The tax gene product of human T-cell leukemia virus type I (HTLV-I) is a potent transcriptional activator that both stimulates viral gene expression and activates an array of cellular genes involved in T-cell growth. Tax acts indirectly by inducing or modifying the action of various host transcription factors, including members of the NF-KB/Rel family of enhancer-binding proteins. In resting T cells, many of these NF-KB/Rel factors are sequestered in the cytoplasm by various ankyrin-rich inhibitory proteins, including IKBot. HTLV-I Tax expression leads to the constitutive nuclear expression of biologically active NF-cB and c-Rel complexes; however, the biochemical mechanism(s) underlying this response remains poorly understood. In this study, we demonstrate that Tax-stimulated nuclear expression of NF-KcB in both HTLV-I-infected and Tax-transfected human T cells is associated with the phosphorylation and rapid proteolytic degradation of IKcBa. In contrast to prior in vitro studies, at least a fraction of the phosphorylated form of IKBa remains physically associated with the NF-KcB complex in vivo but is subject to rapid degradation, thereby promoting the nuclear translocation of the active NF-KcB complex. We further demonstrate that Tax induction of nuclear c-Rel expression is activated by the RelA (p65) subunit of NF-KB, which activates transcription of the c-rel gene through an intrinsic KB enhancer element. In normal cells, the subsequent accumulation of nuclear c-Rel acts to inhibit its own continued production, indicating the presence of an autoregulatory loop. However, the pathologic action of HTLV-I Tax leads to the deregulated and sustained nuclear expression of both NF-cB and c-Rel, a response that may contribute to HTLV-I-induced T-cell transformation.Human T-cell leukemia virus type I (HTLV-I) is a type C retrovirus that primarily produces disease within the CD4+ subset of human T cells (for recent reviews, see references 27 and 88). Infection with HTLV-I has been etiologically linked with an aggressive CD4+ T-cell malignancy termed adult T-cell leukemia (61, 89) and several different nonmalignant diseases (27,88), including a chronic neurodegenerative syndrome termed tropical spastic paraparesis (30) or HTLV-I-associated myelopathy (60). Although the precise mechanism by which HTLV-I transforms human T cells remains unclear, the HTLV-I transactivator protein, Tax, likely plays a central role. Indeed, Tax has been shown to possess transforming potential both in cultured cells (62,75,82) and in transgenic mice (39,57).Tax functions as a potent transcriptional activator that induces the expression of all viral genes controlled by the HTLV-I long terminal repeat (LTR) (21, 76) as well as various cellular genes (1,24,28,44,51,55,73), many of which are involved in T-cell activation and growth. These latter cellular genes include the interleukin-2 (IL-2) gene (51, 73) and the gene encoding the alpha chain of IL-2 receptor (IL-2Ra) (24,44,73 complete the transformation process leading clinically to the adult...

The intrarenal distribution of renin changes markedly during maturation. To determine whether renin gene expression changes along the developing renal vasculature, renin mRNA distribution was assessed using in situ hybridization histochemistry. Fetal, newborn, and adult kidney tissue sections from Wistar-Kyoto rats were hybridized with an oligonucleotide complementary to rat renin mRNA. In fetal kidneys, renin mRNA was found in the vascular pole of juxtamedullary glomeruli and along afferent, interlobular, and arcuate arteries. In kidneys from newborn rats, renin mRNA localized throughout the whole length of afferent arterioles, but was not detected in interlobular or arcuate arteries. In adult kidneys, hybridization signals were less intense and confined to the juxtaglomerular apparatus. Immunolocalization of renin with a polyclonal anti-rat renin antibody paralleled closely the mRNA distribution. Northern blot analyses demonstrated that renin mRNA levels were higher in fetal and newborn (20- and 10-fold, respectively) than in adult kidneys. We conclude the following. 1) The fetal kidney expresses the renin gene. 2) Expression of the renin gene is subjected to developmental changes. 3) As maturation progresses, localization of renin synthesis and storage shifts from large intrarenal arteries to a restricted, classical juxtaglomerular site in the afferent arteriole.

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were subcloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or alpha Gal(1-4)beta Gal-coated latex beads. CBA mice (N = 100) were challenged transurethrally with 10(5), 10(6), 10(7), or 10(9) cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.

Nuclear expression and consequent biological action of the eukaryotic NF-kappa B transcription factor complex are tightly regulated through its cytoplasmic retention by an ankyrin-rich inhibitory protein termed I kappa B alpha. I kappa B alpha specifically binds to and masks the nuclear localization signal of the RelA subunit of NF-kappa B, thereby effectively sequestering this transcription factor complex in the cytoplasm. Specific cellular activation signals lead to the rapid proteolytic degradation of I kappa B alpha and the concomitant nuclear translocation of NF-kappa B. However, the precise biochemical mechanisms underlying the inhibitory effects of I kappa B alpha on RelA and its inducible pattern of degradation remain unclear. By using HeLa cells transfected with various cDNAs end-coding epitope-tagged mutants of I kappa B alpha, our studies demonstrate the following: (i) sequences within the 72-amino-acid N-terminal region of I kappa B alpha are required for tumor necrosis factor alpha (TNF-alpha)-induced degradation but are fully dispensable for I kappa B alpha binding to and inhibition of RelA; (ii) serine residues located at positions 32 and 36 within the N-terminal region of I kappa B alpha represent major sites of induced phosphorylation (substitution of these serine residues with alanine abrogates TNF-alpha-induced degradation of I kappa B alpha); (iii) the C-terminal 40 residues of I kappa B alpha (amino acids 277 to 317), which include a PEST-like domain, are entirely dispensable for TNF-alpha-induced degradation and inhibition of RelA; (iv) a glutamine- and leucine-rich (QL) region of I kappa B alpha located between residues 263 and 277 and overlapping with the sixth ankyrin repeat is required for both inducible degradation and inhibition of RelA function; (v) regulation of I kappa B alpha degradation by this QL-rich region appears to occur independently of phosphorylation at serines 32 and 36. These findings thus indicate that I kappa B alpha is generally organized within distinct modular domains displaying different functional and regulatory properties. These studies have also led to the identification of a novel class of dominant-negative I kappa B alpha molecules that retain full inhibitory function on NF-kappa B yet fail to undergo stimulus-induced degradation. These molecules, which lack N-terminal sequences, potently inhibit TNF-alpha-induced activation of the human immune deficiency virus type 1 kappa B enhancer, thus indicating their possible use as general inhibitors of NF-kappa B.