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981.
Godfrey AC Kupsco JM Burch BD Zimmerman RM Dominski Z Marzluff WF Duronio RJ 《RNA (New York, N.Y.)》2006,12(3):396-409
Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Here we generate null mutations of the single Drosophila U7 gene and demonstrate that U7 snRNA is required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem-loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development. 相似文献
982.
The effect of pH of electrolyte solution on the interfacial tension of lipid membrane formed of phosphatidylcholine (PC, lecithin)–phosphatidylserine (PS) system was studied. In this article, three models describing the H+ and OH− ions adsorption in the bilayer lipid surface are presented. In Model I and Model II, the surface is continuous with uniformly distributed functional groups constituting the centres of H+ and OH− ions adsorption while in the other the surface is built of lipid molecules, free or with attached H+ and OH− ions. In these models contribution of the individual lipid molecule forms to interfacial tension of the bilayer were assumed to be additive. In Model III the adsorption of the H+ and OH− ions at the PC–PS bilayer surface was described in terms of the Gibbs isotherm. Theoretical equations are derived to describe this dependence in the whole pH range. 相似文献
983.
In aqueous solutions, in the presence of double-stranded DNA, chlorophyllin (CHL) forms complexes with each of the three DNA intercalators: acridine orange (AO), quinacrine mustard (QM), and doxorubicin (DOX). The evidence for these interactions was obtained by measurement changes in the absorption and fluorescence spectra of the mixtures containing DNA and intercalators during titration with CHL. A model of simple competition between DNA and CHL for the intercalator was used to define the measured interactions. The concentrations of the complexes estimated based on this model were consistent with the concentrations obtained by actual measurement of the absorption spectra. The present data provide further support for the role of chlorophyllin as an "interceptor" that may neutralize biological activity of aromatic compounds including mutagens and antitumor drugs. 相似文献
984.
The crystal structure of the product of the Bacillus subtilis ykuD gene was solved by the multiwavelength anomalous dispersion (MAD) method and refined using data to 2.0 A resolution. The ykuD protein is a representative of a distinctly prokaryotic and ubiquitous family found among both pathogenic and nonpathogenic Gram-positive and Gram-negative bacteria. The deduced amino acid sequence reveals the presence of an N-terminal LysM domain, which occurs among enzymes involved in cell wall metabolism, and a novel, putative catalytic domain with a highly conserved His/Cys-containing motif of hitherto unknown structure. As the wild-type protein did not crystallize, a double mutant was designed (Lys117Ala/Gln118Ala) to reduce excess surface conformational entropy. As expected, the structure of the LysM domain is similar to the NMR structure reported for an analogous domain from Escherichia coli murein transglycosylase MltD. The molecular model also shows that the 112-residue-long C-terminal domain has a novel tertiary fold consisting of a beta-sandwich with two mixed sheets, one containing five strands and the other, six strands. The two beta-sheets form a cradle capped by an alpha-helix. This domain contains a putative catalytic site with a tetrad of invariant His123, Gly124, Cys139, and Arg141. The stereochemistry of this active site shows similarities to peptidotransferases and sortases, and suggests that the enzymes of the ykuD family may play an important role in cell wall biology. 相似文献
985.
The immunohistochemical expression of the androgen receptor (AR) was investigated in the ovarian atretic follicles and corpora lutea (CL) of pregnant pigs and rats, as well as in porcine uteri and fetuses. Follicular atresia involved either abnormal persistence or depletion of AR in various follicular compartments. Porcine and rat CL expressed nuclear AR. However, in the porcine CL, starting from day 70 of pregnancy, mainly cytoplasmic staining was observed, with exclusively cytoplasmic expression found on day 90. In the CL of pregnant rats, differences in AR distribution within the same CL were observed and decreasing AR expression during luteal regression was found. AR mRNA and protein expression in the porcine uterus depended on the uterine compartment and the day of pregnancy. AR-positive were also testes, ovaries, uteri, kidneys and lungs of fetuses. 相似文献
986.
