Human genes: GALC, galactosylceramidase; ACADM
, acyl-CoA dehydrogenase, C-4 to C-12 straight chain; ACADVL, acyl-CoA dehydrogenase, very long chain; GALT, galactose-1-phosphate uridyl transferase; PAH, phenylalanine hydroxylase; FCGR2A, Fc fragment of IgG, low affinity IIa, receptor (CD32); RPP30, ribonuclease P/MRP 30kDa subunit.
The entire ACADM coding region and exon/intron boundaries were amplified using previously described primers and conditions , and purified PCR products were directly sequenced on ABI PRISM 3130 XL Genetic Analyzer using Big Dye Terminator chemicals (Applied Biosystems, Foster City, CA, USA).
The ACADM gene of 80 healthy control DNA samples was analyzed by sequencing analysis of the fragments containing the new missense mutations identified.
The patients' ACADM gene coding regions and the correspondent exon/intron boundaries were amplified and directly sequenced on both strands.
Three new ACADM nucleotide variants leading to two new amino acid substitutions c.253G>C (p.Gly85Arg) and c.356T>A (p.Val119Asp) and a new nonsense mutation c.823G>T (p.Gly275 *) were identified.
Namely, on day 1 of cold exposure, the HIF-1[alpha] protein level increased in parallel with a decrease in the level of adiponectin, phospho-AMPK[alpha], and important enzymes indicative of oxidative metabolism (PDH, ACADM, ATP synthase, and cytochrome c oxidase), while the reverse was seen after 1 day of cold exposure; the normalization of HIF-1[alpha] was coupled with an increase in adiponectin, activation of AMPK[alpha], and all the above-mentioned metabolic enzymes.
Accordingly, we found that in parallel with the decrease in adiponectin on day 1 of cold exposure there was a decrease in the protein level of phosho-AMPK[alpha] as well as the enzymes involved in oxidative metabolism, PDH, ACADM, ATP synthase, and cytochrome c oxidase.
However, these changes in the above-mentioned molecules were reversed after 3 days of cold exposure: HIF-1[alpha] and GAPDH decreased compared to the control, while increase in adiponectin paralleled the activation of AMPK[alpha].This was followed by a sequential restoration/increase in the enzymes involved in oxidative metabolism (PDH, ACADM, ATP synthase, and cytochrome c oxidase).
(c) Population study of samples retained for interesting genotypes of ACADM
, coagulation (F2, F5, MTHFRR and warfarin susceptibility (VKORC1, CYP2C19), courtesy of Associated Regional and University Pathologists and BioFire Diagnostics.
Primers were designed for PPAR[gamma], FASN, ACADM, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes using Primer Express 3.0 software (PE Applied Biosystems, FosterCity, CA, USA) as shown in Table 1.
Total RNA from different adipose tissues, LD and other organs were analyzed by qRT-PCR for the following genes: PPAR[gamma], FASN, ACADM. RNA integrity and PCR products with the expected size were shown in Figure 1.
PPAR[gamma], FASN, and ACADM mRNA expression in adipose tissue and LD of Yanbian yellow cattle