Nrf2 and c-Jun regulation of antioxidant response element (ARE)-mediated expression and induction of gamma-glutamylcysteine synthetase heavy subunit gene.

gamma-Glutamylcysteine synthetase (gamma-GCS) is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species (ROS). The gamma-GCS gene is expressed ubiquitously and induced coordinately with NAD(P)H:quinone oxidoreductase(1) (NQO1) and glutathione S-transferase Ya (GST Ya) in response to xenobiotics and antioxidants. The antioxidant response element (ARE) is required for expression and induction of these genes. In the current report, we demonstrated that ARE-mediated gamma-GCS gene expression and induction is regulated by similar Nrf and Jun factors as reported earlier for the NQO1 and GST Ya genes. The gamma-GCS gene ARE competed with the binding of nuclear proteins (Nrf + Jun) to the NQO1 gene ARE (hARE). In addition, the overexpression of Nrf2 and Nrf1 with c-Jun significantly up-regulated gamma-GCS ARE-mediated basal expression and beta-naphthoflavone induction of the chloramphenicol acetyltransferase gene in transfected HepG2 cells. Interestingly, Nrf2 + c-Jun was more effective than Nrf1 + c-Jun in the regulation of ARE-mediated gamma-GCS gene expression. Further experiments demonstrated that the c-Jun level within the cells is an important determinant of the level of ARE-mediated gamma-GCS gene expression. Therefore, at higher concentrations of c-Jun, gamma-GCS gene expression is repressed, presumably due to generation of a sufficient amount of c-Jun + c-Fos complex that interferes with the binding of Nrf2 + c-Jun complex to the ARE.

The role of transglutaminase in the mechanism of action of tetanus toxin.

Tetanus toxin is a zinc-dependent metalloendoprotease that cleaves synaptobrevin, a polypeptide found in the membranes of synaptic vesicles. This action is thought to account for toxin-induced blockade of transmitter release. However, Facchiano and Luini (Fachiano, F., and Luini, A. (1992) J. Biol Chem. 267, 13267-13271) have proposed that tetanus toxin can stimulate transglutaminase, and Facchiano et al. (Facchiano, F., Benfenati, F., Valtorta, F., and Luini, A. (1993) J. Biol Chem. 268, 4588-4591) have further proposed that the stimulated enzyme produces cross-linking of synapsin. These actions might also account for toxin-induced blockade of exocytosis. Therefore, a series of experiments were performed to evaluate the possibility that tetanus toxin exerts its effects via transglutaminase. The results indicated that clostridial neurotoxins were poor substrates for the cross-linking effects of transglutaminase, and transglutaminase was a poor substrate for the proteolytic actions of tetanus toxin. In addition, at concentrations relevant to blockade of exocytosis, clostridial neurotoxins did not act on intact cells to stimulate transglutaminase, nor did they act on the isolated enzyme to stimulate cross-linking of putrescine and dimethylcasein. When used as competitive inhibitors of endogenous transglutaminase substrates, glycine methyl ester and monodansylcadaverine did not block toxin action. Furthermore, concentrations of calcium that were too low to support transglutaminase activity did not prevent toxin action. The data suggest that stimulation of transglutaminase is not the principal mechanism by which tetanus toxin blocks exocytosis in nerve cells.

Functional analysis of the N-terminal domain of Tat protein of the human immunodeficiency virus type 1.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent trans-activator of the viral long terminal repeat (LTR). The N-terminal region of Tat is rich in proline and acidic residues analogous to the activation domains of other transcription factors such as GAL-4 and CTF/NF-1. Several basic residues are also present in this region. To investigate the role of these structural features in the Tat-mediated trans-activation, we have chemically synthesized and evaluated Tat analogs with alanine or glutamine replacing one or more of these amino acid residues. Our data show that substitution of Glu-2, His-13, or all the proline in the Pro-Xaa3-Pro triad drastically reduced activity. In contrast, changes at Arg-7, Lys-12 and any one proline residue in the triad moderately reduced, and substitution of Lys-19 showed little effect on, activity. These results show that the native structure of the N-terminal 19 amino acid sequence is essential for Tat function, and that the overall topology of this domain and not the acidic residues alone appears necessary for trans-activation.

Synthetic HIV-1 Tat can dissociate HeLa nuclear protein-TAR RNA complexes in vitro: a novel Tat-nuclear protein interaction.

