Conjugated linoleic acid trans-10, cis-12 increases the expression of genes from cell cycle progression and cis-9, trans-11 stimulates apoptotic genes in different mammary tumor explants of female dogs.

BACKGROUND: The conjugated linoleic acid (CLA) isomer cis-9, trans-11 is an anticarcinogen that inhibits cell proliferation and/or induces apoptosis of tumor cells. The objective of this study was to evaluate the expression of genes responsible for cell cycle regulation and apoptosis in tumor explants of mammary anaplastic carcinoma (AC) and mammary tubulopapillary carcinoma (TC) cultured in vitro with the CLA isomers cis-9, trans-11 and trans-10, cis-12. METHODS: In this study we used mammary explants from two adult female dogs that revealed two types of malignant tumors: (a) anaplastic mammary carcinoma (AC) and (b) mammary tubulopapillary carcinoma (TC). The explants (n = 6 per treatment) had an average weight of 80.0 ± 2.0 mg and were cultured for 24 h in 35 mm culture plates under the following treatments: (a) Control: Culture medium + fatty acid free bovine serum albumin (BSA); (b) Culture medium + cis-9, trans-11 CLA (75 µM) diluted with fatty acid free bovine serum albumin (BSA), and; (c) Culture medium + trans-10, cis-12 CLA (75 µM) diluted with fatty acid free bovine serum albumin (BSA). After that, total RNA was extracted, complementary DNA was synthesized (cDNA), and quantitative analysis by real-time polymerase chain reaction (RT-qPCR) was conducted. Data were analyzed using the MIXED procedure of SAS. RESULTS: Compared with the Control, the CLA trans-10, cis-12 treatment decreased expression of the gene encoding the p53 by 20% (P = 0.02), Caspase-3 by 25% (P = 0.06) and Bax by 51% (P = 0.001) in AC. The CLA cis-9, trans-11 increased the gene expression of proapoptotic protein Bax in TC by 68% (P = 0.01), but increased the expression of the antiapoptotic Bcl2 gene in AC by 72% (P = 0.006). CONCLUSION: The CLA cis-9, trans-11 stimulates apoptotic genes in mammary tubulopapillary carcinoma, but has a contrary effect on the anaplastic carcinoma, and the CLA trans-10, cis-12 stimulates cell cycle progression genes and may have an antiapoptotic effect, mainly in mammary anaplastic carcinoma.

Transcriptional regulation of acetyl-CoA carboxylase α isoforms in dairy ewes during conjugated linoleic acid induced milk fat depression.

Feeding trans-10, cis-12 CLA to lactating ewes reduces milk fat by down-regulating expression of enzymes involved in lipid synthesis in the mammary gland and increases adipose tissue lipogenesis. Acetyl-CoA carboxylase α (ACC-α) is a key regulated enzyme in de novo fatty acid synthesis and is decreased by CLA. In the ovine, the ACC-α gene is expressed from three tissue-specific promoters (PI, PII and PIII). This study evaluated promoter-specific ACC-α expression in mammary and adipose tissue of lactating cross-bred Lacaune/Texel ewes during milk fat depression induced by rumen-unprotected trans-10, cis-12 CLA supplement. In all, 12 ewes arranged in a completely randomized design were fed during early, mid and late lactation one of the following treatments for 14 days: Control (forage+0.9 kg of concentrate on a dry matter basis) and CLA (forage+0.9 kg of concentrate+27 g/day of CLA (29.9% trans-10, cis-12)). Mammary gland and adipose tissue biopsies were taken on day 14 for gene expression analysis by real-time PCR. Milk fat yield and concentration were reduced with CLA supplementation by 27%, 21% and 35% and 28%, 26% and 42% during early, mid and late lactation, respectively. Overall, our results suggest that trans-10, cis-12 CLA down-regulates mammary ACC-α gene expression by decreasing expression from PII and PIII in mammary gland and up-regulates adipose ACC-α gene expression by increasing expression from PI.

Milk fat depression and energy balance in stall-fed dairy goats supplemented with increasing doses of conjugated linoleic acid methyl esters.

