Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds

The promoters of MEA (FIS1), FIS2, and FIE (FIS3), genes that repress seed development in the absence of pollination, were fused to β-glucuronidase (GUS) to study their activity pattern. The FIS2∷GUS product is found in the embryo sac, in each of the polar cell nuclei, and in the central cell nucleus. After pollination, the maternally derived FIS2∷GUS protein occurs in the nuclei of the cenocytic endosperm. Before cellularization of the endosperm, activity is terminated in the micropylar and central nuclei of the endosperm and subsequently in the nuclei of the chalazal cyst. MEA∷GUS has a pattern of activity similar to that of FIS2∷GUS, but FIE∷GUS protein is found in many tissues, including the prepollination embryo sac, and in embryo and endosperm postpollination. The similarity in mutant phenotypes; the activity of FIE, MEA, and FIS2 in the same cells in the embryo sac; and the fact that MEA and FIE proteins interact in a yeast two-hybrid system suggest that these proteins operate in the same system of control of seed development. Maternal and not paternal FIS2∷GUS, MEA∷GUS, and FIE∷GUS show activity in early endosperm, so these genes may be imprinted. When fis2, mea, and fie mutants are pollinated, seed development is arrested at the heart embryo stage. The seed arrest of mea and fis2 is avoided when they are fertilized by a low methylation parent. The wild-type alleles of MEA or FIS2 are not required. The parent-of-origin-determined differential activity of MEA, FIS2, and FIE is not dependent on DNA methylation, but methylation does control some gene(s) that have key roles in seed development.

Pattern of neuronal activity associated with conscious and unconscious processing of visual signals

Following striate cortex damage in monkeys and humans there can be residual function mediated by parallel visual pathways. In humans this can sometimes be associated with a “feeling” that something has happened, especially with rapid movement or abrupt onset. For less transient events, discriminative performance may still be well above chance even when the subject reports no conscious awareness of the stimulus. In a previous study we examined parameters that yield good residual visual performance in the “blind” hemifield of a subject with unilateral damage to the primary visual cortex. With appropriate parameters we demonstrated good discriminative performance, both with and without conscious awareness of a visual event. These observations raise the possibility of imaging the brain activity generated in the “aware” and the “unaware” modes, with matched levels of discrimination performance, and hence of revealing patterns of brain activation associated with visual awareness. The intact hemifield also allows a comparison with normal vision. Here we report the results of a functional magnetic resonance imaging study on the same subject carried out under aware and unaware stimulus conditions. The results point to a shift in the pattern of activity from neocortex in the aware mode, to subcortical structures in the unaware mode. In the aware mode prestriate and dorsolateral prefrontal cortices (area 46) are active. In the unaware mode the superior colliculus is active, together with medial and orbital prefrontal cortical sites.

HCC-2, a human chemokine: Gene structure, expression pattern, and biological activity

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named “HCC-2.” The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKβ8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1γ, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1α. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.

Chilling Delays Circadian Pattern of Sucrose Phosphate Synthase and Nitrate Reductase Activity in Tomato1

Overnight low-temperature exposure inhibits photosynthesis in chilling-sensitive species such as tomato (Lycopersicon esculentum) and cucumber by as much as 60%. In an earlier study we showed that one intriguing effect of low temperature on chilling-sensitive plants is to stall the endogenous rhythm controlling transcription of certain nuclear-encoded genes, causing the synthesis of the corresponding transcripts and proteins to be mistimed when the plant is rewarmed. Here we show that the circadian rhythm controlling the activity of sucrose phosphate synthase (SPS) and nitrate reductase (NR), key control points of carbon and nitrogen metabolism in plant cells, is delayed in tomato by chilling treatments. Using specific protein kinase and phosphatase inhibitors, we further demonstrate that the chilling-induced delay in the circadian control of SPS and NR activity is associated with the activity of critical protein phosphatases. The sensitivity of the pattern of SPS activity to specific inhibitors of transcription and translation indicates that there is a chilling-induced delay in SPS phosphorylation status that is caused by an effect of low temperature on the expression of a gene coding for a phosphoprotein phosphatase, perhaps the SPS phosphatase. In contrast, the chilling-induced delay in NR activity does not appear to arise from effects on NR phosphorylation status, but rather from direct effects on NR expression. It is likely that the mistiming in the regulation of SPS and NR, and perhaps other key metabolic enzymes under circadian regulation, underlies the chilling sensitivity of photosynthesis in these plant species.

