Intron sequences are involved in the plastid- and light-dependent expression of the spinach PsaD gene

OBJECTIVE: To obtain preliminary information about the safety and efficacy of intravenous immune globulin (IVIG: Iveegam, Immuno AG, Vienna) in the treatment of polyarticular juvenile rheumatoid arthritis (poly-JRA) resistant to other forms therapy. METHODS: We used a multicentered, phase I/II blinded-withdrawal design with stratified entry. All patients began by receiving open infusions of IVIG at a dose between 1.5 and 2.0 g/kg/infusion (100 g maximum) bimonthly for the first 2 months, then monthly for up to 6 months. Beginning at Month 3, those who met the criteria for "clinically important improvement" were randomized to receive monthly infusions for 4 months of either placebo or IVIG in a double blind (DB) phase. Patients were permitted nonsteroidal antiinflammatory drugs, slow acting antirheumatic drugs, and low dose (< 10 mg/day) prednisone at constant doses. An "early escape" provision in the DB allowed those who showed "clinically important worsening" to again receive IVIG (if taking placebo) or a higher dose of IVIG (if taking the lower dose of IVIG). RESULTS: Efficacy. Twenty-five children entered the trial and 19 (76%) met the criteria for "clinically important improvement" during the open phase (OP) and entered the DB. Three patients completed the OP but failed to meet the criteria for response, and 3 patients dropped out of the OP, none of whom showed benefit from IVIG. Treatment effect sizes produced by IVIG were moderate to large for all variables in the OP. Patients who continued IVIG in the DB continued to show improvement over that achieved in the OP. Those given placebo showed a rapid loss of efficacy, suggesting IVIG has a limited duration of effect after discontinuation. Safety. No patient developed serious or unexpected adverse side effects in the open or DB phases, and none dropped out of the study due to toxicity or side effects. CONCLUSION: Substantial clinical improvement from IVIG is produced in about three-fourths of patients with poly-JRA during open administration, but the duration of the beneficial effect is short after discontinuation. Those with disease < 3 years' duration may be more likely to respond than those who have had their disease for > 5 years. Short term safety is excellent.     

Induction and expression of seed-specific promoters in Arabidopsis embryo-defective mutants

Our previous study in vivo suggested that vascular smooth muscle cells (VSMCs) in stroke-prone spontaneously hypertensive rats (SHRSP) were vulnerable when plasma components were deficient. Therefore, we cultured VSMCs isolated from normotensive and hypertensive rats to clarify the weakness of VSMCs isolated from hypertensive rats and maintained in plasma-deficient conditions by employing ultrastructural and biochemical analyses. VSMCs, obtained from normotensive rats and cultured without fetal bovine serum (FBS) for 1 week, were intact and well differentiated; without FBS for 2 weeks retained their original structures except for several degenerative changes. VSMCs, obtained from hypertensive rats and cultured without FBS for 2 weeks, were extensively damaged and lost their cell organelles. Apoptotic bodies were frequently observed. We also cultured VSMCs in medium containing a variety of growth factors. VSMCs obtained from normotensive rats and cultured with epidermal growth factor or insulin-like growth factor-1 for 2 weeks were almost intact, as were VSMCs from hypertensive rats, although some degenerative changes of cell organelles were observed. VSMCs from hypertensive rats, maintained with platelet-derived growth factor-BB or basic fibroblast growth factor, were generally in poor condition. Thus VSMCs from hypertensive rats have hereditary weaknesses in cell survival including apoptosis and require specific growth factors for their maintenance.     

Developmental expression of a DNA repair gene in Arabidopsis

As the use of electroconvulsive therapy (ECT) increases, the chance of a practitioner's encountering a patient with significant heart failure, ventricular dysfunction, or valvular heart disease also increases. This article reviews the epidemiology, pathophysiology, and available data on the risk of ECT in these patients. Recommendations are made regarding evaluation and treatment of such patients. Some special situations are identified that may require a modification of routine procedures. Overall, ECT can be performed safely in most patients with underlying cardiac conditions, as long as appropriate precautions are taken to identify these patients ahead of time.     

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.     

Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression

To identify plant defense responses that limit pathogen attack, Arabidopsis eds mutants that exhibit enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 were previously identified. In this study, we show that each of four eds mutants (eds5-1, eds6-1, eds7-1, and eds9-1) has a distinguishable phenotype with respect to the degree of susceptibility to a panel of bacterial phytopathogens and the ability to activate pathogenesis-related PR-1 gene expression after pathogen attack. None of the four eds mutants exhibited observable defects in mounting a hypersensitive response. Although all four eds mutants were also capable of mounting a systemic acquired resistance response, enhanced growth of P. s. maculicola ES4326 was still apparent in the secondarily infected leaves of three of the eds mutants. These data indicate that eds genes define a diverse set of previously unknown defense responses that affect resistance to virulent pathogens.     

Nutrient regulation of gene expression: a methodological strategy

Nutrients should be viewed as the earliest of the hormone signals which allowed an organism to respond to changes in its nutrient environment. Defining nutrient function at the nuclear level will permit us to understand how our dietary environment is related to the development of nutritionally related pathophysiologies such as diabetes, heart disease, and cancer. Moreover, through the application of molecular techniques, we will begin to understand how specific nutrients govern the developmental pattern of specific organs such as the kidney. In this review, the fatty acid synthase gene is employed as a model to demonstrate how one sequentially approaches questions pertaining to the regulation of gene expression by a nutrient, and the article presents a nuclear explanation for how dietary polyunsaturated fats decrease blood triglycerides. More importantly, studies of this nature provide a basis for screening genetically susceptible populations and information that will allow the nutritionists of the 21st century to customize a diet for patients at risk.     

Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion

A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.     

age Mutants of Arabidopsis exhibit altered auxin-regulated gene expression

An Arabidopsis transgenic line was constructed expressing beta-glucuronidase (GUS) via the auxin-responsive domains (AuxRDs) A and B (BA-GUS) of the PS-IAA4/5 gene in an indoleacetic acid (IAA)-dependent fashion. GUS expression was preferentially enhanced in the root elongation zone after treatment of young seedlings with 10(-7) M IAA. Expression of the BA-GUS gene in the axr1, axr4, and aux1 mutants required 10- to 100-fold higher auxin concentration than that in the wild-type background. GUS expression was nil in the axr 2 and axr 3 mutants. The transgene was used to isolate mutants exhibiting altered auxin-responsive gene expression (age). Two mutants, age1 and age2, were isolated and characterized. age1 showed enhanced sensitivity to IAA, with strong GUS expression localized in the root elongation zone in the presence of 10(-8) M IAA. In contrast, age2 exhibited ectopic GUS expression associated with the root vascular tissue, even in the absence of exogenous IAA. Morphological and molecular analyses indicated that the age1 and age2 alleles are involved in the regulation of gene expression in response to IAA.     

Cadherin-6 in the developing mouse brain: expression along restricted connection systems and synaptic localization suggest a potential role in neuronal circuitry

Multiple subtypes of the cadherin homophilic cell-cell adhesion molecule are expressed differentially in developing and mature brains, each being expressed in restricted neuronal groups. Cadherin-6 (cad6) is one of such cadherins. Recent studies of cad6 mRNA expression in the postnatal mouse forebrain showed that it occurs in neurons constituting a specific subset of thalamocortical connections. Here we analyzed the localization of cad6 mRNA as well as its protein in the entire central nervous system and also in cranial ganglia of mice at late embryonic to postnatal stages. Our results showed that cad6 is expressed by a limited population of neurons or their precursors, which are synaptically connected to one another, throughout the perinatal stages, and that this expression delineates restricted neuronal circuits from the central to peripheral nervous systems, which include subpathways of the auditory, somatosensory, solitary, vestibular, and olivocerebellar systems. cad6 proteins were detected in these cad6 mRNA-positive neurons on the surface of their cell bodies or dendrites as well as in the cytoplasm. Confocal microscopic analysis revealed that the cad6 protein distribution overlapped that of synaptotagmin in synapse forming areas, suggesting that homotypic cad6 interactions are involved in synaptic connections between neurons expressing this protein. These findings support the idea that cadherin-mediated cell-cell adhesions take part in specific interneuronal connections.     

A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.     

