CD1d function is regulated by microsomal triglyceride transfer protein

CD1d is a major histocompatibility complex (MHC) class I-related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-alpha chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell-mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.     

Efficient cloning and engineering of entire mitochondrial genomes in Escherichia coli and transfer into transcriptionally active mitochondria

We have devised an efficient method for replicating and stably maintaining entire mitochondrial genomes in Escherichia coli and have shown that we can engineer these mitochondrial DNA (mtDNA) genome clones using standard molecular biological techniques. In general, we accomplish this by inserting an E.coli replication origin and selectable marker into isolated, circular mtDNA at random locations using an in vitro transposition reaction and then transforming the modified genomes into E.coli. We tested this approach by cloning the 16.3 kb mouse mitochondrial genome and found that the resulting clones could be engineered and faithfully maintained when we used E.coli hosts that replicated them at moderately low copy numbers. When these recombinant mtDNAs were replicated at high copy numbers, however, mtDNA sequences were partially or fully deleted from the original clone. We successfully electroporated recombinant mouse mitochondrial genomes into isolated mouse mitochondria devoid of their own DNA and detected robust in organello RNA synthesis by RT-PCR. This approach for modifying mtDNA and subsequent in organello analysis of the recombinant genomes offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a first step towards true in vivo engineering of mammalian mitochondrial genomes.     

TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.     

The role of hypothalamic input on corticotroph maturation in fetal sheep

Corticotropin-releasing hormone receptor type 1 (CRH-R1) expression and vasopressin type 1b (V1b) receptor protein decrease in late-gestation fetal sheep. Because hypothalamo-pituitary disconnection (HPD) has been demonstrated to prevent the morphological maturation of corticotrophs, we hypothesized that hypothalamic input is necessary for the maturational changes in CRH-R1 and V1b receptor levels. We measured CRH-R1 and V1b receptor expression in the anterior pituitaries of fetuses at 140 days gestational age (dGA) that underwent HPD or sham surgery at 120 dGA. CRH-R1 mRNA decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. However, CRH-R1 protein levels were elevated in HPD fetuses compared with sham and were not different from 120 dGA values. V1b protein levels decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. We conclude that hypothalamic input to the pituitary is necessary for the decrease in CRH-R1 receptor protein levels in late-gestation fetal sheep. However, hypothalamic input is not necessary for the decrease in V1b receptor expression seen in late gestation.     

The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-tolerant soybean expressed in Escherichia coli shows no severe allergenicity

The recombinant gene was amplified from the chromosomal DNA of genetically-modified (GM) soybeans and identified as epsps encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which renders glyphosate resistance. The epsps structural gene was introduced in the pET28(a) plasmid for its expression in Escherichia coli BL21(DE3). It was confirmed that the maximal productivity of the EPSPS protein was achieved when cultivating the recombinant strain in a LB broth for 2 h after supplementing 1 mM isopropylbeta-D-thiogalactopyranoside (IPTG) in a 2 h-culture broth. Since the expressed EPSPS protein was found as an insoluble form in the inclusion body, it was extracted by 6 M urea after sonication, and then purified through immobilized nickel-affinity column chromatography to isolate EPSPS having a molecular mass of 57 kDa. When incubated in simulated gastric fluid containing pepsin at pH 1.5, the purified EPSPS protein was completely digested within 1 min. In addition, the passive cutaneous anaphylaxis reaction of the purified EPSPS protein was not observed in the Sprague Dawley rat system that was administered either orally or subcutaneously. Furthermore, treatment of the EPSPS protein to the culture of the sensitized peritoneal mast cells, or unsensitized but antisera-labeled mast cells, showed neither a remarkable change in the histamine release nor a cytokine production, including interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). Thus, it can be concluded that the EPSPS protein in the GM soybean showed no significant allergenicity in the Sprague Dawley rats.     

Effects of Aroclor 1254 on the expression of rat placental PRL-family genes

This study was performed to investigate the effects of Aroclor 1254 (A1254), a commercial polychlorinated biphenyl mixture, on the expression of rat placental prolactin (PRL) family genes and reproductive activity. Placental lactogen-Iv and -II, and prolactin-like protein-A and -C mRNA levels were significantly decreased in the placentas of A1254-treated rats in a dose-dependent manner. The mRNA levels of Pit-1alpha and beta isotypes, which are involved in the regulation of PRL family gene expression, were also decreased in the A1254-treated rat placenta. In the rat placental junctional zone, high-dose A1254 (25 mg/kg B.W.) treatment reduced the number of spongiotrophoblasts, cells in which the PRL family genes are expressed. Finally, maternal exposure to A1254 was shown to have significant toxic effects on reproductive activity, including embryonic and placental growth retardation, delay of parturition, and reduction of the number of pups per litter. The results of the present study indicated that A1254 has an inhibitory effect on PRL family, Pit-1alpha, and beta gene expression in the rat placenta, leading to significant toxic effects on reproductive activity in rats.     

Is the Voltage Gate of Connexins CO<Subscript>2</Subscript>-sensitive? Cx45 Channels and Inhibition of Calmodulin Expression

The sensitivity of Cx45 channels to CO₂, transjunctional voltage (V _j) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage clamp. Cx45 channels are very sensitive to V _j and close with V _j preferentially by the slow gate, likely to be the same as the chemical gate. With a CO₂-induced drop in junctional conductance (G _j), both the speed of V _j-dependent inactivation of junctional current (I _j) and V _j sensitivity increased. With 40-mV V _j-pulses, the of single exponential I _j decay reversibly decreased by 40% during CO₂ application, and G_{j steady state}/G_{j peak} decreased multiphasically, indicating that both kinetics and V _j sensitivity of chemical/slow V _j gating are altered by changes in [H⁺]_i and/or [Ca²⁺]_i. CaM expression was inhibited with oligonucleotides antisense to CaM mRNA. With 15 min CO₂, relative junctional conductance (G _jt/G _jt0) dropped to 0% in controls, but only by 17% in CaM-antisense oocytes. Similarly, V _j sensitivity was significantly lessened in CaM-antisense oocytes. The data indicate that both the speed and sensitivity of V _j-dependent inactivation of the junctional current of Cx45 channels are affected by CO₂ application, and that CaM plays a key role in channel gating.     

The MHC class I-like IgG receptor controls perinatal IgG transport,IgG homeostasis,and fate of IgG-Fc-coupled drugs

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.