Modulation of cognition-specific cortical activity by gonadal steroids: A positron-emission tomography study in women

There is considerable evidence from animal studies that gonadal steroid hormones modulate neuronal activity and affect behavior. To study this in humans directly, we used H215O positron-emission tomography to measure regional cerebral blood flow (rCBF) in young women during three pharmacologically controlled hormonal conditions spanning 4–5 months: ovarian suppression induced by the gonadotropin-releasing hormone agonist leuprolide acetate (Lupron), Lupron plus estradiol replacement, and Lupron plus progesterone replacement. Estradiol and progesterone were administered in a double-blind cross-over design. On each occasion positron-emission tomography scans were performed during (i) the Wisconsin Card Sorting Test, a neuropsychological test that physiologically activates prefrontal cortex (PFC) and an associated cortical network including inferior parietal lobule and posterior inferolateral temporal gyrus, and (ii) a no-delay matching-to-sample sensorimotor control task. During treatment with Lupron alone (i.e., with virtual absence of gonadal steroid hormones), there was marked attenuation of the typical Wisconsin Card Sorting Test activation pattern even though task performance did not change. Most strikingly, there was no rCBF increase in PFC. When either progesterone or estrogen was added to the Lupron regimen, there was normalization of the rCBF activation pattern with augmentation of the parietal and temporal foci and return of the dorsolateral PFC activation. These data directly demonstrate that the hormonal milieu modulates cognition-related neural activity in humans.

Developmental disorder associated with increased cellular nucleotidase activity

Four unrelated patients are described with a syndrome that included developmental delay, seizures, ataxia, recurrent infections, severe language deficit, and an unusual behavioral phenotype characterized by hyperactivity, short attention span, and poor social interaction. These manifestations appeared within the first few years of life. Each patient displayed abnormalities on EEG. No unusual metabolites were found in plasma or urine, and metabolic testing was normal except for persistent hypouricosuria. Investigation of purine and pyrimidine metabolism in cultured fibroblasts derived from these patients showed normal incorporation of purine bases into nucleotides but decreased incorporation of uridine. De novo synthesis of purines and cellular phosphoribosyl pyrophosphate content also were moderately decreased. The distribution of incorporated purines and pyrimidines did not reveal a pattern suggestive of a deficient enzyme activity. Assay of individual enzymes in fibroblast lysates showed no deficiencies. However, the activity of cytosolic 5′-nucleotidase was elevated 6- to 10-fold. Based on the possibility that the observed increased catabolic activity and decreased pyrimidine salvage might be causing a deficiency of pyrimidine nucleotides, the patients were treated with oral pyrimidine nucleoside or nucleotide compounds. All patients showed remarkable improvement in speech and behavior as well as decreased seizure activity and frequency of infections. A double-blind placebo trial was undertaken to ascertain the efficacy of this supplementation regimen. Upon replacement of the supplements with placebo, all patients showed rapid regression to their pretreatment states. These observations suggest that increased nucleotide catabolism is related to the symptoms of these patients, and that the effects of this increased catabolism are reversed by administration of uridine.

The DNA Helicase Activity of BLM Is Necessary for the Correction of the Genomic Instability of Bloom Syndrome Cells

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern.

Kinase Activity-dependent Nuclear Export Opposes Stress-induced Nuclear Accumulation and Retention of Hog1 Mitogen-activated Protein Kinase in the Budding Yeast Saccharomyces cerevisiae

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1–green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1–GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1–GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.

Fibroblast Growth Factor (FGF) Soluble Receptor 1 Acts as a Natural Inhibitor of FGF2 Neurotrophic Activity during Retinal Degeneration

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

Frequency of migrants and migratory activity are genetically correlated in a bird population: Evolutionary implications

Most migratory bird populations are composed of individuals that migrate and individuals that remain resident. While the role of ecological factors in maintaining this behavioral dimorphism has received much attention, the importance of genetic constraints on the evolution of avian migration has not yet been considered. Drawing on the recorded migratory activities of 775 blackcaps (Sylvia atricapilla) from a partially migratory population in southern France, we tested two alternative genetic models about the relationship between incidence and amount of migratory activity. The amount of migratory activity could be the continuous variable “underlying” the phenotypic expression of migratory urge, or, alternatively, the expression of both traits could be controlled by two separate genetic systems. The distributions of migratory activities in five different cohorts and the inheritance pattern derived from selective breeding experiments both indicate that incidence and amount of migratory activity are two aspects of one trait. Thus, all birds without measurable activity have activity levels at the low end of a continuous distribution, below the limit of expression or detection. The phenotypic dichotomy “migrant–nonmigrant” is caused by a threshold which may not be fixed but influenced both genetically and environmentally. This finding has profound implications for the evolution of migration: the transition from migratoriness to residency should not only be driven by selection favoring resident birds but also by selection for lower migratory activity. This potential for selection on two aspects, residency and migration distance, of the same trait may enable extremely rapid evolutionary changes to occur in migratory behavior.

