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.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonist fails to overcome trans-10, cis-12 conjugated linoleic acid (CLA) inhibition of milk fat in dairy sheep.

The trans-10, cis-12 conjugated linoleic acid (CLA) causes milk fat depression by downregulating expression of genes and transcription factors involved in lipogenesis and it has been proposed that peroxisome proliferator-activated receptor gamma (PPARγ) can be inhibited by trans-10, cis-12 CLA. The PPARγ is a nuclear receptor activated by natural or synthetic ligands and promotes expression of lipogenic genes and its effect on mammary lipogenesis and the interaction with trans-10, cis-12 CLA in lactating ewes was evaluated using thiazolidinedione (TZD), a chemical PPARγ agonist. A total of 24 lactating ewes were randomly assigned to one of the following treatments for 7 days: (1) Control (5 ml/day of saline solution); (2) TZD (4 mg/kg of BW/day in 5 ml of saline solution); (3) CLA (27 g/day with 29.9% of trans-10, cis-12); (4) TZD+CLA. Compared with Control, milk fat content was not changed by TZD, but was decreased 22.3% and 20.5% by CLA and TZD+CLA treatments. In the mammary gland, TZD increased PPARγ gene expression by 174.8% and 207.8% compared with Control and TZD+CLA treatments, respectively. Conjugated linoleic acid reduced sterol regulatory element-binding transcription protein 1 (SREBP1) gene expression 89.2% and 75.3% compared with Control and TZD+CLA, respectively, demonstrating that TZD fails to overcome CLA inhibition of SREBP1 signaling. In adipose tissue, the expression of SREBP1 and stearoyl CoA desaturase 1 (SCD1) genes were increased by the TZD+CLA treatment, compared with the other treatments. Conjugated linoleic acid decreased milk fat concentration and expression of lipogenic genes, while TZD had no effect on milk fat concentration, expression of lipogenic enzymes or regulators in the mammary gland and failed to overcome the inhibition of these by CLA. Therefore, CLA inhibition of milk fat synthesis was independent of the PPARγ pathway in lactating dairy ewes.

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.

Coat protein RNAs-mediated protection against Andean potato mottle virus in transgenic tobacco.

The expression of translatable sequences of either one of the two Andean potato mottle virus (APMoV) coat protein (CP) genes (CP22 and CP42) and of the nontranslatable sequence of CP42 in transgenic tobacco provided protection against APMoV. Resistance was mediated by CP transgene RNAs rather than the protein, as an inverse correlation between resistance and the accumulation levels of CPs transgene mRNAs was observed. These data indicated that a post-transcriptional gene silencing (PTGS) mechanism is likely involved in the APMoV CP RNA-mediated protection. Moreover, the HindIII-AccI restriction pattern of the CP22 transgene was different in susceptible and resistant transgenic plants, suggesting the involvement of methylation in PTGS. Southern blot experiments also revealed that CPs transgene insertion loci and organisation in the plant genome may play a role in determining the degree of protection.

Co-suppression mediated virus resistance in transgenic tobacco plants harboring the 3'-untranslated region of Andean potato mottle virus.

Nicotiana tabacum plants were transformed with the 3'-untranslated region of the Andean Potato Mottle Virus (APMoV) genome RNA-2. Three strategies were used: the introduction of this region in sense and in antisense orientation and of a fragment comprising the entire 3'-untranslated region from RNA-2 and part of the CP22 coat protein sequence. The transgenic lines were inoculated with the virus and different responses were observed, ranging from susceptibility to APMoV to complete immunity to virus infection, in which neither the virion nor viral RNA was detected in the inoculated leaf and leaves that emerged after inoculation. The R1 progeny from different R0 lines also showed an array of virus resistance phenotypes, which was not correlated with the zygotic state of the transgene. Resistance was positively correlated with low levels of transgene mRNA accumulation, indicating a co-suppression-mediated mechanism towards the transgenic transcripts and APMoV viral RNA.

Plant glycine-rich proteins: a family or just proteins with a common motif?

Twelve years ago a set of glycine-rich proteins (GRP) of plants were characterized and since then a wealth of new GRPs have been identified. The highly specific but diverse expression pattern of grp genes, taken together with the distinct sub-cellular localisation of some GRP groups, clearly indicate that these proteins are implicated in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, studies conducted with these proteins have provided new and interesting insights on the molecular and cell biology of plants. Complex regulated promoters and distinct mechanisms of gene expression regulation have been demonstrated. New protein targeting pathways, as well as the exportation of GRPs from different cell types have been discovered. These data show that GRPs can be useful as markers and/or models to understand distinct aspects of plant biology. In this review, the structural and functional features of this family of plant proteins will be summarised. Special emphasis will be given to the gene expression regulation of GRPs isolated from different plant species, as it can help to unravel their possible biological functions.

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.

Efficient shoot organogenesis of eggplant (Solanum melongena L.) induced by thidiazuron.

Morphogenesis from several explant types excised from in-vitro-grown plantlets of a Brazilian eggplant (Solanum melongena L.) variety (F-100) was evaluated in response to thidiazuron (TDZ). Leaves and cotyledons were found to be the most responsive explants. Optimal shoot bud induction rates (75-100 buds/explant) were achieved in the presence of 0.2 µM TDZ. Organogenic calli were transferred to growth regulator free MS medium before shoot excision. Rooting was induced on half-strength MS medium supplemented with 0.6 µM IAA.

Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris.

Endochitinases are widely distributed among higher plants, including a number of important crop species. They are generally considered to be involved in plant defence against potential pathogens. We have cloned a class IV chitinase gene (AtchitIV) from Arabidopsis thaliana. Southern blot analysis allowed the detection of two cross-hybridising genes in the A. thaliana genome. AtchitIV transcripts are detected in seedpods, but not in roots, inflorescence stems, leaves and flowers of healthy plants. The transcripts accumulated very rapidly in leaves after inoculation with Xanthomonas campestris. Maximum mRNA accumulation was reached one hour after infection and decreased to very low levels 72 hours after induction. This result suggests an involvement of AtchitIV in the initial events of the hypersensitive reaction. Nevertheless, A. thaliana plants transformed with the gus gene under the control of a class IV chitinase bean promoter, showed GUS activity in seed embryos. These data, together with the constitutive expression of the endogenous gene in the seedpods, points to additional physiological roles for this protein.

Inflorescence-specific genes from Arabidopsis thaliana encoding glycine-rich proteins.

Genomic and cDNA clones for three inflorescence-specific genes from Arabidopsis thaliana were isolated and characterized. The genes are tandemly organized in the genome on a 10 kb fragment. The expression of these genes is coordinately regulated in a developmental and organ-specific pattern. They are expressed predominantly in anthers at the later stage of flower development. The primary structure of the encoded gene products exhibits comparable features consisting of a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Little homology is observed either between the glycine-rich domain of the three genes or with previously described glycine-rich proteins from other plant species.

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.

Differential expression of five Arabidopsis genes encoding glycine-rich proteins.

Five cDNA clones coding for glycine-rich proteins in Arabidopsis thaliana were isolated. The corresponding genes are present in the genome as single copies. The derived protein sequences contain highly repetitive glycine-rich motifs. There is, however, little homology among them, nor with previously described glycine-rich proteins from other species. All five genes are expressed in leaves and stems of 6-week-old plants but show different patterns of expression in other organ systems. Analysis of the effect of different external stimuli on the expression pattern showed that, in most cases, the transcript levels were moderately but selectively affected. With flooding stress, the accumulated level of the transcript from one of the genes was remarkably increased.

Trehalose and maltose metabolism in yeast transformed by a MAL4 regulatory gene cloned from a constitutive donor strain.

A 6.8 kb fragment of DNA containing the regulatory sequence MAL4p has been cloned from a genomic library prepared from Saccharomyces cerevisiae strain 1403-7A which ferments maltose constitutively. The library was prepared by ligation of 5-20 kb Sau3AI restriction fragments of total yeast DNA into the BamH1 restriction site of shuttle vector YEp13. A restriction map of the cloned fragment indicates that it encompasses a 2.6 kb segment which closely resembles the regulatory MAL6 gene previously identified (Needleman et al. 1984). The hybrid plasmid, p(MAL4p)4, could transform maltose-nonfermenting strains which contain cryptic alpha-glucosidase and maltose permease genes (malp MALg), but could not transform strains containing a functional regulatory sequence and a defective maltase-permease region (MAlp malg). A correlated absence of maltase and permease DNA from the cloned fragment was indicated by the restriction map. Although the cloned DNA fragment was derived from a constitutive strain, maltose fermentation and alpha-glucosidase formation by yeast transformed with p(MAL4p)4 was largely inducible by maltose and sensitive to catabolite repression. Moreover, the active trehalose accumulation pattern (TAC(+) phenotype) linked to the complete MAL4 locus in strain 1403-7A and other constitutive MAL strains (Oliveira et al. 1981b) was not found in p(MAL4p)4 transformants. It may be concluded that constitutivity of maltose fermentation and the associated active trehalose accumulation are not merely consequences of a cis-dominant mutation causing constitutive formation of the MALp regulatory product. Moreover, constitutivity may not be caused solely by a mutation within the structural region of the MALp gene.

Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae : II. Effect of Constitutive MAL Genes.

A pattern of active accumulation of trehalose during growth on glucose medium, TAC(+) phenotype, is controlled by a polymeric series of maltose fermentation (MAL) genes. An essential requirement for expression of the TAC(+) phenotype is that the MAL gene be in the constitutive state, MAL (c). Mutation of a constitutive MAL allele to a maltose- inducible or nonfermenting (mal) state, alters the pattern of trehalose metabolism so that little or no trehalose accumulation occurs during growth on glucose medium. The TAC(+) phenotype is obtained in MAL (c) strains whether or not α-glucosidase formation is sensitive or resistant to carbon catabolite repression. However, trehalose accumulation is sensitive to glucose levels even in MAL (c) strains in which α-glucosidase formation is insensitive to catabolite repression. The effects of constitutive MAL genes on trehalose accumulation cannot be accounted for by an increase in trehalose-6 phosphate synthase or a decrease in trehalase as determined in vitro. A mechanism is proposed in which the gene-product of a MAL gene serves as a common positive regulator for expression of four genes coding respectively for maltose permease, maltase, α-methylglucosidase and a component of the trehalose accumulation system.