Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus.

The HIV-1-specific cytotoxic T lymphocyte (CTL) response is temporally associated with the decline in viremia during primary HIV-1 infection, but definitive evidence that it is of importance in virus containment has been lacking. Here we show that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp 160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL. The magnitude (> 100-fold), kinetics (30-72 days from onset of symptoms) and genetic pathways of virus escape from CTL pressure were comparable to virus escape from antiretroviral therapy, indicating the biological significance of the CTL response in vivo. One aim of HIV-1 vaccines should thus be to elicit strong CTL responses against multiple codominant viral epitopes.

First Report of Bacterial Spot of Tomato Caused by Xanthomonas gardneri in Pennsylvania.

Recent investigation of bacteria isolated from samples submitted to the Plant Disease Diagnostic Laboratory, Pennsylvania Department of Agriculture indicated that in 1995, Xanthomonas gardneri (ex Sutic 1957) (2) caused a leaf spot on tomato plants (Lycopersicon esculentum Mill.). In 1995, we examined 185 tomato and 36 pepper samples (13 field, 2 garden center, 38 greenhouse, 4 residence, 16 field-grown transplant, and 148 greenhouse-grown transplant samples). A processing company representative collected samples showing symptoms of bacterial spot of tomato on a hybrid, whole pack processing tomato, from a 16-ha field in Northumberland County, PA exhibiting almost 50% crop infection. Symptoms consisted of circular- to irregularly shaped, dark brown spots, <5 mm in diameter, and frequently with chlorotic haloes on leaves and stems. The center of a spot may be raised and scabby. Several spots on a single leaflet may coalesce and a portion or the entire leaflet may turn yellow or die. These symptoms were indistinguishable from those of bacterial spot caused by X. euvesicatoria, X. vesicatoria, and X. perforans. Bacterial streaming from lesions was evident under dark-field microscopy. Aerobic, gram-negative, yellow-pigmented, mucoid bacteria were isolated from the leaf spots and purified and stored in nutrient broth with 10% glycerol at -80°C. The 16S rRNA gene from a strain (PDA80951-95) typical of the cultures from these samples was sequenced (GenBank Accession No. GU573763). A BlastN search of GenBank revealed 100% nucleotide identity with the type strain of X. gardneri (XCGA2; No. AF123093). This strain also exhibited repetitive sequence-based (rep)-PCR profiles (4) identical to profiles of X. gardneri type strain XCGA2 DNA and produced a ~425-bp PCR product with BSX primers, a genetic marker indicative of X. gardneri (1). The strain was not amylolytic or pectolytic (2) and failed to utilize maltose, gentiobiose, and melezitose (3). For pathogenicity tests, inoculum was grown in nutrient broth with shaking for 24 h at 28°C. Inoculum was centrifuged, resuspended in sterile tap water, and adjusted to 2.5 × 108 CFU/ml. Lower leaf surfaces of tomato (cvs. Bonnie Best and Walter) and pepper (cvs. California Wonder and Early Niagara) plants were gently rubbed with sterile cheesecloth that was moistened with the inoculum. Strain PDA80951-95 caused leaf spots, with chlorotic haloes and occasional coalescence on both tomato and pepper, within 2 weeks at 15 s of mist per 20 min at 20 to 35°C in a secured greenhouse chamber. X. gardneri was only reisolated from symptomatic plants and its identity was confirmed by rep-PCR and absence of amylolytic and pectolytic activities. Negative controls consisting of X. campestris pv. campestris and sterile tap water did not show symptoms. A known type strain of X. gardneri was not included as a positive control for pathogenicity studies because this species is not known to occur in the United States (2). To our knowledge, this is the first report of bacterial spot on tomato plants caused by X. gardneri in Pennsylvania and the United States. Since the first occurrence in 1995, bacterial spot caused by X. gardneri reoccurred in Pennsylvania tomato fields in 2001 and consecutively from 2003 to 2009. Reference: (1) D. A. Cuppels et al. Plant Dis. 90:451, 2006. (2) J. B. Jones et al. Syst. Appl. Microbiol. 27:755, 2004. (3) A. M. Quezado-Duval et al. Plant Dis. 88:15, 2004. (4) D. J. Versalovic et al. Methods Mol. Cell Biol. 5:25, 1994.

Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I.

