of the deduced amino acid (aa) sequences of pb-1-pb-3 showed that these three CYPs belong to the same family with > 40% aa sequence similarity, and pb-1 and pb-3 are in the same subfamily, with > 55% aa sequence similarity. Furthermore, pb-1-pb-3 appeared to be a subfamily of CYP63A (CYP63A1-CYP63A4), found in Phanerochaete chrysosporium. The phylogenetic tree constructed by 500 bootstrap replications using the neighbor-joining method showed that the evolutionary distance between pb-1 and pb-3 was shorter than that between pb-2 and pb-1 (or pb-3). Exon-intron analysis of pb-1 and pb-3 showed that both genes have nearly the same number, size and order of exons and the types of introns, also indicating both genes appear to be evolutionarily close. It is interesting that the transcription level of pb-3 was evidently increased above the pb-1 transcription level by exposure to 12 coplanar PCB congeners and 2,3,7,8-tetrachlorodibenzo-p-dioxin, though the two genes were evolutionarily close

Amino Acid Transport System N: Molecular Structure, Distribution and Functional Analysis of Canine SLC38A5 (SNAT5) in Lens Epithelial Cells.

Na-dependent of neutral amino acid transport activity in canine lens epithelial cells (LEC) line was investigated. The transporter activity of glutamine was 11.17 ± 3.17 nmol/mg protein/min, and it was reduced by 75% in the absence of sodium. The full-length cDNA sequence of canine sodium-dependent neutral amino acid transporter 5 (SNAT5) was 2151 bp long and was predicted to encode the 536 amino acid polypeptides. The deduced amino acid sequence of canine SNAT5 showed >80% similarities with those of human and mouse. The RT-PCR analysis indicated that SNAT5 was expressed in liver, kidney and LEC, but not in heart and skin

Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney

BACKGROUND: Carbonic anhydrase VI (CA-VI) is produced by the salivary gland and is secreted into the saliva. Although CA-VI is found in the epithelial cells of distal straight tubule of swine kidneys, the exact function of CA-VI in the kidneys remains unclear. RESULTS: CA-VI was located in the epithelial cells of distal straight tubule of swine kidneys. A full-length cDNA clone of CA-VI was generated from the swine parotid gland by reverse transcription polymerase chain reaction, using degenerate primers designed based on conserved regions of the same locus in human and bovine tissues. The cDNA sequence was 1348 base pairs long and was predicted to encode a 317 amino acid polypeptide with a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI was most similar (77.4%) to that of human CA-VI. CA-VI expression was confirmed in both normal and nephritic kidneys, as well as parotid. As the primers used in this study spanned two exons, the influence of genomic DNA was not detected. The expression of CA-VI was demonstrated in both normal and nephritic kidneys, and mRNA of CA-VI in the normal kidneys which was the normalised to an endogenous β–actin was 0.098 ± 0.047, while it was significantly lower in the diseased kidneys (0.012 ± 0.007). The level of CA-VI mRNA in normal kidneys was 19-fold lower than that of the parotid gland (1.887). CONCLUSIONS: The localisation of CA-VI indicates that it may play a specialised role in the kidney

Aquaporin 1 expression in tissues of canines possessing inherited high K+ erythrocytes

We investigated the expression of aquaporin 1 (AQP1) in tissues from canines with an inherited anomaly that causes their erythrocytes to have high K+. Northern blot analysis revealed abundant AQP1 expression in lung and kidney, though little expression was found in spleen. Using anti-C-terminus for dog AQP1, abundant expression was shown in kidney, trachea, and eye, but little expression was shown in pancreas and cerebrum, indicating that AQP1 expression in canine tissues is similar to that noted in other mammals

イヌのグルコース輸送体4（GluT4）の分子構造と組織発現

イヌのグルコース輸送体4（GluT4）の分子構造と組織発現を調べた。イヌGLUT4のcDNA配列全長1637bpであり，510のアミノ酸から構成され，ヒトとマウスより1アミノ酸大きいことが明らかとなった。このアミノ酸はヒトGluT4と95.2％，マウスGluT4と93.7％の相同性を示した。RT-PCRの結果，心臓・骨格筋・脂肪組織に発現がみられた。ウェスタンブロット解析により，GluT4タンパクは50kDaの質量をもつことが示された。イヌのインシュリン感受性グルコース輸送体4の構造が判明したことにより，イヌの糖尿病の病態生化学の理解に寄与するものと考えられる。Molecular structure and distribution of canine glucose transporter 4(GluT4)　were investigated. Canine GluT4 was consisted of 1637 nucleotides encoding 510 amino acids, which is one amino acid longer than human and mouse. Canine GluT4 exhibited 95.2% and 93.7% identity to human and mouse, respectively RT-PCR analysis indicated that its expression was confirmed in heart, skeletal muscle and lipocytes. Westernblot analysis showed the molecular weight of canine GluT4 was 50kDa. Molecular structure and distribution of insulin-sensitive GluT4 will contribute to a better understanding of the patho-physiology of canine diabetes

Purification of chicken carbonic anhydrase isozyme-III (CA-III) and its measurement in White Leghorn chickens

<p>Abstract</p> <p>Background</p> <p>The developmental profile of chicken carbonic anhydrase-III (CA-III) blood levels has not been previously determined or reported. We isolated CA-III from chicken muscle and investigated age-related changes in the levels of CA-III in blood.</p> <p>Methods</p> <p>CA-III was purified from chicken muscle. The levels of CA-III in plasma and erythrocytes from 278 female chickens (aged 1-93 weeks) and 68 male chickens (aged 3-59 weeks) were determined by ELISA.</p> <p>Results</p> <p>The mean level of CA-III in female chicken erythrocytes (1 week old) was 4.6 μg/g of Hb, and the CA-III level did not change until 16 weeks of age. The level then increased until 63 weeks of age (11.8 μg/g of Hb), decreased to 4.7 μg/g of Hb at 73 weeks of age, and increased again until 93 weeks of age (8.6 μg/g of Hb). The mean level of CA-III in erythrocytes from male chickens (3 weeks old) was 2.4 μg/g of Hb, and this level remained steady until 59 weeks of age. The mean plasma level of CA-III in 1-week-old female chickens was 60 ng/mL, and this level was increased at 3 weeks of age (141 ng/mL) and then remained steady until 80 weeks of age (122 ng/mL). The mean plasma level of CA-III in 3-week-old male chickens was 58 ng/mL, and this level remained steady until 59 weeks of age.</p> <p>Conclusion</p> <p>We observed both developmental changes and sex differences in CA-III concentrations in White Leghorn (WL) chicken erythrocytes and plasma. Simple linear regression analysis showed a significant association between the erythrocyte CA-III level and egg-laying rate in WL-chickens 16-63 weeks of age (p < 0.01).</p