The homologous regulators ANR of Pseudomonas aeruginosa and FNR of Escherichia coli have overlapping but distinct specificities for anaerobically inducible promoters.

The anaerobic transcriptional regulator ANR induces the arginine deiminase and denitrification pathways in Pseudomonas aeruginosa during oxygen limitation. The homologous activator FNR of Escherichia coli, when introduced into an anr mutant of P. aeruginosa, could functionally replace ANR for anaerobic growth on nitrate but not for anaerobic induction of arginine deiminase. In an FNR-positive E. coli strain, the ANR-dependent promoter of the arcDABC operon, which encodes the enzymes of the arginine deiminase pathway, was not expressed. To analyse systematically these distinct induction patterns, a lacZ promoter-probe, broad-host-range plasmid containing various -40 regions (the ANR/FNR recognition sequences) and -10 promoter sequences was constructed. These constructs were tested in P. aeruginosa and in E. coli expressing either ANR or FNR. In conjunction with the consensus -10 hexamer of E. coli sigma 70 RNA polymerase (TATAAT), the consensus FNR site (TTGAT ..... ATCAA) was recognized efficiently by ANR and FNR in both hosts. By contrast, when promoters contained the Arc box (TTGAC .... ATCAG), which is found in the arcDABC promoter, or a symmetrical mutant FNR site (CTGAT .... ATCAG), ANR was a more effective activator than was FNR. Conversely, an extended 22 bp, fully symmetrical FNR site allowed better activation with FNR than with ANR. Combination of the arc promoter -10 sequence (CCTAAT) with the Arc box or the consensus FNR site resulted in good ANR-dependent expression in P. aeruginosa but gave practically no expression in E. coli, suggesting that RNA polymerase of P. aeruginosa differs from the E. coli enzyme in -10 recognition specificity. In conclusion, ANR and FNR are able to activate the RNA polymerases of P. aeruginosa and E. coli when the -40 and -10 promoter elements ae identical or close to the E. coli consensus sequences.

IB1 reduces cytokine-induced apoptosis of insulin-secreting cells.

IB1/JIP-1 is a scaffold protein that interacts with upstream components of the c-Jun N-terminal kinase (JNK) signaling pathway. IB1 is expressed at high levels in pancreatic beta cells and may therefore exert a tight control on signaling events mediated by JNK in these cells. Activation of JNK by interleukin 1 (IL-1beta) or by the upstream JNK constitutive activator DeltaMEKK1 promoted apoptosis in two pancreatic beta cell lines and decreased IB1 content by 50-60%. To study the functional consequences of the reduced IB1 content in beta cell lines, we used an insulin-secreting cell line expressing an inducible IB1 antisense RNA that lead to a 38% IB1 decrease. Reducing IB1 levels in these cells increased phosphorylation of c-Jun and increased the apoptotic rate in presence of IL-1beta. Nitric oxide production was not stimulated by expression of the IB1 antisense RNA. Complementary experiments indicated that overexpression of IB1 in insulin-producing cells prevented JNK-mediated activation of the transcription factors c-Jun, ATF2, and Elk1 and decreased IL-1beta- and DeltaMEKK1-induced apoptosis. These data indicate that IB1 plays an anti-apoptotic function in insulin-producing cells probably by controlling the activity of the JNK signaling pathway.

DNA sequence of the gene encoding the Zc1 protein from Zea mays W64A

The Zein-2 component named Zc 1 corresponds to a storage protein of an apparent M.W. of 16 kDa present in maize endosperm.

DNA sequence of the gene encoding the Zc1 protein from Zea mays W64A

The Zein-2 component named Zc 1 corresponds to a storage protein of an apparent M.W. of 16 kDa present in maize endosperm.

Sequence analysis of a genomic clone encoding a Zc2 protein from Zea mays W64A

A genomic clone (p268c) coding for the 28 kD storage protein Zc2 from maize endosperm has been isolated and sequenced.

Sequence analysis of a genomic clone encoding a Zc2 protein from Zea mays W64A

A genomic clone (p268c) coding for the 28 kD storage protein Zc2 from maize endosperm has been isolated and sequenced.

The design and characterization of receptor-selective APRIL variants.

A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily with an important role in humoral immunity, is also implicated in several cancers as a prosurvival factor. APRIL binds two different TNF receptors, B cell maturation antigen (BCMA) and transmembrane activator and cylclophilin ligand interactor (TACI), and also interacts independently with heparan sulfate proteoglycans. Because APRIL shares binding of the TNF receptors with B cell activation factor, separating the precise signaling pathways activated by either ligand in a given context has proven quite difficult. In this study, we have used the protein design algorithm FoldX to successfully generate a BCMA-specific variant of APRIL, APRIL-R206E, and two TACI-selective variants, D132F and D132Y. These APRIL variants show selective activity toward their receptors in several in vitro assays. Moreover, we have used these ligands to show that BCMA and TACI have a distinct role in APRIL-induced B cell stimulation. We conclude that these ligands are useful tools for studying APRIL biology in the context of individual receptor activation.

The peroxisome proliferator-activated receptor (PPAR) β/δ agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3 (STAT3) activation and insulin resistance in human liver cells.