Feeding dietary supplements containing trans-10, cis-12-conjugated linoleic acid (t10,c12-CLA) has been shown to induce milk fat depression in cows, ewes and goats. However, the magnitude of the response is apparently less pronounced in lactating goats. The objective of this study was to evaluate the effects of increasing doses of CLA methyl esters (CLA-ME) on milk production, composition and fatty-acid profile of dairy goats. Eight Toggenburg goats were separated in two groups (four primiparous and four multiparous) and received the following dietary treatments in a 4×4 Latin Square design: CLA0: 45 g/day of calcium salts of fatty acids (CSFA); CLA15; 30 g/day of CSFA+15 g/day of CLA-ME; CLA30: 15 g/day of CSFA+30 g/day of CLA-ME; and CLA45: 45 g/day of CLA-ME. The CLA-ME supplement (Luta-CLA 60) contained 29.9% of t10,c12-CLA; therefore, the dietary treatments provided 0, 4.48, 8.97 and 13.45 g/day of t10,c12-CLA, respectively. Feed intake, milk production, concentration and secretion of milk protein and lactose, body condition score and body weight were unaffected by the dietary treatments. Milk fat secretion was reduced by 14.9%, 30.8% and 40.5%, whereas milk fat concentration was decreased by 17.2%, 33.1% and 40.7% in response to CLA15, CLA30 and CLA45, respectively. Secretions of both de novo synthesized and preformed fatty acids were progressively reduced as the CLA dose increased, but the magnitude of the inhibition was greater for the former. There was a linear reduction in most milk fat desaturase indexes (14:1/14:0, 16:1/16:0, 17:1/17:0 and 18:1/18:0). Milk fat t10,c12-CLA concentration and secretion increased with the CLA dose, and its apparent transfer efficiency from diet to milk was 1.18%, 1.17% and 1.21% for CLA15, CLA30 and CLA45 treatments, respectively. The estimated energy balance was linearly improved in goats fed CLA.

Characterization of early induced genes in Arabidopsis thaliana responding to bacterial inoculation: identification of centrin and of a novel protein with two regions related to kinase domains.

The early response to bacterial inoculation has been investigated and two Arabidopsis genes, ap3.3a and ap4.3a have been characterized. The AP3.3A protein showed high identity to centrin, a ubiquitous cytoskeletal protein first identified in unicellular green alga. Amino-acid sequence analyses of the AP4.3A protein indicates that the second gene characterized encodes an unusual protein with two putative kinase domains. Expression of ap3.3a and ap4.3a was rapidly induced after pathogen inoculation. A role of ap3.3a in plant defense could be postulated based on its preferential induction during the incompatible interactions analyzed. In contrast, activation of ap4.3a was not specific and could be related to a more general stress response.

Molecular cloning and sequence analysis of a segment from Andean potato mottle virus B RNA encoding the putative RNA polymerase.

Andean potato mottle virus (APMV), an endemic South American comovirus, has a bipartite genome consisting of two plus-strand RNA molecules (M and B RNA). We have cloned the 3' half of the B RNA and identified the complete sequence of the putative APMV RNA polymerase. The RNA polymerase gene is part of a large polyprotein-encoding open reading frame. The putative, mature RNA polymerase, as deduced by comparison with the related cowpea mosaic virus (CPMV), type member of the comovirus group, is 703 amino acids long and shows a large degree of similarity with CPMV and other RNA polymerases.

Nucleotide sequence analysis of an Andean potato mottle virus middle component RNA cDNA clone: comparisons of the encoded proteins with those of other comoviruses.

Andean potato mottle virus (APMV) is a comovirus whose genomic structure consists of two plus-strand RNA molecules (M- and B-RNA). Here we report the nucleotide sequence analysis of an APMV M-RNA cDNA clone with 3,669 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end, covering approximately 99% of the APMV M-RNA. The first initiation codon in register translates from nt 194 to 3185 a polyprotein of 997 amino acid (aa) residues. A second initiation codon in register, beginning at nt position 416, translates a polyprotein of 923 aa. The cleavage sites used in the processing of polyprotein were identified in the long open reading frame by N-terminal microsequencing of the large coat protein (LCP) and the small coat protein (SCP). These dipeptide cleavage sites are Q/M for the LCP and Q/F for the SCP. In a comparison of the deduced APMV polyprotein aa sequence with those of four other comoviruses, the coding regions for the putative movement protein, LCP and SCP, were found similar in length in all five species. Multiple alignment of the M-RNA sequences for each of the three genes from the five comoviruses revealed different degrees of homology. APMV was always the least homologous of the five comoviruses, showing significant aa substitutions in positions where the other comoviruses have identical residue or conservative substitutions.