Changes in protein interactions of cell cycle-related genes during the dormancy-to-growth transition in pea axillary buds

Apical dominance is a phenomenon in which a terminal bud grows predominantly and the growth of the axillary buds is suppressed. Here, we investigated the molecular mechanisms associated with cell cycle control that occur in pea axillary buds as a result of decapitation. Proliferating cell nuclear antigen (PCNA) protein was detected in both dormant and growing buds, while PCNA mRNA was absent in dormant buds. Pissa; CycB1;2 and Cdc2 proteins were undetectable during dormancy. To analyze an interaction between PCNA and Pissa;CycD3;1, we performed anti-PCNA immunoaffinity column chromatography. Pissa;CycD3;1 protein was detected in the eluate prepared from the dormant buds, but not in the eluate prepared from the growing buds. Furthermore, we performed anti-Pissa;CycD3;1 immunoaffinity column chromatography. PCNA protein was detected in the eluate prepared from the dormant buds, but not in the eluate prepared from the growing buds. These results indicated that PCNA associated with Pissa;CycD3;1 only during dormancy. In addition, the interaction between PCNA and Pissa;CycD3;1 was confirmed by a yeast two-hybrid system.     

Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+. In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.     

Cloning and expression of a chicken alpha-amylase gene

The effects of cabbage leaf protein concentrate (CLPC) on serum and liver lipid concentrations were determined in rats fed cholesterol-enriched and cholesterol-free diets. In rats fed the cholesterol-enriched diet with CLPC, total cholesterol, triacylglycerol and phospholipid concentrations in both the serum and liver, as well as the atherogenic index diet were significantly lower than those of the rats fed a casein diet. A supplement of methionine to the CLPC diet raised serum HDL-cholesterol and body weight gain, indicating that the addition of methionine to the CLPC diet is not only available to improve the nutritive value of CLPC but also to lower the atherogenic index. In rats fed the cholesterol-free diet, the liver total cholesterol and triacylglycerol concentrations of the CLPC-fed rats also showed lower values than those of the casein-fed rats, however, the serum total cholesterol concentration of the CLPC-fed rats did not differ from that of the casein-fed rats.     

Cloning and expression of the Borrelia burgdorferi lon gene

BACKGROUND: We studied the morphological changes occurring in neurons from the dorsal lateral geniculate nucleus (dLGN) during aging by analysing the size and shape of cell bodies and nuclei. METHODS: Male albino Wistar rats, aged 3, 18, 24, and 30 months, were used. After appropriate tissue preparation and following the usual histological procedure, the profiles of 1,920 neuronal bodies and nuclei were drawn using a camera lucida. Data was later recorded and processed with a semiautomatic image analyser. RESULTS AND CONCLUSIONS: We observed that dLGN neurons do not change in size from the age of 3-24 months. Between 24 and 30 months, the soma and nucleus of the cell undergo hypertrophy, 32.8% and 35.6%, respectively, when compared to those from 3-month-old animals (P < 0.01). Furthermore, we found a high correlation between cell body size/nucleus size, which does not disappear with age. The r values (correlation coefficient) were 0.7998, 0.8662, 0.8433 and 0.7304, and R2 (determination coefficient) was equal to 0.6397, 0.7504, 0.7112, and 0.5335. These latter values show that in 63.97%, 75.04%, 71.12%, and 53.35% of cases, respectively, modifications in somata size were accompanied by similar changes in nucleus size, and vice-versa. The study of the shape of the soma and nucleus of the cell revealed that both structures have a rounded-oval configuration that does not change in a significant way from adulthood to old age.     

Cloning and expression of the Mycobacterium fortuitum superoxide dismutase gene

In this paper we report the cloning, sequencing and expression of the superoxide dismutase (sod) gene from Mycobacterium fortuitum. A single gene was found to code for superoxide dismutase activity with its identity being confirmed by expression in M. aurum. The amino acid sequence was found to be similar to that of superoxide dismutases of several other origins. A region downstream of the sod gene also showed similarities to the corresponding sequences of the two main mycobacterial pathogens: M. leprae and M. tuberculosis. Analysis of enzymatic activity showed this enzyme in M. fortuitum required manganese as cofactor.     

Regulation of E2F-1 gene expression in avian cells

Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.     

Cloning of chicken interferon regulatory factor-2 (IRF-2) cDNA: expression and mapping of the IRF-2 gene

The Visual Evoked Potential (VEP) is an electrophysiological commonly used test in investigating various neurophysiological disorders. Through the years many methods have been developed, but there are not many objective criteria in distinguishing a normal VEP waveform from an abnormal. In this communication we use the phase characteristics of the power spectrum as a criterion of distinguishing normals and abnormals. From our analysis, it was shown that the phase spectrum of a VEP has a certain periodicity in the 0- to 40-Hz region. By studying these periodicities we were able to determine the range of the period that characterizes normal and abnormal populations and to establish an experimental method for objectively examining any kind of VEP waveforms.     