Proteinase inhibitors I and II from potatoes block UVB-induced AP-1 activity by regulating the AP-1 protein compositional patterns in JB6 cells

Proteinase inhibitor I (Inh I) and proteinase inhibitor II (Inh II) from potato tubers are effective proteinase inhibitors of chymotrypsin and trypsin. Inh I and Inh II were shown to suppress irradiation-induced transformation in mouse embryo fibroblasts suggesting that they possess anticarcinogenic characteristics. We have previously demonstrated that Inh I and Inh II could effectively block UV irradiation-induced activation of transcription activator protein 1 (AP-1) in mouse JB6 epidermal cells, which mechanistically may explain their anticarcinogenic actions. In the present study, we investigated the effects of Inh I and Inh II on the expression and composition pattern of the AP-1 complex following stimulation by UV B (UVB) irradiation in the JB6 model. We found that Inh I and Inh II specifically inhibited UVB-induced AP-1, but not NFκB, activity in JB6 cells. Both Inh I and Inh II up-regulated AP-1 constituent proteins, JunD and Fra-2, and suppressed c-Jun and c-Fos expression and composition in bound AP-1 in response to UVB stimulation. This regulation of the AP-1 protein compositional pattern in response to Inh I or Inh II may be critical for the inhibition of UVB-induced AP-1 activity by these agents found in potatoes.

Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: Identification of functional themes

The genetic basis for virulence in influenza virus is largely unknown. To explore the mutational basis for increased virulence in the lung, the H3N2 prototype clinical isolate, A/HK/1/68, was adapted to the mouse. Genomic sequencing provided the first demonstration, to our knowledge, that a group of 11 mutations can convert an avirulent virus to a virulent variant that can kill at a minimal dose. Thirteen of the 14 amino acid substitutions (93%) detected among clonal isolates were likely instrumental in adaptation because of their positive selection, location in functional regions, and/or independent occurrence in other virulent influenza viruses. Mutations in virulent variants repeatedly involved nuclear localization signals and sites of protein and RNA interaction, implicating them as novel modulators of virulence. Mouse-adapted variants with the same hemagglutinin mutations possessed different pH optima of fusion, indicating that fusion activity of hemagglutinin can be modulated by other viral genes. Experimental adaptation resulted in the selection of three mutations that were in common with the virulent human H5N1 isolate A/HK/156/97 and that may be instrumental in its extreme virulence. Analysis of viral adaptation by serial passage appears to provide the identification of biologically relevant mutations.

A Gene Coding for Tomato Fruit β-Galactosidase II Is Expressed during Fruit Ripening : Cloning, Characterization, and Expression Pattern

β-Galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes characterized by their ability to hydrolyze terminal, nonreducing β-d-galactosyl residues from β-d-galactosides. Several β-galactosidases, sometimes referred to as exo-galactanases, have been purified from plants and shown to possess in vitro activity against extracted cell wall material via the release of galactose from wall polymers containing β(1→4)-d-galactan. Although β-galactosidase II, a protein present in tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan, has been purified, cloning of the corresponding gene has been elusive. We report here the cloning of a cDNA, pTomβgal 4 (accession no. AF020390), corresponding to β-galactosidase II, and show that its corresponding gene is expressed during fruit ripening. Northern-blot analysis revealed that the β-galactosidase II gene transcript was detectable at the breaker stage of ripeness, maximum at the turning stage, and present at decreasing levels during the later stages of normal tomato fruit ripening. At the turning stage of ripeness, the transcript was present in all fruit tissues and was highest in the outermost tissues (including the peel). Confirmation that pTomβgal 4 codes for β-galactosidase II was derived from matching protein and deduced amino acid sequences. Furthermore, analysis of the deduced amino acid sequence of pTomβgal 4 suggested a high probability for secretion based on the presence of a hydrophobic leader sequence, a leader-sequence cleavage site, and three possible N-glycosylation sites. The predicted molecular mass and isoelectric point of the pTomβgal 4-encoded mature protein were similar to those reported for the purified β-galactosidase II protein from tomato fruit.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers.

Several epidemiologic studies indicate that NAT2-related slow N-acetylation increases bladder cancer risk among workers exposed to aromatic amines, presumably because N-acetylation is important for the detoxification of these compounds. Previously, we showed that NAT2 polymorphisms did not influence bladder cancer risk among Chinese workers exposed exclusively to benzidine (BZ), suggesting that NAT2 N-acetylation is not a critical detoxifying pathway for this aromatic amine. To evaluate the biologic plausibility of this finding, we carried out a cross-sectional study of 33 workers exposed to BZ and 15 unexposed controls in Ahmedabad, India, to evaluate the presence of BZ-related DNA adducts in exfoliated urothelial cells, the excretion pattern of BZ metabolites, and the impact of NAT2 activity on these outcomes. Four DNA adducts were significantly elevated in exposed workers compared to controls; of these, the predominant adduct cochromatographed with a synthetic N-(3'- phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine standard and was the only adduct that was significantly associated with total BZ urinary metabolites (r = 0.68, P < 0.0001). To our knowledge this is the first report to show that BZ forms DNA adducts in exfoliated urothelial cells of exposed humans and that the predominant adduct formed is N-acetylated, supporting the concept that monofunctional acetylation is an activation, rather than a detoxification, step for BZ. However, because almost all BZ-related metabolites measured in the urine of exposed workers were acetylated among slow, as well as rapid, acetylators (mean +/- SD 95 +/- 1.9% vs. 97 +/- 1.6%, respectively) and NAT2 activity did not affect the levels of any DNA adduct measured, it is unlikely that interindividual variation in NAT2 function is relevant for BZ-associated bladder carcinogenesis.