The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of ␣-eleostearic (18:3⌬ 9cis,11trans,13trans ) and ␣-parinaric (18:4⌬ 9cis,11trans,13trans,15cis ) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of ␣-eleostearic and ␣-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the ⌬ 12 -oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of ␣-eleostearic and ␣-parinaric acids, neither of which is present in untransformed soybean embryos. ␣-Eleostearic and ␣-parinaric acids together accounted for as much as 17% (wt͞wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for ⌬ 12 -oleic acid desaturase-type enzymes that we have termed ''conjugase.'' H undreds of unusual fatty acid structures are known to occur in the seed oils of various plant species (1). The biosynthetic pathways of many of these fatty acids are unknown or have not been well characterized. One such class consists of fatty acids with double bonds that are conjugated. This structural configuration is in contrast to that of linoleic (18:2⌬ 9cis,12cis ) and ␣-linolenic (18:3⌬ 9cis,12cis,15cis ) acids, the typical polyunsaturated fatty acids of plant seed oils, which contain double bonds that are separated by methylene (ϪCH 2 Ϫ) groups (Fig. 1). Among the fatty acids with conjugated double bonds that occur in the plant kingdom are ␣-eleostearic acid (18:3⌬ 9cis,11cis,13trans) and ␣-parinaric acid (18:4⌬ 9cis,11trans,13trans,15cis ) ( Fig. 1). ␣-Eleostearic acid accounts for Ͼ65% (wt͞wt) of the total fatty acids of tung oil, a high-value drying oil obtained from seeds of Aleurites fordii (2). Other sources of this fatty acid include the seed oil of Momordica charantia, which contains approximately 65% (wt͞wt) ␣-eleostearic acid (2). In addition, ␣-parinaric acid composes 30 to 65% (wt͞wt) of the seed oils of plants such as Parinarium laurinum and Impatiens species (1, 3, 4).The presence of conjugated double bonds in fatty acids markedly increases their rate of oxidation relative to polyunsaturated fatty acids with methylene-interrupted double bonds (5). This property makes seed oils, such as tung oil, that are enriched in fatty acids with conjugated double bonds...

Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARα) activate expression of genes involved in fatty acid and glucose oxidation including carnitine palmitoyltransferase-1A (CPT-1A) and pyruvate dehydrogenase kinase 4 (PDK4). CPT-1A catalyzes the transfer of long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes and is an initiating step in the mitochondrial oxidation of long chain fatty acids. PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex (PDC) which catalyzes the conversion of pyruvate to acetyl-CoA in the glucose oxidation pathway. The activity of CPT-1A is modulated both by transcriptional changes as well as by malonyl-CoA inhibition. In the liver, CPT-1A and PDK4 gene expression are induced by starvation, high fat diets and PPARα ligands. Here, we characterized a binding site for PPARα in the second intron of the rat CPT-1A gene. Our studies indicated that WY14643 and long chain fatty acids induce CPT-1A gene expression through this element. In addition, we found that mutation of the PPARα binding site reduced the expression of CPT-1A-luciferase vectors in the liver of fasted rats. We had demonstrated previously that CPT-1A was stimulated by the peroxisome proliferator activated receptor gamma coactivator (PGC-1α) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1α did not enhance CPT-1A transcription through the PPARα binding site in the second intron. Following knockdown of PGC-1α with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARα and PGC-1α stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene.

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-controlling step in the pathway of mitochondrial fatty acid oxidation. Thyroid hormone will stimulate the expression of the liver isoform of CPT-I (CPT-I␣). This induction of CPT-I␣ gene expression requires the thyroid hormone response element in the promoter and sequences within the first intron. The peroxisomal proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣) is a coactivator that promotes mitochondrial biogenesis, mitochondrial fatty acid oxidation, and hepatic gluconeogenesis. In addition, PGC-1␣ will stimulate the expression of CPT-I␣ in primary rat hepatocytes. Here we report that thyroid hormone will increase PGC-1␣ mRNA and protein levels in rat hepatocytes. In addition, overexpression of PGC-1␣ will enhance the thyroid hormone induction of CPT-I␣ indicating that PGC-1␣ is a coactivator for thyroid hormone. By using chromatin immunoprecipitation assays, we show that PGC-1␣ is associated with both the thyroid hormone response element in the CPT-I␣ gene promoter and the first intron of the CPT-I␣ gene. Our data demonstrate that PGC-1␣ participates in the stimulation of CPT-I␣ gene expression by thyroid hormone and suggest that PGC-1␣ is a coactivator for thyroid hormone.