To investigate the mechanism by which immune activation augments replication of the human immunodeficiency virus type 1 (HIV-1) in infected T cells, four different classes of T cell mitogens were evaluated for their effects on the HIV-1 long terminal repeat (LTR). Phytohemagglutinin (PHA), a mitogenic lectin; phorbol 12-myristic 13-acetate, a tumor promoter; ionomycin, a calcium ionophore; and tat-1, the trans-activator protein from the human T cell leukemia/lymphoma virus type I (HTLV-I) each stimulated the HIV-1 LTR. Studies of deleted forms of the LTR supported a central role in these responses for the HIV-1 enhancer, which alone was sufficient for mitogen inducibility, but also suggested that other 5' positive and negative regulatory elements contribute to the overall magnitude of the response. Synergistic activation of the HIV-1 LTR (up to several thousandfold) was observed with combinations of these mitogens and the HIV-1--derived tat-III protein. Cyclosporin A, an immunosuppressive agent, inhibited PHA-mediated activation of the HIV-1 LTR but was without effect in the presence of other mitogens. Thus, HIV-1 gene expression and replication appear to be regulated, via the HIV-1 LTR, by the same mitogenic signals that induce T cell activation.

Trans-activator gene of HTLV-II induces IL-2 receptor and IL-2 cellular gene expression.

The human T-lymphotropic viruses types I and II (HTLV-I and -II) have been etiologically linked with certain T-cell leukemias and lymphomas that characteristically display membrane receptors for interleukin-2. The relation of these viruses to this growth factor receptor has remained unexplained. It is demonstrated here that introduction of the trans-activator (tat) gene of HTLV-II into the Jurkat T-lymphoid cell line results in the induction of both interleukin-2 receptor and interleukin-2 gene expression. The coexpression of these cellular genes may play a role in the altering T-cell growth following retroviral infection.

Antisense and antigene properties of peptide nucleic acids.

Peptide nucleic acids (PNAs) are polyamide oligomers that can strand invade duplex DNA, causing displacement of one DNA strand and formation of a D-loop. Binding of either a T10 PNA or a mixed sequence 15-mer PNA to the transcribed strand of a G-free transcription cassette caused 90 to 100 percent site-specific termination of pol II transcription elongation. When a T10 PNA was bound on the nontranscribed strand, site-specific inhibition never exceeded 50 percent. Binding of PNAs to RNA resulted in site-specific termination of both reverse transcription and in vitro translation, precisely at the position of the PNA.RNA heteroduplex. Nuclear microinjection of cells constitutively expressing SV40 large T antigen (T Ag) with either a 15-mer or 20-mer PNA targeted to the T Ag messenger RNA suppressed T Ag expression. This effect was specific in that there was no reduction in beta-galactosidase expression from a coinjected expression vector and no inhibition of T Ag expression after microinjection of a 10-mer PNA.

Structure of the human interleukin-2 receptor gene.

The gene encoding the human interleukin-2 (IL-2) receptor consists of 8 exons spanning more than 25 kilobases on chromosome 10. Exons 2 and 4 were derived from a gene duplication event and unexpectedly also are homologous to the recognition domain of human complement factor B. Alternative messenger RNA (mRNA) splicing may delete exon 4 sequences, resulting in a mRNA that does not encode a functional IL-2 receptor. Leukemic T cells infected with HTLV-I and normal activated T cells express IL-2 receptors with identical deduced protein sequences. Receptor gene transcription is initiated at two principal sites in normal activated T cells. Adult T cell leukemia cells infected with HTLV-I show activity at both of these sites, but also at a third transcription initiation site.

Isolation and expression of complementary DNAs encoding the human interleukin 2 receptor.

Complementary DNAs corresponding to the human receptor for interleukin 2 (IL-2) have been molecularly cloned, sequenced, and expressed in COS-1 cells. The human genome appears to contain a single structural gene for this receptor; however, when transcribed at least two messenger RNAs (mRNAs) are produced which vary in length due to the use of different polyadenylation signals. Sequence analysis of the cloned complementary DNAs indicates an alternate pathway of mRNA processing for this receptor. Splicing of a 216 base pairs segment contained within the protein coding region results in an mRNA unable to code for the IL-2 receptor. In contact complementary DNAs corresponding to unspliced mRNA encode membrane receptors which bind both IL-2 and anti-Tac (monoclonal anti-IL-2 receptor antibody). Analysis of the deduced amino acid sequence reveals that the receptor is composed of 272 amino acids including a signal peptide 21 amino acids in length. Hydrophobicity analysis suggests a single 19 amino acid transmembrane domain. A short intracytoplasmic domain composed of 13 amino acids is present at the carboxy terminus and contains three potential phosphate acceptor sites (serine and threonine but not tyrosine) and typical positively charged amino acids presumably involved in cytoplasmic anchoring. Two sites for N-linked glycosylation sites and numerous extracytoplasmic O-linked glycosylation sites are present.

