Purification and Molecular Genetic Characterization of ZPU1, a Pullulanase-Type Starch-Debranching Enzyme from Maize1

This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.

Dynamic properties of the Ras switch I region and its importance for binding to effectors

We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)⋅GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. 31P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. 31P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.

γ-Aminobutyric acid type A receptors modulate cAMP-mediated long-term potentiation and long-term depression at monosynaptic CA3–CA1 synapses

cAMP induces a protein-synthesis-dependent late phase of long-term potentiation (LTP) at CA3–CA1 synapses in acute hippocampal slices. Herein we report cAMP-mediated LTP and long-term depression (LTD) at monosynaptic CA3–CA1 cell pairs in organotypic hippocampal slice cultures. After bath application of the membrane-permeable cAMP analog adenosine 3′,5′-cyclic monophosphorothioate, Sp isomer (Sp-cAMPS), synaptic transmission was enhanced for at least 2 h. Consistent with previous findings, the late phase of LTP requires activation of cAMP-dependent protein kinase A and protein synthesis. There is also an early phase of LTP induced by cAMP; the early phase depends on protein kinase A but, in contrast to the later phase, does not require protein synthesis. In addition, the cAMP-induced LTP is associated with a reduction of paired-pulse facilitation, suggesting that presynaptic modification may be involved. Furthermore, we found that Sp-cAMPS induced LTD in slices pretreated with picrotoxin, a γ-aminobutyric acid type A (GABAA) receptor antagonist. This form of LTD depends on protein synthesis and protein phosphatase(s) and is accompanied by an increased ratio of failed synaptic transmission. These results suggest that GABAA receptors can modulate the effect of cAMP on synaptic transmission and thus determine the direction of synaptic plasticity.

Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor α (TGFα), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

Antiviral activities of the soluble extracellular domains of type I interferon receptors

Alternative splicing leads to the expression of multiple isoforms of the subunits (IFNAR1 and IFNAR2) of the type I IFN receptor. Here we describe two transcripts representing extracellular forms of ovine IFNAR1 and show that soluble extracellular forms of both IFNAR2 and IFNAR1, prepared in recombinant form in Escherichia coli, have antiviral (AV) activity in the absence of IFN. Exposure of Madin-Darby bovine kidney cells to the extracellular domain (R2E) of IFNAR2 at concentrations as low as 10 nM afforded complete protection against vesicular stomatitis virus and led to the rapid activation of the transcription factors ISGF3 and GAF. Although R2E can bind IFN (Kd ≈70 nM), activity was observed irrespective of whether or not ligand was present. R2E was inactive on mouse L929 cells but active on L929 cells expressing a membraneanchored, ovine/human chimeric IFNAR2 with an ovine extracellular domain. The data suggest that AV activity is conferred by the ability of soluble R2E to associate with the transfected IFNAR2 subunit rather than resident murine IFNAR1. Soluble extracellular forms of IFNAR1 have lower AV activity than R2E on Madin-Darby bovine kidney cells but are less species-specific and protect wild-type L929 cells as efficiently as the transfected cell line, presumably by interacting with one of the murine receptor subunits.

Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases.

We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.

A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation.

The interleukin 2 (IL-2) gene is subject to two types of regulation: its expression is T-lymphocyte-specific and it is acutely dependent on specific activation signals. The IL-2 transcriptional apparatus integrates multiple types of biochemical information in determining whether or not the gene will be expressed, using multiple diverse transcription factors that are each optimally activated or inhibited by different signaling pathways. When activation of one or two of these factors is blocked IL-2 expression is completely inhibited. The inability of the other, unaffected factors to work is explained by the striking finding that none of the factors interacts stably with its target site in the IL-2 enhancer unless all the factors are present. Coordinate occupancy of all the sites in the minimal enhancer is apparently maintained by continuous assembly and disassembly cycles that respond to the instantaneous levels of each factor in the nuclear compartment. In addition, the minimal enhancer undergoes specific increases in DNase I accessibility, consistent with dramatic changes in chromatin structure upon activation. Still to be resolved is what interaction(s) conveys T-lineage specificity. In the absence of activating signals, the minimal IL-2 enhancer region in mature T cells is apparently unoccupied, exactly as in non-T lineage cells. However, in a conserved but poorly studied upstream region, we have now mapped several novel sites of DNase I hypersensitivity in vivo that constitutively distinguish IL-2 producer type T cells from cell types that cannot express IL-2. Thus a distinct domain of the IL-2 regulatory sequence may contain sites for competence- or lineage-marking protein contacts.

Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy.

This study evaluated hippocampal inhibitory function and the level of expression of gamma-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the alpha 2 and alpha 5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding alpha 1, beta 2, or gamma 2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of alpha CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.

Molecular characterization of a FKBP-type immunophilin from higher plants.

Immunophilins are intracellular receptors for the immunosuppressants cyclosporin A, FK506, and rapamycin. In addition to their use in organ transplantation, these natural products have been used to investigate signaling pathways in yeast, plant, and mammalian cells. We have recently described the identification of an immunosuppressant-sensitive signaling pathway in and the purification of several immunophilins from Vicia faba plants. We now report the molecular characterization of a 15 kDa FK506- and rapamycin-binding protein from V. faba (VfFKBP15). The amino acid sequence deduced from the cDNA starts with a signal peptide of 22 hydrophobic amino acids. The core region of VfFKBP15 is most similar to yeast and mammalian FKBP13 localized in the endoplasmic reticulum (ER). In addition, VfFKBP15 has a carboxyl-terminal sequence that is ended with SSEL, a putative ER retention signal. These findings suggest that VfFKBP15 is a functional homolog of FKBP13 from other organisms. Interestingly, two distinct cDNAs corresponding to two isoforms of FKBP15 have been cloned from Arabidopsis and also identified from rice data base, suggesting that pFKBP15 (plant FKBP15) is encoded by a small gene family in plants. This adds to the diversity of plant FKBP members even with the same subcellular localization and is in contrast with the situation in mammalian and yeast systems in which only one FKBP13 gene has been found. Like the mammalian and yeast FKBP13, the recombinant VfFKBP15 protein has rotamase activity that is inhibited by both FK506 and rapamycin with a Ki value of 30 nM and 0.9 nM, respectively, illustrating that VfFKBP15 binds rapamycin in preference over FK506. The mRNA of VfFKBP15 is ubiquitously expressed in various plant tissues including leaves, stems, and roots, consistent with the ER localization of the protein. Levels of VfFKBP15 mRNA are elevated by heat shock, suggesting a possible role for this FKBP member under stress conditions.

Cell cycle regulation and cell type-specific localization of the FtsZ division initiation protein in Caulobacter.

Many genes involved in cell division and DNA replication and their protein products have been identified in bacteria; however, little is known about the cell cycle regulation of the intracellular concentration of these proteins. It has been shown that the level of the tubulin-like GTPase FtsZ is critical for the initiation of cell division in bacteria. We show that the concentration of FtsZ varies dramatically during the cell cycle of Caulobacter crescentus. Caulobacter produce two different cell types at each cell division: (i) a sessile stalked cell that can initiate DNA replication immediately after cell division and (ii) a motile swarmer cell in which DNA replication is blocked. After cell division, only the stalked cell contains FtsZ. FtsZ is synthesized slightly before the swarmer cells differentiate into stalked cells and the intracellular concentration of FtsZ is maximal at the beginning of cell division. Late in the cell cycle, after the completion of chromosome replication, the level of FtsZ decreases dramatically. This decrease is probably mostly due to the degradation of FtsZ in the swarmer compartment of the predivisional cell. Thus, the variation of FtsZ concentration parallels the pattern of DNA synthesis. Constitutive expression of FtsZ leads to defects in stalk biosynthesis suggesting a role for FtsZ in this developmental process in addition to its role in cell division.

prlA suppressors in Escherichia coli relieve the proton electrochemical gradient dependency of translocation of wild-type precursors.

The SecY protein of Escherichia coli is an integral membrane component of the protein export apparatus. Suppressor mutations in the secY gene (prlA alleles) have been isolated that restore the secretion of precursor proteins with defective signal sequences. These mutations have never been shown to affect the translocation of wild-type precursor proteins. Here, we report that prlA suppressor mutations relieve the proton-motive force (pmf) dependency of the translocation of wild-type precursors, both in vivo and in vitro. Furthermore, the proton-motive force dependency of the translocation of a precursor with a stably folded domain in the mature region was suppressed by prlA mutations in vitro. These data show that prlA mutations cause a general relaxation of the export apparatus rather than a specific change that results in bypassing of the recognition of the signal sequence. In addition, these results are indicative for a mechanism in which the proton-motive force stimulates translocation by altering the conformation of the translocon.

Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice.

High-risk human papillomaviruses (HPVs), including type 16, have been identified as factors in cervical carcinogenesis. However, the presence and expression of the virus per se appear to be insufficient for carcinogenesis. Rather, cofactors most likely are necessary in addition to viral gene expression to initiate neoplasia. One candidate cofactor is prolonged exposure to sex hormones. To examine the possible effects of estrogen on HPV-associated neoplasia, we treated transgenic mice expressing the oncogenes of HPV16 under control of the human keratin-14 promoter (K14-HPV16 transgenic mice) and nontransgenic control mice with slow release pellets of 17beta-estradiol. Squamous carcinomas developed in a multistage pathway exclusively in the vagina and cervix of K14-HPV16 transgenic mice. Estrogen-induced carcinogenesis was accompanied by an incremental increase in the incidence and distribution of proliferating cells solely within the cervical and vaginal squamous epithelium of K14-HPV16 mice. Expression of the HPV transgenes in untreated transgenic mice was detectable only during estrus; estrogen treatment resulted in transgene expression that was persistent but not further upregulated, remaining at low levels at all stages of carcinogenesis. The data demonstrate a novel mechanism of synergistic cooperation between chronic estrogen exposure and the oncogenes of HPV16 that coordinates squamous carcinogenesis in the female reproductive tract of K14-HPV16 transgenic mice.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Immunocytochemical localization of the endogenous neuroexcitotoxin quinolinate in human peripheral blood monocytes/macrophages and the effect of human T-cell lymphotropic virus type I infection.

Quinolinate (Quin), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that appears to act through the N-methyl-D-aspartate receptor system, was localized in cultured human peripheral blood monocytes/macrophages (PBMOs) by using a recently developed immunocytochemical method. Quin immunoreactivity (Quin-IR) was increased in gamma interferon (IFN-gamma)-stimulated monocytes/macrophages (MOs). In addition, the precursors, tryptophan and kynurenine, significantly increased Quin-IR. Infection of MOs by human T-cell lymphotropic virus type I (HTLV-I) in vitro substantially increased both the number of Quin-IR cells and the intensity of Quin-IR. At the peak of the Quin-IR response, about 40% of the cells were Quin-IR positive. In contrast, only about 2-5% of the cells were positive for HTLV-I, as detected by both immunofluorescence for the HTLV-I antigens and PCR techniques for the HTLV-I Tax gene. These results suggest that HTLV-I-induced Quin production in MOs occurs by an indirect mechanism, perhaps via cytokines produced by the infection but not directly by the virus infection per se. The significance of these findings to the neuropathology of HTLV-I infection is discussed.

Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.

Two chemokine (chemoattractant cytokines) beta peptides, macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta), were induced in human monocyte cultures following infection with the human immunodeficiency virus type 1 (HIV-1). Induction depended on productive viral infection: not only did the kinetics of MIP-1 peptide induction closely follow those of viral replication, but monocyte cultures inoculated with heat-inactivated virus or infected in the presence of AZT failed to produce these chemokine beta peptides. In addition, HIV infection markedly altered the pattern of beta chemokine expression elicited by tumor necrosis factor (TNF), itself a potent proinflammatory cytokine upregulated during the development of AIDS. Reverse transcription (RT)-PCR and RT-in situ PCR studies on brain tissue from patients with AIDS dementia demonstrated elevated MIP-1 alpha and MIP-1 beta mRNA expression relative to comparable samples from HIV-1-infected patients without dementia. Cells expressing chemokines in HIV-1-infected brains were identified morphologically as microglia and astrocytes. As MIP-1 alpha and MIP-1 beta are potent chemoattractants for both monocytes and specific subpopulations of lymphocytes, this dysregulation of beta chemokine expression may influence the trafficking of leukocytes during HIV infection. These data, taken together, suggest a mechanism by which HIV-1-infected monocytes might recruit uninfected T cells and monocytes to sites of active viral replication or inflammation, notably the brain and lymph nodes.