Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Genetics of α-amylases from rye endosperm

Summary Fifteen inbred lines of rye, F₁ and F₂ progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis     

Endosperm-specific demethylation and activation of specific alleles of α-tubulin genes of Zea mays L.

We have investigated the methylation status of the -tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the -tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub2 and tub4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two -tubulin genes, tub3 and tub4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

Post-translational modifications of α-tubulin in<Emphasis Type="Italic"> Zea mays</Emphasis> L. are highly tissue specific

To further understand post-translational modifications (PTMs) of plant -tubulin, post-translationally modified -tubulin isoforms from selected tissues of Zea mays L. were examined using two-dimensional electrophoresis and immunoblotting. Except for polyglycylated tubulin, tyrosinated, detyrosinated, acetylated and polyglutamylated -tubulin isoforms were all present in maize tissues. Tyrosinated -tubulin was the predominant variant in all cases, with isoforms 1–4 (5) being the most common components. Leaves exhibited a striking difference in PTM patterns of -tubulin isoforms compared to other tissues examined. In leaves, several major specific isoforms were highly modified by detyrosination, acetylation and polyglutamylation. In pollen and anthers, only the most abundant isoform 3 was acetylated to an appreciable extent, and no acetylated isoform was found in roots. Similarly, in pollen, anthers and roots, only 3 was appreciably polyglutamylated. Additionally, a detyrosinated isoform 6 was present in anthers and in leaves, while the tyrosinated isoform 6 seemed to be pollen specific. These results indicate that certain types of PTM of plant -tubulin preferentially occur in a tissue-specific way.Abbreviations 1-, 2-D one-, two-dimensional - MT microtubule - PTM post-translational modification     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

Cloning and nucleotide sequence of the α-galactosidase cDNA from Cyamopsis tetragonoloba (guar)

Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the -galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH₂ terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH₂ terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/ factor and killer factor. A comparison of the derived amino acid sequence of this -galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the -galactosidase from Escherichia coli.     

Effect of anticalmodulin drugs on the action of yeast α factor pheromone

Saccharomyces cerevisiae factor pheromone arrest growth of cells of the a mating type (MAT a) at the G1 phase of the cell cycle. When treatment of MAT a cells with factor was carried out in the presence of anticalmodulin drugs, trifluoperazine or chlorpromazine, the extent of cell growth arrest induced by factor was reduced or even became undetectable. These results lend support to the hypothesis that calmodulin plays a role as mediator in the action of factor on MAT a cells.Abbreviation MAT mating type     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Kinetic changes of αB crystallin expression in neoplastic cells and syngeneic rat fibroblasts at various subculture stages

B crystallin, a structural protein of the mammalian lens essential for the maintenance of lens transparency, is also expressed, at variable levels, in many extraocular tissues where it plays a protective role in stress conditions. In fact, heat or toxic shocks, as well as pathological states, increase B crystallin levels in many cell types. Here we show that B crystallin expression is also modulated in subcultures of rat fibroblasts and Galliera sarcoma cells. Western blots analysis with anti B crystallin antibodies reveals the presence of the protein in both cell populations, although the kinetic pattern of expression is different. Galliera fibroblasts constitutively express the protein up to the 70th subculture and afterwards the synthesis ceases. On the other hand, Galliera sarcoma cells do not contain B crystallin in the early stages of the culture, but there is a progressive increases between the 20th and 40th cell subculture. Differences also exist concerning the intracellular distribution: B crystallin is diffusely localized in the cytoplasm of fibroblasts while in sarcoma cells it localizes mainly to the perinuclear region. B crystallin is totally recovered as soluble protein in the supernatants obtained after low speed centrifugation of fibroblast homogenates, while in sarcoma cells a portion of the protein is also recovered in the insoluble pellet. Intracellular pH measurements show an alkaline cytosol in sarcoma cells compared to fibroblasts. Heat shock treatment of fibroblast subcultures constitutively expressing B crystallin induces an over-expression of the protein, while in fibroblasts whose biosynthetic capacity is lost, heat shock is unable to activate the crystallin gene. Correlation between B crystallin expression and proliferative rate shows that highly proliferating fibroblasts do not express B crystallin, while neoplastic cells do.     

rol genes of Agrobacterium rhizogenes cucumopine strain: sequence,effects and pattern of expression

By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA-which we refer to as rol, and -were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5 non-coding region of rol and rol was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.     