Design of ruthenium-cytochrome c derivatives to measure electron transfer to cytochrome c peroxidase.

A new technique has been introduced to measure interprotein electron transfer which involves photoexcitation of a tris(bipyridine)ruthenium (Ru) complex covalently attached to one of the proteins. Four different strategies have been developed to specifically attach Ru to protein lysine amino groups, histidine imidazole groups, and cysteine sulhydryl groups. These strategies have been used to prepare more than 20 different singly-labeled Ru-cytochrome c derivatives. The new ruthenium photoexcitation technique has been used to study the mechanism for electron transfer between cytochrome c and cytochrome c peroxidase. Laser excitation of a complex between Ru-cytochrome c and cytochrome c peroxidase compound I results in formation of Ru(II*) which is a strong reducing agent, and rapidly transfers an electron to heme c Fe(III) to form Fe(II). The heme c Fe(II) then rapidly transfers an electron to the Trp-191 radical cation in CMPI. The rate constant for this reaction is 6 x 10(4) s-1 for a horse Ru-cytochrome c derivative labeled at lysine 27, and greater than 10(6) s-1 for yeast Ru-cytochrome c derivatives. A second laser flash results in electron transfer from heme c to the oxyferryl heme in cytochrome c peroxidase compound II with a rate constant of 350 s-1. The ruthenium photoreduction technique has been used to study the interaction domain between the two proteins, the pathway for electron transfer to the radical cation and the oxyferryl heme, and the specific residues in the heme crevice which control the electron transfer properties of the Trp-191 radical cation and the oxyferryl heme.

MAF1, a novel plant protein interacting with matrix attachment region binding protein MFP1, is located at the nuclear envelope.

The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.

Lymphocytes isolated from the intestinal lamina propria of normal nonhuman primates have increased expression of genes associated with T-cell activation.

Synthesis of interleukin-2 (IL-2) and expression of interleukin-2 receptors play central roles in T-cell activation, proliferation, and differentiation. The state of activation of T lymphocytes in the intestinal lamina propria was compared with that of circulating lymphocytes and lymphocytes isolated from the spleen or mesenteric lymph nodes of normal nonhuman primates. Lamina propria lymphocytes (LPL) had significantly higher proliferation in response to recombinant IL-2 compared with the other populations. In agreement with this finding, LPL had a significantly higher percentage of interleukin-2 receptor-positive (IL-2R+) cells as determined by staining with fluoresceinated monoclonal anti-IL-2R antibody. Two-color immunofluorescence staining showed that both CD4+ and CD8+ lamina propria T cells were IL-2R+. It was also found that the percentage of major histocompatibility complex class II-positive T cells was higher in the lamina propria. Northern blot analysis with a cDNA specific for the IL-2R showed that unstimulated LPL had easily detectable IL-2R mRNA, whereas no IL-2R mRNA was found in unstimulated lymphocyte populations from other sites. The activation of the IL-2R gene in LPL was not associated with the activation of other cellular genes (actin, major histocompatibility complex class I). Although no IL-2 bioactivity was measured in culture supernatants of unstimulated lymphocytes, concanavalin A-stimulated LPL produced significantly more IL-2 than other lymphocytes. This finding was confirmed at the molecular level as IL-2 mRNA was not detected in unstimulated LPL but was found in concanavalin A-stimulated LPL. Thus, normal lamina propria T lymphocytes have selective expression of genes associated with cell activation.

Reconstitution of high affinity IL-2 receptor expression in a human T-cell line using a retroviral cDNA expression vector.

