Lyophilization as a method to store samples of whole blood.

We have investigated the feasibility of using lyophilization as a method of preserving whole blood samples for HLA class II molecular typing. Genomic DNA extracted from lyophilized blood appears similar in size to that extracted from fresh or frozen blood and is suitable for both PCR amplification and Southern blot analysis.

Cold-induced alterations in the binding of adrenomedullary nuclear proteins to the promoter region of the tyrosine hydroxylase gene.

It is well documented that cold stress induces a rapid trans-synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5' flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides -246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK-N-BE(2)-C, hepatoma HepG2, and rat pheochromocytoma PC-12 cells. Results of this analysis indicate that the proximal 5' flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5' flanking region of the TH gene. The most striking cold stress-induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides -246 to -189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold-induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1.

NK cells are able to lyse a variety of virally infected and neoplastic cells in an MHC-unrestricted manner. The cell-surface protein NKR-P1 is thought to play a key role in this process. NKR-P1, initially identified in rat IL-2 activated NK cells, is encoded in the mouse by at least three similar, but not identical, genes. We previously reported the isolation and characterization of three different NKR-P1 cDNA, termed cDNA 2, 34, and 40, from IL-2 activated mouse NK cells. This report describes the structure of the gene encoding NKR-P1 cDNA 2, the smallest of these three cDNA. Gene 2 is composed of six exons spanning approximately 14 kb of genomic DNA. The first exon encodes the N-terminal intracellular domain, and exons 4, 5, and 6 contain the sequences coding for the CRD. This organization is similar to that of other genes that encode C-type animal lectins. The expression of the NKR-P1 genes in A-LAK cells from 13 mouse strains was examined by Northern blot analysis. NKR-P1 expression appears to coincide with that of the NK1.1 Ag. This observation further supports the hypothesis that the NK1.1 Ag is encoded by one of the NKR-P1 genes. Nucleotide sequence analysis of the promoter region of the three NKR-P1 genes in BALB/c and C57BL/6 mice suggests that differences in the level of expression probably do not result from alterations in the upstream regions of these genes, but may be caused by the expression of strain-specific transacting factors.

Effects of cold exposure on rat adrenal tyrosine hydroxylase: an analysis of RNA, protein, enzyme activity, and cofactor levels.

Long-term cold exposure (5-7 days) is known to induce concomitant increases in the levels of adrenomedullary tyrosine hydroxylase (TH) RNA, protein, and enzyme activity. In this report, we compare the time courses of these changes and investigate the effects of cold exposure on the levels of biopterin, the cofactor required for tyrosine hydroxylation. After only 1 h of cold exposure, TH mRNA abundance increased 71% compared with nonstressed controls. Increases in total cellular TH RNA levels were maximal (threefold over control values) within 3-6 h of cold exposure and remained elevated throughout the duration of the experiment (72 h). TH protein levels increased rapidly after 24 h of cold exposure and reached a maximal value threefold above that of controls at 48-72 h. Despite the relatively rapid and large elevations in TH RNA and protein content, only modest increases in TH activity were detected during the initial 48 h of cold exposure. Adrenomedullary biopterin increased rapidly after the onset of cold exposure, rising to a level approximately twofold that of the nonstressed controls at 24 h, and remained at this level throughout the duration of the stress period. Taken together, the results of this time course study indicate that cold-induced alterations in adrenal TH activity are mediated by multiple cellular control mechanisms, which may include pre- and posttranslational regulation. Our findings also suggest that cold stress-induced increases in the levels of the TH cofactor may represent another key event in the sympathoadrenal system's response to cold stress.

Isolation of a rat adrenal cDNA clone encoding phenylethanolamine N-methyltransferase and cold-induced alterations in adrenal PNMT mRNA and protein.

