Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat.

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.

Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is involved in trafficking of lymphocytes to mucosal endothelium. Expression of MAdCAM-1 is induced in the murine endothelial cell line bEnd.3 by tumor necrosis factor alpha (TNF-alpha), interleukin 1, and bacterial lipopolysaccharide. Here we show that TNF-alpha enhances expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter, confirming transcriptional regulation of MAdCAM-1. Mutational analysis of the promoter indicates that a DNA fragment extending from nt -132 to nt +6 of the gene is sufficient for TNF-alpha inducibility. Two regulatory sites critical for TNF-alpha induction were identified in this region. DNA-binding experiments demonstrate that NF-kappa B proteins from nuclear extracts of TNF-alpha-stimulated bEnd.3 cells bind to these sites, and transfection assays with promoter mutants of the MAdCAM-1 gene indicate that occupancy of both sites is essential for promoter function. The predominant NF-kappa B binding activity detected with these nuclear extracts is a p65 homodimer. These findings establish that, as with other endothelial cell adhesion molecules, transcriptional induction of MAdCAM-1 by TNF-alpha requires activated NF-kappa B proteins.

The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection. Vpr protein induced nuclear translocation of Rip-1, as did glucocorticoid receptor (GR)-II-stimulating steroids. Importantly, Vpr and Rip-1 coimmunoprecipitated with the human GR as part of an activated receptor complex. Vpr complementation of a vpr mutant virus was also mimicked by GR-II-stimulating steroids. Vpr and GR-II actions were inhibited by mifepristone, a GR-II pathway inhibitor. Together these data directly link the activity of the vpr gene product to the glucocorticoid steroid pathway and provide a biochemical mechanism for the cellular and viral activity of Vpr, as well as suggest that a unique class of antivirals, which includes mifepristone (RU486), may influence HIV-1 replication.

Quantitative assessment of sense and antisense transcripts from genes involved in antigenic variation (vsp genes) and encystation (cwp 1 gene) of Giardia lamblia clone GS/M-83-H7.

Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed predominated in subvariant-type trophozoites. Interestingly, the two trophozoite populations exhibited a similar relative distribution regarding the vsp H7 and vsp IVg antisense RNA molecules. An analogous sense versus antisense RNA pattern was also observed when the transcripts of gene cwp 1 (encoding cyst wall protein 1) were investigated. Here, both types of RNA molecules only appeared after cwp 1 had been induced through in vitro encystation of the parasite. These findings for the first time demonstrated that giardial antisense RNA production did not occur in a constitutive manner but was directly linked to complementary sense RNA production after activation of the respective gene systems.

Hyposulfatemia, growth retardation, reduced fertility, and seizures in mice lacking a functional NaSi-1 gene

inorganic sulfate is required for numerous functions in mammalian physiology, and its circulating levels are proposed to be maintained by the Na+-SO42- cotransporter, (NaSi-1). To determine the role of NaSi-1 in sulfate homeostasis and the physiological consequences in its absence, we have generated a mouse lacking a functional NaSi-1 gene, Nas1. Serum sulfate concentration was reduced by >75% in Nas1(-/-) mice when compared with Nas1(+/+) mice. Nas1(-/-) mice exhibit increased urinary sulfate excretion, reduced renal and intestinal Na+-SO42- cotransport, and a general growth retardation. Nas1(-/-) mouse body weight was reduced by >20% when compared with Nas1(+/+) and Nas1(+/-) littermates at 2 weeks of age and remained so throughout adulthood. Nas1(-/-) females had a lowered fertility, with a 60% reduction in litter size. Spontaneous clonic seizures were observed in Nas1(-/-) mice from 8 months of age. These data demonstrate NaSi-1 is essential for maintaining sulfate homeostasis, and its expression is necessary for a wide range of physiological functions.

Etr1-1 gene expression alters regeneration patterns in transgenic lettuce stimulating root formation

We have evaluated the transformation efficiency of two lettuce ( Lactuca sativa L.) cultivars, LE126 and Seagreen, using Agrobacterium tumefaciens- mediated gene transfer. Six- day- old cotyledons were co- cultivated with Agrobacterium cultures carrying binary vectors with two different genetic constructs. The first construct contained the beta- glucuronidase gene ( GUS) under the control of the cauliflower mosaic virus 35S promoter ( CaMV 35S), while the second construct contained the ethylene mutant receptor etr1- 1, which confers ethylene insensitivity, under the control of a leaf senescence- specific promoter ( sag12). Tissues co- cultivated with the GUS construct showed strong regeneration potential with over 90% of explants developing callus masses and 85% of the calli developing shoots. Histochemical GUS assays showed that 85.7% of the plants recovered were transgenic. Very different results were observed when cotyledon explants were co- cultivated with Agrobacteria carrying the etr1- 1 gene. There was a dramatic effect on the regeneration properties of the cultured explants with root formation taking place directly from the cotyledon tissue in 34% of the explants and no callus or shoots observed initially. Eventually callus formed in 10% of cotyledons and some organogenic shoots were obtained ( 2.86%). These results indicate that the ethylene insensitivity conferred by the etr1- 1 gene alters the normal pattern of regeneration in lettuce cotyledons, inhibiting the formation of shoots and stimulating root formation during regeneration.

No disease-associated mutations found in the coding sequence of the canine polycystic kidney disease gene 1 in Bull Terriers with polycystic kidney disease

The aim of this study was to identify possible disease-associated mutations in the canine homologue of the polycystic kidney disease gene 1 (PKD1) in Bull Terriers with autosomal dominant polycystic kidney disease. Messenger RNA was obtained from the blood or renal tissue of five Bull Terriers with the disease and four close relatives without the disease. Reverse transcription, PCR and 3' rapid amplification of cDNA ends were used to amplify the coding and 3' untranslated regions of this transcript. Comparison of PKD1 sequence between the affected and unaffected Bull Terriers, revealed six polymorphisms, but no disease-associated mutations.