A recombinant amphotropic retrovirus was used to introduce the protein-coding region of the IL-2 receptor cDNA derived from HUT-102 cells into human CEM leukemic T-cells that lack these receptors. CEM T-cells that contained the virus expressed functional IL-2 receptors that transiently mediated five- to tenfold increases in [3H]thymidine incorporation following the addition of picomolar quantities of IL-2. Although IL-2 responsiveness was subsequently lost, it could be reinduced by cellular activation with the OKT11 monoclonal antibody. This phenotype also proved unstable with progressive time in culture. Despite the loss of IL-2 responsiveness, the infected CEM T-cells continued to express Tac antigen and displayed 50 to 200 high-affinity IL-2 receptors per cell that bound IL-2 with a dissociation constant of 4.3 pM. This affinity is fully equivalent to that detected on activated normal T-cells (2 to 50 pM). The apparent molecular size of the Tac antigen on these cells (55,000 to 60,000 daltons) was comparable to that on normal activated T-cells but 5000 daltons larger than the aberrant IL-2 receptors on HUT-102 cells. These data demonstrate that expression of a human IL-2 receptor cDNA in human T-cells results in high-affinity IL-2 receptor display that transiently imparts an IL-2 responsive state of growth. These results also raise the possibility that the T11 surface receptor may play an important regulatory role in high-affinity IL-2 receptor expression.

Interleukin 2 receptor gene expression in normal human T lymphocytes.

We have used cDNAs for the human interleukin 2 (IL-2) receptor to study IL-2 receptor gene expression in normal activated T cells. Resting T cells do not contain detectable IL-2 receptor mRNA. Within 1 hr after stimulation with phytohemagglutinin (PHA), a large, presumably nuclear precursor RNA species is seen, which then gradually disappears. Mature IL-2 receptor mRNA forms appear within 8 hr after stimulation, reach peak levels between 8 and 24 hr, and then decline. Thus, in PHA-activated lymphocytes the rise and fall in IL-2 receptor mRNA levels precede by more than 24 hr the peak and decline of IL-2 receptor protein expression occurring at the cell surface. 4 beta-Phorbol 12-myristate 13-acetate (PMA) also stimulates IL-2 receptor mRNA and protein expression by T cells. Combinations of optimal concentrations of PHA and PMA produce an additive effect on IL-2 receptor mRNA levels, suggesting that PHA and PMA may induce IL-2 receptor gene expression through different, complementary mechanisms. Nuclease S1-protection assays indicate that IL-2 receptor mRNAs may differ in length due to the use of three different polyadenylylation signals. Further, these assays demonstrate the presence of transcripts that lack a 216-base segment within the protein-coding region and thus do not encode a functional IL-2 receptor. Nuclear transcription assays indicate that the increase in IL-2 receptor mRNA is reflected at the level of transcription. Thus, IL-2 receptor gene regulation controls IL-2 receptor expression at the cell surface and is intimately linked to the control of T-cell proliferation.

The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for v-Rel-mediated repression.

Molecular cloning of the polypeptide component of the Rel-related human p75 nucleoprotein complex has revealed its identity with the 65-kDa (p65) subunit of NF-kappa B. Functional analyses of chimeric proteins composed of NF-kappa B p65 C-terminal sequences linked to the DNA-binding domain of the yeast GAL4 polypeptide have indicated that the final 101 amino acids of NF-kappa B p65 comprise a potent transcriptional activation domain. Transient transfection of human T cells with an expression vector encoding NF-kappa B p65, but not NF-kappa B p50, produced marked transcriptional activation of a basal promoter containing duplicated kappa B enhancer motifs from the long terminal repeat of type 1 human immunodeficiency virus. These stimulatory effects of NF-kappa B p65 were synergistically enhanced by coexpression of NF-kappa B p50 but were completely inhibited by coexpression of the v-rel oncogene product. Together, these functional studies demonstrate that NF-kappa B p65 is a transactivating subunit of the heterodimeric NF-kappa B complex and serves as one cellular target for v-Rel-mediated transcriptional repression.

Sequence requirements for ligand binding and cell surface expression of the Tac antigen, a human interleukin-2 receptor.

Discrete peptide domains within the primary sequence of cell-surface receptor glycoproteins are believed to regulate not only their function but also their targeting to the cell membrane. To identify sequence elements required for intracellular transport and ligand binding by the human Tac interleukin-2 (IL-2) receptor, we prepared expression plasmids encoding a series of artificially mutated or naturally occurring variants of the Tac cDNA. In particular, we sought to further delineate the functional role of the sequences contributed by each of the eight exons that together encode the Tac protein. Deletion of exons 5 through 8 of the receptor had no detectable effect on IL-2 binding or intracellular transport of the Tac protein, and resulted in secreted forms of this IL-2-binding protein. Removal of sequences corresponding to all of exon 4 ablated IL-2 binding activity yet still permitted transport to the cell surface. In contrast, partial deletion of exon 4 sequences resulted in proteins that not only lacked IL-2 binding activity but also were sequestered within the endoplasmic reticulum. Removal of one or both of the N-linked glycosylation sites present in the Tac protein did not impair receptor transport or ligand binding. These results demonstrate that exon 4 of the Tac gene encodes amino acid residues that play an important role in regulating both the intracellular transport and function of this IL-2 receptor.

Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein.

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.

Induction of interleukin-2 receptor-alpha gene expression is regulated by post-translational activation of kappa B specific DNA binding proteins.

T cell mitogens induce the expression of specific trans-acting DNA binding proteins that in turn regulate the expression of the interleukin-2 receptor-alpha (IL-2R alpha) gene. To investigate whether de novo protein synthesis is required for the activation of these transacting factors and the induced expression of this receptor gene, Jurkat T cells were incubated with various inhibitors of protein synthesis prior to stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA). Despite the presence of cycloheximide or anisomycin at concentrations sufficient to block greater than 97% of cellular protein synthesis, phytohemagglutinin and phorbol 12-myristate 13-acetate effectively induced the expression of the IL-2R alpha gene as measured at the mRNA level. Similarly, gel retardation, DNA footprinting, and DNA-protein cross-linking studies revealed that these mitogens induced the activation of two predominant DNA binding proteins (50-55 and 80-90 kDa) in the presence or absence of cycloheximide and anisomycin. Both of these proteins specifically interacted with a kappa B-like binding site present in the IL-2R alpha promoter (-267 to -256) that is requisite for mitogen-induced expression of this receptor gene. These findings support a post-translational mechanism of induction of pre-existing, but inactive, DNA binding proteins which in turn bind to and activate the IL-2R alpha gene.

Strand-invasion of duplex DNA by peptide nucleic acid oligomers.

Polyamide oligomers, termed peptide nucleic acids (PNAs), bind with high affinity to both DNA and RNA and offer both antisense and antigene approaches for regulating gene expression. When a PNA binds to a complementary sequence in a double-stranded DNA, one strand of the duplex is displaced, and a stable D-loop is formed. Unlike oligodeoxynucleotides for which binding polarity is determined by the deoxyribose sugar, the unrestrained polyamide backbone of the PNA could permit binding to a DNA target in an orientation-independent manner. We now provide evidence that PNAs can, in fact, bind to their complementary sequence in DNA independent of the DNA-strand polarity--that is, a PNA binds to DNA in both "parallel" and "antiparallel" fashion. With a mixed-sequence 15-mer PNA, kinetic studies of PNA.DNA interactions revealed that D-loop formation was rapid and the complex was stable for several hours. However, when measured either by gel-mobility-shift analysis or RNA polymerase II-elongation termination, D-loop formation was salt dependent, but PNA-strand dissociation was not salt dependent. We observed that D-loop-containing DNA fragments had anomalous gel mobilities that varied as a function of the position of the D-loop relative to the DNA termini. On the basis of permutation analysis, the decreased mobility of the PNA.DNA complex was attributed to a bend in the DNA at or near the D-loop.

Role of configurational gating in intracomplex electron transfer from cytochrome c to the radical cation in cytochrome c peroxidase.

Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.

The v-rel oncogene encodes a kappa B enhancer binding protein that inhibits NF-kappa B function.

Studies of NF-kappa B suggest that this enhancer binding activity corresponds to a family of at least four proteins (p50, p55, p75, and p85) differentially induced with biphasic kinetics during T cell activation. While p55 and p50 are closely related to the 50 kd DNA binding subunit of NF-kappa B, p75 and p85 exhibit DNA binding properties that distinguish them from this 50 kd polypeptide and its regulatory subunits I kappa B and p65. All four members of this kappa B-specific protein family are structurally related to the v-Rel oncoprotein and one, p85, appears identical to human c-Rel. v-Rel, but not nontransforming v-Rel mutants, binds to the kappa B enhancer and inhibits NF-kappa B-activated transcription from the IL-2 receptor alpha promoter and HIV-1 LTR. These findings suggest a Rel-related family of kappa B enhancer binding proteins and raise the possibility that the transforming activity of v-Rel is linked to its inhibitory action on cellular genes under NF-kappa B control.