Cold stress is known to increase the synthesis and release of catecholamines in the sympathoadrenal system. Previously, we have demonstrated that cold exposure results in a 3- to 4-fold increase in adrenomedullary tyrosine hydroxylase (TH) activity, which is mediated by concomitant alterations in TH mRNA and protein levels. To further investigate the effects of stress on the expression of the catecholamine biosynthetic enzymes, we have isolated a rat cDNA clone encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). The cDNA clone is 905 nucleotides in length and contains a single open reading frame corresponding to 270 amino acids. The amino acid sequence predicted from this nearly full-length cDNA is 89% and 86% identical to that of bovine and human PNMT, respectively. Using the rat PNMT cDNA as a hybridization probe, we have measured the effects of cold stress on the relative abundance of adrenomedullary PNMT mRNA. Levels of PNMT protein were also estimated using an immunoblot analysis. As in the case of TH, cold exposure resulted in a rapid and prolonged increase in PNMT mRNA abundance, followed by concomitant increases in PNMT immunoreactivity. However, there appear to be quantitative and qualitative differences in the adaptive response of TH and PNMT to cold stress.

Isolation and nucleotide sequence of a cDNA clone encoding bovine adrenal tyrosine hydroxylase: comparative analysis of tyrosine hydroxylase gene products.

Investigations into the structure and mechanisms regulating the expression of the genes involved in catecholamine biosynthesis have led to the isolation of a cDNA coding for bovine adrenal tyrosine hydroxylase (TH). The 1,722 bp cDNA contains the complete coding sequence and 3' untranslated region of the TH mRNA. The nucleotide sequence of the cDNA and the deduced amino acid sequence were compared to those reported for rat and human TH. Bovine TH shares 85% and 84% amino acid sequence identity with that of rat and human TH, respectively. Alignment of the amino acid sequences of rat, bovine, and human TH reveals that 79% of the residues are identical in all three species, indicating a strong evolutionary conservation of enzyme structure. Moreover, three of the four putative phosphorylation sites located in the N-terminal region of TH are conserved in these animal species. There are, however, some interspecies differences in TH gene products. The 3' untranslated region of bovine TH mRNA is 56 and 97 nucleotides shorter than rat and human TH mRNA, respectively. Additionally, the bovine protein is 7 and 6 amino acids smaller than its rat and human homologues. All of the absent amino acid residues of bovine TH are missing from an alanine-rich region in the N-terminal portion of the rat and human proteins (amino acids 51-68). Comparison of the size of bovine and rat TH mRNA and protein by northern blot and immunoblot analyses yielded differences consistent with those predicted from the nucleotide sequence data.

Purification and partial amino acid sequence of bovine adrenal phenylethanolamine N-methyltransferase: a comparison of nucleic acid and protein sequence data.

Recently, we have reported the isolation and characterization of a putative genomic DNA clone encoding bovine adrenal phenylethanolamine N-methyltransferase (PNMT) (Batter et al., 1988). However, the lack of primary amino-acid sequence data for this enzyme precluded a definitive proof of the authenticity of this clone. To establish identity, the amino acid sequence of several peptides generated by chemical and enzymatic hydrolysis of purified PNMT was compared to that predicted from the nucleotide sequence of the exons of the putative PNMT gene. Bovine adrenomedullary PNMT was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Treatment with 70% formic acid cleaved the protein at a single Asp-Pro bond near the N-terminus. The purified protein was also cleaved at a single methionine residue near the C-terminus by treatment with cyanogen bromide. N-terminal amino acid sequence analysis identified 8 and 10 amino acid residues, respectively, following each of the scissile peptide bonds. Four tryptic peptides, generated by complete enzymatic digestion, were isolated by reverse-phase HPLC and subjected to sequence analysis. Combined, the amino acid sequences of these six peptides represent 20% of the PNMT protein. These amino acid sequences matched exactly the sequences predicted from the exons of the putative PNMT genomic clone.

Purification and characterization of GTP cyclohydrolase I from Drosophila melanogaster.

The enzyme GTP cyclohydrolase I, which catalyzes the first step in the pteridine biosynthetic pathway, has been purified by at least 4400-fold from Drosophila melanogaster. The active complex has an apparent molecular mass of 575,000 daltons, as estimated from gel filtration. This high molecular mass complex appears to be composed of a number of 39,000-dalton subunits. A polyspecific antiserum generated against the active complex has been used to identify this polypeptide as being severely affected by mutations in Punch, the structural gene for GTP cyclohydrolase. Enzyme activity is inhibited by divalent cations and high ionic strength buffers. No cofactors have been demonstrated to be required for enzyme activity. The enzyme displays positive cooperativity in phosphate buffer, a Hill number of 2.1, but only slight cooperativity in Tris buffer, a Hill number of 1.2.