Increased duodenal expression of divalent metal transporter 1 and iron-regulated gene 1 in cirrhosis

Hepatic hemosiderosis and increased iron absorption are common findings in cirrhosis. It has been proposed that a positive relation exists between intestinal iron absorption and the development of hepatic hemosiderosis. The current study investigated the duodenal expression of the iron transport molecules divalent metal transporter 1 (DMT1 [IRE]), iron-regulated gene 1 (Ireg1 [ferroportin]), hephaestin, and duodenal cytochrome b (Dyctb) in 46 patients with cirrhosis and 20 control subjects. Total RNA samples were extracted from duodenal biopsy samples and the expression of the iron transport genes was assessed by ribonuclease protection assays. Expression of DMT1 and Ireg1 was increased 1.5 to 3-fold in subjects with cirrhosis compared with iron-replete control subjects. The presence of cirrhosis per se and serum ferritin (SF) concentration were independent factors that influenced the expression of DMT1. However, only SF concentration was independently associated with Iregl expression. In cirrhosis, the expression of DMT1 and Iregl was not related to the severity of liver disease or cirrhosis type. There was no correlation between the duodenal expression of DMT1 and Iregl and the degree of hepatic siderosis. In conclusion, the presence of cirrhosis is an independent factor associated with increased expression of DMT1 but not Iregl. The mechanism by which cirrhosis mediates this change in DMT1 expression has yet to be determined. Increased expression of DMT1 may play an important role in the pathogenesis of cirrhosis-associated hepatic iron overload.

Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys

Vascular endothelial growth factor (VEGF) is one of the major mediators of retinal ischemia-associated neovascularization. We have shown here that adeno-associated virus (AAV)-mediated expression of sFIt-1, a soluble form of the Flt-1 VEGF receptor, was maintained for up to 8 and 17 months postinjection in mice and in monkeys, respectively. The expression of sFIt-1 was associated with the long-term (8 months) regression of neovascular vessels in 85% of trVEGF029 eyes. In addition, it resulted in the maintenance of retinal morphology, as the majority of the treated trVEGF029 eyes (75%) retained high numbers of photoreceptors, and in retinal function as measured by electroretinography. AAV-mediated expression of sFIt-1 prevented the development of laser photocoagulation-incluced choroidal neovascularization in all treated monkey eyes. There were no clinically or histologically detectable signs of toxicity present in either animal model following AAV.sFlt injection. These results suggest that AAV-mediated secretion gene therapy could be considered for treatment of retinal and choroidal neovascularizations.

The TAF5L gene on chromosome 1q42 is associated with type 1 diabetes in Russian affected patients

Type 1 diabetes (T1D) is a multifactorial autoimmune disease, with strong genetic component. Several susceptibility loci contribute to genetic predisposition to T1D. One of these loci have been mapped to chromosome 1q42 in UK and US joined affected family data sets but needs to be replicated in other populations. In this study, we evaluated sixteen microsatellites located on 1q42 for linkage with T1D in 97 Russian affected sibling pairs. A 2.7-cm region of suggestive linkage to T1D between markers D1S1644 and D1S225 was found by multipoint linkage analysis. The peak of linkage was shown for D1S2847 (P = 0.0005). Transmission disequilibrium test showed significant undertransmission of the 156-bp allele of D1S2847 from parents to diabetic children (28 transmissions vs. 68 nontransmissions, P = 0.043) in Russian affected families. A preferential transmission from parents to diabetic offspring was also shown for the T(-25) and T1362 alleles of the C/T(-25) and C/T1362 dimorphisms, both located at the TAF5L gene, which is situated 103 kb from D1S2847. Together with the A/C744 TAF5L SNP, these markers share common T(-25)/A744/T1362 and C(-25)/C744/T1362 haplotypes associated with higher and lower risk of diabetes (Odds Ratio = 2.15 and 0.62, respectively). Our results suggest that the TAF5L gene, encoding TAF5L-like RNA polymerase II p300/CBP associated factor (PCAF)-associated factor, could represent the susceptibility gene for T1D on chromosome 1q42 in Russian affected patients.

The human stress-activated protein kinase-interacting 1 gene encodes JNK-binding proteins

The orthologous proteins of the stress-activated protein kinase-interacting 1 (Sin1) family have been implicated in several different signal transduction pathways. In this study, we have investigated the function of the full-length human Sin1 protein and a C-terminally truncated isoform, Sin 1 alpha, which is produced by alternative splicing. Immunoblot analysis using an anti-Sin 1 polyclonal antibody showed that full-length Sin I and several smaller isoforms are widely expressed. Sin 1 was demonstrated to bind to c-Jun N-terminal kinase (JNK) in vitro and in vivo, while no interaction with p38- or ERK1/2-family MAPKs was observed. The Sin1 alpha isoform could also form a complex with JNK in vivo. Despite localizing in distinct compartments within the cell, both Sin1 and Sin1 alpha co-localized with JNK, suggesting that the Sin1 proteins could recruit JNK. Over-expression of full-length Sin1 inhibited the activation of JNK by UV-C in DG75 cells, as well as basal JNK-activity in HEK293 cells. These data suggest that the human Sin1 proteins may act as scaffold molecules in the regulation of signaling by JNK. (c) 2004 Elsevier Inc. All rights reserved.

Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn(1) mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn(1) was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn(1) mutation.