Expressed sequence tags of radish flower buds and characterization of a CONSTANS LIKE 1 gene

Glutaric acidemia type I (GAI) (McKusick 231670) is an autosomal recessive disease affecting the catabolism of the amino acids lysine, hydroxylysine and tryptophan, caused by a defect in the gene encoding glutaryl-coenzyme A dehydrogenase (GCDH) and associated with severe neurological symptoms. Several pathogenic mutations in GCDH have been reported to cause GAI. One mutation, R402W, is more common than the others, which seem to be private" mutations. Here we report the entire sequences of introns 1, 2, 3, 6, 7, 8 and 9, and part of those of introns 4, 5 and 10 as well as 21 different mutations in 20 patients with GAI, corresponding to 38 out of 40 alleles.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyltransferase (FTase) gene was examined using transgenic expression of the beta-glucuronidase (GUS) gene fused to a 3.2 kb 5' upstream sequence of the gene encoding the pea FTase beta subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase beta subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

Shh expression in developing and regenerating limb buds of Xenopus laevis

Analysis of the kinetic behaviour of a two-enzyme-system carrying out two consecutive reactions was investigated in macroheterogeneous biphasic media (octane/buffer pH 9.6, v/v = 1:1). The lipase-catalysed hydrolysis of trilinolein and the subsequent lipoxygenation of the liberated linoleic acid, were coupled in a modified Lewis cell with a well-defined liquid/liquid interfacial area. Trilinolein was dissolved in the organic phase and hydrolysed in the presence of Mucor javanicus lipase at the organic/aqueous interface. Linoleic acid, liberated after hydrolysis was transferred to the aqueous phase and reacted with lipoxygenase. This reaction consumed linoleic acid and produced hydroperoxides, which favoured the transfer of residual linoleic acid, since they possess surface active properties. Catalysis and transfer influenced each other reciprocally. At low substrate concentrations, cooperativity phenomena were observed in the experimental and also the modelled two-enzyme systems. When the initial substrate concentration was high, the kinetic behaviour of the two-enzyme system in a compartmentalised medium, seemed to be independent of the substrate concentration, unlike that observed in homogeneous monophasic enzymology. The numerical integration program used to model the two-enzyme system was based on results obtained in separate studies of the following three phenomena: (1) trilinolein hydrolysis in biphasic medium. (2) linoleic acid transfer across a liquid/liquid interface and (3) lipoxygenation in an aqueous media. Results obtained by modelling were similar to the results observed experimentally.     

Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1

2'-Deoxyuridylate hydroxymethylase (dUMP-hmase) from phage SPO1 has been cloned and expressed in Escherichia coli. In crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported. The enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography. The subunits of dUMP-hmase are 45 kDa by SDS-PAGE and form dimers with a molecular mass of 89.2 kDa by analytical centrifugation. In addition to the normal reaction, dUMP-hmase catalyzes the 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate)-independent tritium exchange of [5-3H]dUMP for protons of water and dehalogenation of 5-bromo-2'-deoxy-uridine-5'-monophosphate; the enzyme also forms a covalent binary adduct with pyridoxal 5'-monophosphate and a covalent ternary complex with 5-fluoro-2'-deoxyuridine-5'-monophosphate and CH2H4folate. Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect on the catalysis of cofactor-independent tritium exchange.     

Cloning and sequencing of a gene encoding D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 and expression of the gene in Escherichia coli

The gene encoding the D-aminoacylase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete nucleotide sequence was identified. The D-aminoacylase structural gene consists of 1452 nucleotides and encodes 484 amino acid residues. The molecular weight of D-aminoacylase was calculated to be 51,918. This value agreed well with the apparent molecular weight of 52,000 found for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence (NH2-SQSDSQPFDLLRAG-) predicted by the nucleotide sequence exactly matched those of the purified D-aminoacylase both from Alcaligenes A-6 and from cloned Escherichia coli (E. coli), with the exception of the removal of the N-terminal methionine processed after translation. The purified recombinant enzyme showed almost the same enzymatic properties as the native enzyme from Alcaligenes A-6. Alcaligenes A-6 D-aminoacylase showed 25-29% homology with L-aminoacylases from Bacillus stearothermophilus, porcine and humans.     

Cell-density-dependent expression of Cyp1a2 gene in monolayer-cultured adult mouse hepatocytes

The effects of PAF antagonists, of substances which influence the arachidonic acid metabolism, and of dexamethasone and ketotifen were evaluated in an acute PAF-induced mortality model in female NMRI mice. We established a dependence of sensitivity to PAF on strain (AB mice showed no dose dependence) and on sex of the animals as well as on the PAF charges used in our experiments. PAF produced resistance in surviving animals against the PAF-induced death on repeated application. The PAF antagonists, WEB 2170 and WEB 2086, provided the best dose-dependent protection against PAF toxicity, followed by dexamethasone, by the COX/LOX synthetase inhibitor X 86 (a BW 755 C-analogue) and by the PAF receptor antagonist BN 52021. Particularly remarkable was the excellent prevention by aspirin. Aspirin may not only inhibit the cyclooxygenase pathway but also endogenous PAF synthesis. Other drugs, i.e. indomethacin, the thromboxane receptor antagonist, BM 13177, the thromboxane synthetase inhibitor, HOE 944, as well as the lipoxygenase inhibitors (NDGA, esculetin, SHAM and phenidone) exerted a dose-dependent protection only at high doses.