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK) inhibits its activity. The expression of the pyruvate dehydrogenase kinase 4 (PDK4) gene is increased in fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. Transcription of the PDK4 gene is elevated by glucocorticoids and inhibited by insulin. In this study, we have investigated the factors involved in the regulation of the PDK4 gene by these hormones. Glucocorticoids stimulate PDK4 through two glucocorticoid receptor (GR) binding sites located more than 6,000 base pairs upstream of the transcriptional start site. Insulin inhibits the glucocorticoid induction in part by causing dissociation of the GR from the promoter. Previously, we found that the estrogen related receptor alpha (ERRα) stimulates the expression of PDK4. Here, we determined that one of the ERRα binding sites contributes to the insulin inhibition of PDK4. A binding site for the forkhead transcription factor (FoxO1) is adjacent to the ERRα binding sites. FoxO1 participates in the glucocorticoid induction of PDK4 and the regulation of this gene by insulin. Our data demonstrate that glucocorticoids and insulin each modulate PDK4 gene expression through complex hormone response units that contain multiple factors.

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Tetraspanin CD82 has been implicated in integrinmediated functions such as cell motility and invasiveness. Although tetraspanins associate with integrins, it is unknown if and how CD82 regulates the functionality of integrins. In this study, we found that Du145 prostate cancer cells underwent morphogenesis on the reconstituted basement membrane Matrigel to form an anastomosing network of multicellular structures. This process entirely depends on integrin ␣6, a receptor for laminin. After CD82 is expressed in Du145 cells, this cellular morphogenesis was abolished, indicating a functional cross-talk between CD82 and ␣6 integrins. Interestingly, antibodies against other tetraspanins expressed in Du145 cells such as CD9, CD81, and CD151 did not block this integrin ␣6-dependent morphogenesis. We further found that CD82 significantly inhibited cell adhesion on laminin 1. Notably, the level of ␣6 integrins on the cell surface was down-regulated upon CD82 expression, although total cellular ␣6 protein levels remained unchanged in CD82-expressing cells. This down-regulation indicates that the diminished cell adhesiveness of CD82-expressing Du145 cells on laminin likely resulted from less cell surface expression of ␣6 integrins. As expected, CD82 physically associated with the integrin ␣6 in Du145-CD82 transfectant cells, suggesting that the formation of the CD82-integrin ␣6 complex reduces ␣6 integrin cell surface expression. Finally, the internalization of cell surface integrin ␣6 is significantly enhanced upon CD82 expression. In conclusion, our results indicate that 1) CD82 attenuates integrin ␣6 signaling during a cellular morphogenic process; 2) the decreased surface expression of ␣6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of ␣6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin ␣6 upon CD82 expression correlates with the down-regulation of cell surface integrin ␣6.CD82 belongs to the tetraspanin superfamily, in which members are involved in biological events ranging from cell fusion, cell adhesion, and cell migration to cell proliferation, synapse formation, and neurite outgrowth (1-5). Originally, CD82 was identified as either a membrane protein that could induce the intracellular calcium mobilization in lymphocytes, an accessory molecule in T-cell activation, or the target of a monoclonal antibody (mAb) 1 that inhibits human T-cell leukemia virus-induced syncytium formation (6 -8). Studies have indicated that CD82 regulates cell aggregation (9 -12), cell motility (10, 11, 13-17), cancer metastasis (11,15,18), and apoptosis (13,19). Clustering CD82 on the plasma membrane with its mAb induces the tyrosine phosphorylation of Vav-1, SLP76, and Cas-L (20, 21), actin cytoskeletal rearrangement (20,22), and dendritic cellular protrusions (8, 22). These observations strongly suggest that CD82 directly or indirectly solicits outside-in signaling to modulate cellular behavior...