Multiple binding of Tat and nuclear protein(s) to HIV-1 TAR RNA appears to be essential for the Tat-mediated trans-activation. As synthetic Tat-(1-47), which lacks the basic domain and does not bind TAR RNA in vitro, efficiently transactivated HIV-1 LTR in HeLa nuclear extracts, we hypothesized that Tat might trans-activate by interaction with TAR RNA via a host nuclear protein. The role of nuclear proteins in Tat-TAR interaction was examined through evaluation of several synthetic Tat peptides for ability to bind TAR RNA in vitro both in the presence and in the absence of HeLa nuclear proteins. Our data show that both Tat-(1-47) and Tat-(1-86) interact with TAR RNA-bound nuclear proteins, leading to dissociation of the nuclear protein-TAR RNA complexes; the N-terminal sequence of Tat appears to be involved in this interaction. Thus, after binding to TAR RNA, Tat can interact with a proximal TAR-bound nuclear protein and the resulting Tat-nuclear protein complex, now displaced from TAR, may initiate a facile and rapid assembly of the RNA polymerase II transcription complex. This study thus recognizes a novel interaction between Tat and a nuclear protein(s). Here we propose that the interaction of Tat with a nuclear protein(s) occurring on TAR RNA may be one of several steps in the mechanism of Tat-mediated trans-activation of the HIV-1 LTR.

Activity of synthetic tat peptides in human immunodeficiency virus type 1 long terminal repeat-promoted transcription in a cell-free system.

The tat protein encoded by the human immunodeficiency virus type 1 is a potent trans-activator of gene expression from the viral long terminal repeat. The domains that are essential for trans-activation, a Pro-Xaa3-Pro triad, a cysteine-rich metal-binding sequence motif, and a cluster of basic residues, are present within the N-terminal 57 residues of tat. To determine the structural requirements for tat function and the role of metal binding at the transcription level alone, tat-(1-86) (full-length tat peptide), tat-(1-57), and tat-(1-47) were chemically synthesized. These peptides as well as the Cd2+ and Zn2+ complexes of tat-(1-86) and tat-(1-57) were evaluated for stimulation of transcription from the human immunodeficiency virus type 1 long terminal repeat by using cell-free in vitro methods. All three peptides produced a 7- to 9-fold increase over the basal level of transcription at a peptide concentration of 0.4 microM. Interestingly, at 4 microM, both tat-(1-57) and tat-(1-86) inhibited even the basal level of transcription. In contrast, tat-(1-47), which lacks the basic domain (residues 49-57), exhibited full stimulatory activity at 4 microM. Our data suggest, therefore, that the basic region may be responsible for the observed inhibitory activity of tat-(1-86) and tat-(1-57). Furthermore, binding to Zn2+ and not to Cd2+ ions only slightly augments (approximately 2-fold) the activity of the tat peptides.

Fc and complement receptor integrity of polymorphonuclear (PMN) cells following thermal injury.

In patients sustaining thermal injury, a sequential study was formulated to evaluate Fc and complement receptor expression of polymorphonuclear cells (PMN). Additionally, the role of factors in burn sera and maturity of PMN cells in the circulation were studied. The salient features of the study were: Marked reduction in Fc receptor expression by the 5th day of injury in both survivors and non-survivors. Thereafter levels gradually increased in survivors, though they were still below the normal range. In non-survivors, the depression was severe and persistent. In contrast to Fc receptor expression, complement receptor integrity was not grossly affected in both survivors and non-survivors. Burn sera collected from survivors on the 5th and 13th post-burn day showed reduction in Fc receptor expression of normal PMN cells, whereas sera obtained a month after the injury exhibited no inhibitory effect. Non-survivors sera inhibited Fc receptor expression of normal PMN cells on 5th, 13th and 21st post-burn days. The appearance, increase and disappearance of immature PMN cells in the circulation was correlated with the clinical progress of the patient. Mechanisms involved in the aberrations and its implications are discussed.

Audiological and haematological studies on the Todas of Nilgiris.

A detailed audiological study combined with certain haematological investigations which were never previously done was undertaken on Todas, a small vanishing tribe of Nilgiris, South India. Ten per cent of the Todas studied exhibited otosclerosis which, though lower than that of a previous study, is still a very high percentage when compared to the general population. The incidence of chronic suppurative otitis media in Todas is markedly low (1-6 per cent) and 13-3 per cent of Todas had sensorineural hearing loss. Evaluation of hearing thresholds at various frequencies in the different age groups did not show much variation from that of the general population. The incidence of presbyacusis is not found to be low as reported earlier. Blood pressure recordings did not show any age-related increase. The majority of Todas studied belonged to the blood group 'B' (63 per cent) and 16-6 per cent belonged to the 'AB' group, which is distinctly different from the distribution pattern seen in the general Indian population. There was no significant variation in the serum cholesterol level in Todas.