AIM/HYPOTHESIS: IL-6 induces insulin resistance by activating signal transducer and activator of transcription 3 (STAT3) and upregulating the transcription of its target gene SOCS3. Here we examined whether the peroxisome proliferator-activated receptor (PPAR)β/δ agonist GW501516 prevented activation of the IL-6-STAT3-suppressor of cytokine signalling 3 (SOCS3) pathway and insulin resistance in human hepatic HepG2 cells. METHODS: Studies were conducted with human HepG2 cells and livers from mice null for Pparβ/δ (also known as Ppard) and wild-type mice. RESULTS: GW501516 prevented IL-6-dependent reduction in insulin-stimulated v-akt murine thymoma viral oncogene homologue 1 (AKT) phosphorylation and in IRS-1 and IRS-2 protein levels. In addition, treatment with this drug abolished IL-6-induced STAT3 phosphorylation of Tyr⁷⁰⁵ and Ser⁷²⁷ and prevented the increase in SOCS3 caused by this cytokine. Moreover, GW501516 prevented IL-6-dependent induction of extracellular-related kinase 1/2 (ERK1/2), a serine-threonine protein kinase involved in serine STAT3 phosphorylation; the livers of Pparβ/δ-null mice showed increased Tyr⁷⁰⁵- and Ser⁷²⁷-STAT3 as well as phospho-ERK1/2 levels. Furthermore, drug treatment prevented the IL-6-dependent reduction in phosphorylated AMP-activated protein kinase (AMPK), a kinase reported to inhibit STAT3 phosphorylation on Tyr⁷⁰⁵. In agreement with the recovery in phospho-AMPK levels observed following GW501516 treatment, this drug increased the AMP/ATP ratio and decreased the ATP/ADP ratio. CONCLUSIONS/INTERPRETATION: Overall, our findings show that the PPARβ/δ activator GW501516 prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 phosphorylation and preventing the reduction in phospho-AMPK levels. These effects of GW501516 may contribute to the prevention of cytokine-induced insulin resistance in hepatic cells.

Spatio-temporal sequence of cross-regulatory events in root meristem growth.

A central question in developmental biology is how multicellular organisms coordinate cell division and differentiation to determine organ size. In Arabidopsis roots, this balance is controlled by cytokinin-induced expression of SHORT HYPOCOTYL 2 (SHY2) in the so-called transition zone of the meristem, where SHY2 negatively regulates auxin response factors (ARFs) by protein-protein interaction. The resulting down-regulation of PIN-FORMED (PIN) auxin efflux carriers is considered the key event in promoting differentiation of meristematic cells. Here we show that this regulation involves additional, intermediary factors and is spatio-temporally constrained. We found that the described cytokinin-auxin crosstalk antagonizes BREVIS RADIX (BRX) activity in the developing protophloem. BRX is an auxin-responsive target of the prototypical ARF MONOPTEROS (MP), a key promoter of vascular development, and transiently enhances PIN3 expression to promote meristem growth in young roots. At later stages, cytokinin induction of SHY2 in the vascular transition zone restricts BRX expression to down-regulate PIN3 and thus limit meristem growth. Interestingly, proper SHY2 expression requires BRX, which could reflect feedback on the auxin responsiveness of SHY2 because BRX protein can directly interact with MP, likely acting as a cofactor. Thus, cross-regulatory antagonism between BRX and SHY2 could determine ARF activity in the protophloem. Our data suggest a model in which the regulatory interactions favor BRX expression in the early proximal meristem and SHY2 prevails because of supplementary cytokinin induction in the later distal meristem. The complex equilibrium of this regulatory module might represent a universal switch in the transition toward differentiation in various developmental contexts.

Long sequence duplications, repeats, and palindromes in HIV-1 gp120: length variation in V4 as the product of misalignment mechanism.

We have shown that indels in gp120 V4 are associated to the presence of duplicated and palindromic sequences, suggesting that they may be produced by strand-slippage misalignment mechanism. Indels in V4 involved region-specific duplications 9 to 15 bp long, and repeats of various lengths, associated to trinucleotides AAT. No duplications were found in V3 and C3. The frequency of palindromic sequences in individual genes was found to be significantly higher in gp120 (p < or = 3.00E-7), and significantly lower in Tat (p < or = 9.00E-7) than the average frequency calculated over the full genome. The finding of elements of misalignment in association with indels in V4 suggests that these mutations may occur in proviral DNA after integration of HIV into the host genome. It also implies that occurrence of large indels in gp120 is not random but is directed by the presence and distribution of elements of misalignment in the HIV genome.

The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that down-regulates viral transcription.

The RNA genome of the human T-cell leukemia virus type 1 (HTLV-1) codes for proteins involved in infectivity, replication, and transformation. We report in this study the characterization of a novel viral protein encoded by the complementary strand of the HTLV-1 RNA genome. This protein, designated HBZ (for HTLV-1 bZIP factor), contains a N-terminal transcriptional activation domain and a leucine zipper motif in its C terminus. We show here that HBZ is able to interact with the bZIP transcription factor CREB-2 (also called ATF-4), known to activate the HTLV-1 transcription by recruiting the viral trans-activator Tax on the Tax-responsive elements (TxREs). However, we demonstrate that the HBZ/CREB-2 heterodimers are no more able to bind to the TxRE and cyclic AMP response element sites. Taking these findings together, the functional inactivation of CREB-2 by HBZ is suggested to contribute to regulation of the HTLV-1 transcription. Moreover, the characterization of a minus-strand gene protein encoded by HTLV-1 has never been reported until now.

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

Cooccurrence of predominant Panton-Valentine leukocidin-positive sequence type (ST) 152 and multidrug-resistant ST 241 Staphylococcus aureus clones in Nigerian hospitals.

Ninety-six clinical isolates of Staphylococcus aureus from Nigeria were characterized phenotypically and genetically. Twelve multidrug-resistant methicillin (meticillin)-resistant S. aureus (MRSA) isolates carrying a new staphylococcal cassette chromosome mec element and a high proportion of Panton-Valentine leukocidin (PVL)-positive methicillin-susceptible S. aureus (MSSA) isolates were observed. The cooccurrence of multidrug-resistant MRSA and PVL-positive MSSA isolates entails the risk of emergence of a multidrug-resistant PVL-positive MRSA clone.