Purification, properties and possible gene assignment of an α1,3-fucosyltransferase expressed in human liver

1,3-Fucosyltransferase solubilized from human liver has been purified 40 000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with 1,3-fucosyltransferase activity and had a specific activity of 5–6 µmol min^–1 mg^–1 and anM _r 44 000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal1-4GlcNAc, NeuAc2-3Gal1-4GlcNAc and Fuc1-2Gal1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc1-2Gal1-4Glc and the Type 1 compound Gal1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc2-6Gal1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with theFuc-TVI cDNA, suggests a provisional identification ofFuc-TVI as the major 1,3-fucosyltransferase gene expressed in human liver.Died June, 1991     

Increased alpha3-fucosylation of alpha1-acid glycoprotein in Type I diabetic patients is related to vascular function

Diabetic mellitus is attended by the development of endothelial dysfunction which is suggested to be accompanied with a chronic low-degree of inflammation. During a chronic hepatic inflammatory response, specific changes in glycosylation of the acute phase protein ₁-acid glycoprotein (AGP) can be detected. In this report we studied the changes in glycosylation of AGP in more detail and evaluated the relation between a change in glycosylation of AGP and urinary albumin secretion in Type I diabetic patients. The glycosylation of AGP, studied by crossed affinity immunoelectrophoresis (CAIE) and high pH anion exchange chromatography with pulse amperometric detection (HPAEC-PAD), showed an increase in 3-fucosylation. Staining with an antibody against sialyl Lewis^x (sLe^x) implied that part of the 3-fucosylation was present in a sLe^x-conformation. In the group of Type I diabetic patients with increased urinary albumin excretion, a significant increase in 3-fucosylation of AGP (p[emsp4 ]<[emsp4 ]0.0005) could be detected. Therefore, the increased 3-fucosylation of AGP can be used as an additional marker for the development of vascular complications in Type I diabetic patients.     

The effect of abscisic acid on the differential expression of α-amylase isozymes in barley aleurone layers

Summary The treatment of barley aleurone layers with gibberellic acid (GA₃) results in the synthesis of two groups of -amylase isozymes. Addition of abscisic acid (ABA) at the same time as GA₃ inhibited the synthesis of both groups of isozymes. However, midcourse ABA addition (12 h or later after GA₃) had a more inhibitory effect on the high pI -amylase group than on the low pI -amylase group. This midcourse inhibition was detectable within 2 h of ABA addition. Northern analysis results using cDNA probes for the high pI and low pI -amylase groups paralleled the protein synthesis results for both isozyme groups. High pI -amylase mRNA levels began to decrease within 2 h of midcourse ABA treatment and were less than 10% of the original level by 4 h. The levels of low pI -amylase mRNA were decreased less by midcourse ABA addition than were high pI mRNA levels. Cordycepin and cycloheximide blocked the effects of midcourse ABA addition on -amylase mRNA. These observations indicate that ABA inhibits -amylase expression at the pretranslational level and that protein and RNA synthesis are required for midcourse ABA action to occur. Our results also show that -amylase mRNA, which has been thought to be very stable, is degraded after midcourse ABA treatment.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

3α-Hydroxysteroid Dehydrogenase in Animal and Human Tissues

This review analyzes data on the biological role of 3-hydroxysteroid dehydrogenase (3-HSD) in animal and human tissues and describes its main characteristics, mechanism of action, and regulation of activity. Based on published data, a scheme for the actions of androgen, progestin, and glucocorticoids involving the participation of 3-HSD is proposed. According to this scheme, in the mechanism of steroid action 3-HSD not only regulates the concentration of the main effector androgen, 5-dihydrotestosterone, in target cells, but also switches androgen, progestin, and glucocorticosteroid genomic activity to non-genomic activity.     

Evidence for a non-antioxidant,dose-dependent role of α -lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as -lipoic acid and -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with -lipoic acid and -tocopherol, alone or in combination, prior to incubation with H₂O₂ or staurosporine. -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H₂O₂ and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with -lipoic acid and/or -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with -lipoic acid and -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of -lipoic acid as an antioxidant.