Zbigniew Mikulski Petra Hartmann Gitte Jositsch Zbigniew Zas?ona Katrin S Lips Uwe Pfeil Hjalmar Kurzen Jürgen Lohmeyer Wolfgang G Clauss Veronika Grau Martin Fronius Wolfgang Kummer 《Respiratory research》2010,11(1):133
Background
Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.Methods
Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted.Results
Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium.Conclusions
Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential. 相似文献987.
Sasha M Daskalova Josiah E Radder Zbigniew A Cichacz Sam H Olsen George Tsaprailis Hugh Mason Linda C Lopez 《BMC biotechnology》2010,10(1):1-18
Background
Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.Results
The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit: H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.Conclusion
Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics. 相似文献988.
Alicja Kluczyk Magdalena Rudowska Piotr Stefanowicz Zbigniew Szewczuk 《Journal of peptide science》2010,16(1):31-39
Microwave‐assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off‐resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC‐MS and MS/MS experiments. In a 5 min microwave‐assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW‐assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI‐MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
989.
Krzysztof Grabski Anna Aksmann Piotr Mucha Zbigniew Tukaj 《Journal of applied phycology》2010,22(4):517-524
Conditioned medium (CM) containing algal exudates released by cells of Desmodesmus subspicatus (green microalga) enhanced proliferation of the producer in a dilution-dependent manner, and twofold diluted CM (CM/2) appeared
to be the most effective. Asynchronous and synchronized by light/dark regime (14/10 h) cultures of D. subspicatus were used to explain the effects of CM/2 on photosynthesis as well as growth and reproductive processes of cell cycle of
the producer. During the light period, all control cells attained three commitment points (CP) triggering the reproductive
processes and 20% of them, additionally the fourth one. This resulted in the formation of mainly eight and partly 16 autospores
released from the parent cell during the dark period of the cell cycle. CM/2 markedly increased the number of cells that attained
the fourth CP. Chlorophyll a fluorescence parameters, measured by OJIP test (O, J, I, and P-different steps of fluorescence induction curve), were applied
to explain stimulatory effect of CM/2 on the rate of oxygen evolution. The number of cells as well as the parameter of their
“vitality” (P.I) determined by OJIP analysis were equally enhanced by CM/2. The maximum yield of electron trapping and transport,
as well as the fraction of active reaction centers, was also significantly higher in CM/2. The cells quickly produced and
released CM factor (CMF) to the culture medium; its highest activity was observed in the middle light phase of the cell cycle.
Dialysis, ion exchange chromatography, and solid-phase extraction with C18 allowed reproducible extraction of the active compound from CM. Subjecting the extract to reverse-phase high-performance
liquid chromatography led to one active fraction eluted as a peak with the shortest retention time indicating its hydrophilicity. 相似文献
990.
Das S Das U Sakagami H Hashimoto K Kawase M Gorecki DK Dimmock JR 《Bioorganic & medicinal chemistry letters》2010,20(22):6464-6468
The concept of sequential cytotoxicity, which states that successive chemical attacks on cellular constituents can be more deleterious to neoplasms than normal cells, was evaluated using a series of 3,5-bis(benzylidene)-1-diethylphosphono-4-oxopiperidines 1 and related phosphonic acids 2, which were screened against a panel of malignant and normal cell lines. The compounds proved to be not only potent cytotoxins (71% of the CC(50) figures are submicromolar) but to display greater cytotoxicity to the neoplastic cells. QSAR revealed that both cytotoxic potencies and selective toxicity were increased by a rise in the electron-withdrawing properties and a decrease in the hydrophobicity of the aryl substituents. Utilisation of the PL10 concept and evaluation of druglike properties revealed 1c as the lead tumour-specific cytotoxin. This molecule activated caspase-3 in HL-60 cells but not in the HSC-2 cell line. While 1c caused internucleosomal DNA fragmentation in HL-60 cells, it did not elicit this effect in either HSC-2 and HSC-4 cells. Clearly 1c exerts its cytotoxic potencies by different mechanisms and such pleiotropy is likely the principal reason for the remarkable display of preferential toxicity towards malignant cells of the compounds in series 1 and 2. 相似文献