Cross-talk between orphan nuclear hormone receptor RZRalpha and peroxisome proliferator-activated receptor alpha in regulation of the peroxisomal hydratase-dehydrogenase gene

The genes encoding the peroxisomal beta-oxidation enzymes enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) and fatty acyl-CoA oxidase (AOx) are coordinately regulated by peroxisome proliferator-activated receptor alpha (PPARalpha)/9-cis-retinoic acid receptor (RXRalpha) heterodimers that transactivate these genes in a ligand-dependent manner via upstream peroxisome proliferator response elements (PPRE). Here we demonstrate that the monomeric orphan nuclear hormone receptor, RZRalpha, modulates PPARalpha/RXRalpha-dependent transactivation in a response-element dependent manner. Electrophoretic mobility shift analysis showed that RZRalpha bound specifically as a monomer to the HD-PPRE but not the AOx-PPRE. Determinants in the HD-PPRE for binding of RZRalpha were distinct from those required for interaction with PPARalpha/RXRalpha heterodimers. In transient transfections, RZRalpha stimulated ligand-mediated transactivation by PPARalpha from an HD-PPRE luciferase reporter in the absence of exogenously added RXRalpha, but did not affect PPARalpha-dependent transactivation of an AOx-PPRE reporter gene. These data illustrate cross-talk between the RZRalpha and PPARalpha signaling pathways at the level of the HD-PPRE in the regulation of the HD gene and characterize additional factors governing the regulation of peroxisomal beta-oxidation.     

trithorax and the regulation of homeotic gene expression in Drosophila: a historical perspective

To investigate whether interferons (IFNs) selectively suppress the growth of solid tumor cells with elevated protein tyrosine kinase (PTK) activity, we evaluated the effect of recombinant IFN-alpha2a and IFN-gamma on the proliferative and neoplastic potentials triggered by p60v-src using v-src-transformed HAG-1 human epithelial cells. When compared with control cells harboring the pSV2neo gene, the monolayer growth of v-src-transformed cell lines was inhibited by both recombinant IFNs, in a dose-dependent manner, whereas growth of ras-transfected cell lines was not affected. Moreover, IFNs markedly reduced the clonogenic growth of v-src-transformed cells in soft-agar rather than monolayer growth, suggesting the preferential activity of IFNs on anchorage-independent growth. Pretreatment of cells with Src or the Src-like PTK inhibitor herbimycin A or radicicol, alleviated dose-dependently the growth-inhibitory activity of IFN-alpha2a against v-src-transformed cells, suggesting that IFNs may share a common inhibitory pathway with Src PTK inhibitors. Accordingly, like herbimycin A, IFNs were found to reduce tyrosine phosphorylation of p60v-src and suppressed in vitro p60v-src kinase activity in v-src-transformed cells. Our data, together with the fact that IFNs inhibit the growth potential driven by Src but not by activated Ras, suggest that inhibition of signal transduction pathway through Src to downstream transduction events may be a primary mechanism of IFN-induced anti-prolifeative and anti-tumoral activity.     

The regulation of pac gene expression by CRP and FNR

Effects of white and monochromatic (blue-434 nm, green-548 nm, and red-614 nm) lights on the nighttime retinal and pineal NAT activity were examined in chicks. The potency of the tested lights to suppress NAT activity was similar for the retina and pineal gland, with a following rank order: white > green > blue > or = red. The studied tissues of chick were far less sensitive to pulses of monochromatic light than the rat pineal gland. The potency of light to decrease pineal NAT activity of rat was: white > green > blue > red. In chicks, the suppression of the nocturnal NAT activity produced by a short 5-min pulse of monochromatic light was completely reversible in the pineal gland, and partially reversible in the retina. Our data suggest the existence of some differences between birds and mammals in terms of sensitivity and mechanisms involved in the light-induced suppression of melatonin biosynthesis.     

Activation of c-myc gene expression by tumor-derived p53 mutants requires a discrete C-terminal domain

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Here we report that (i) mutant p53 can regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter; (ii) the region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3' end of exon 1; (iii) the mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer; and (iv) transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells. A possible mechanism for transactivation of the c-myc gene by mutant p53 is proposed.     

Biotin synthase from Arabidopsis thaliana. cDNA isolation and characterization of gene expression

The full-length BIO2 cDNA from Arabidopsis thaliana was isolated using an expressed sequence tag that was homologous to the Escherichia coli biotin synthase gene (BioB). Comparisons of the deduced amino acid sequence from BIO2 with bacterial and yeast biotin synthase homologs revealed a high degree of sequence similarity. The amino terminus of the predicted BIO2 protein contains a stretch of hydrophobic residues similar in composition to transit peptide sequences. BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants. Expression of BIO2 was higher in the light relative to dark and was induced 5-fold during biotin-limited conditions. These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and bio-chemical stimuli.