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Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quaternary structure formation and activity.
Get PDFArabidopsis thaliana AtNUDT7, a homodimeric Nudix hydrolase active on ADP-ribose and NADH, exerts negative control on the major signaling complex involved in plant defense activation and programmed cell death. The structural and functional consequences of altering several amino-acid residues of the AtNUDT7 protein have been examined by site-directed mutagenesis, far-UV circular dichroism (CD), attenuated total reflection-Fourier transform infrared (ATR-FTIR) and photon correlation (PCS) spectroscopy, biochemical analysis and protein-protein interaction studies. Alanine substitutions of F73 and V168 disallowed dimer formation. Both the F73A- and V168A-mutated proteins displayed no observable enzymatic activity. Alanine substitution of the V69 residue did not significantly alter the enzyme activity and had no influence on dimer arrangement. The non-conserved V26 residue, used as a negative control, did not contribute to the enzyme quaternary structure or activity. Detailed biophysical characterization of the wild-type and mutant proteins indicates that the mutations do not considerably alter the secondary structure of the enzyme but they affect dimer assembly. In addition, mutating residues V69, F73 and V168 disrupted the binding of AtNUDT7 to the regulatory 14.3.3 protein. These are the first studies of the structure-function relationship of AtNUDT7, a Nudix hydrolase of important regulatory function
An integrated LC-ESI-MS platform for quantitation of serum peptide ladders. Application for colon carcinoma study.
No full textMounting evidence indicates that MS analysis of the human blood peptidome allows to distinguish between cancer and non-cancer samples, giving promise for a new MS-based diagnostic tool. However, several aspects of already published work have been criticized and demand for more methodical approach has been formulated. Motivated by this we undertook a systematic study of the plasma and serum peptidome using an integrated ESI-LC-MS-based platform, equipped with new data analysis tools for relative and absolute peptide quantitation. We used a high resolution LC-ESI-MS to analyze well-separated MS signals corresponding to peptides, and measured the variability of >1000 peptide signal amplitudes across a set of plasma and serum samples from healthy individuals. By spiking serum samples with known amounts of isotopically labeled versions of a selected set of peptides we measured the variability of their absolute concentration in this sample set and demonstrated a strong influence of clotting time on the concentration of these peptides in serum. Finally, we used this new LC-ESI-MS analytical platform for the differential analysis of healthy versus colon cancer serum samples and found that it was possible to distinguish the two groups with 89.8% sensitivity and 94.6% specificity
A novel splicing variant encoding putative catalytic alpha subunit of maize protein kinase CK2
Get PDFA cDNA highly homologous to the known catalytic alpha subunit of protein kinase CK2 was cloned from maize (Zea mays). It was designated ZmCK2 alpha-4 (accession no. AAF76187). Sequence analysis shows that ZmCK2 alpha-4 and the previously identified ZmCK2 alpha-1 (accession no. X61387) are transcribed from the same gene, ZmPKCK2AL (accession no. Y11649), but at different levels in various maize organs and at different stages of development. The cDNA encoding ZmCK2 alpha-4 has three potential translation initiation sites. The three putative variants of ZmCK2 alpha-4 were expressed in Escherichia coli as GST-fusion proteins and purified from bacterial extracts. In contrast to the previously characterized ZmCK2 alpha s, the obtained GST:ZmCK2 alpha-4 proteins were catalytically inactive as monomers or in the presence of equimolar amounts of the human CK2 beta. However, GST:ZmCK2 alpha-4 did phosphorylate casein in the presence of a large excess of the beta subunit. The activity of ZmCK2 alpha-4 toward casein could also be stimulated by increasing ATP concentration. Modeling studies have shown that there is no interaction between the N-terminal segment of ZmCK2 alpha-4 and the activation loop responsible for constitutive catalytic activity of CK2 alpha. Preliminary results suggest that ZmCK2 alpha-4 may function as a negative regulator of other CK2s, and at certain circumstances as a holoenzyme which catalytic activity is stimulated by specific regulatory subunit(s)
e-LiSe--an online tool for finding needles in the '(Medline) haystack'.
Get PDFUNLABELLED
Using literature databases one can find not only known and true relations between processes but also less studied, non-obvious associations. The main problem with discovering such type of relevant biological information is 'selection'. The ability to distinguish between a true correlation (e.g. between different types of biological processes) and random chance that this correlation is statistically significant is crucial for any bio-medical research, literature mining being no exception. This problem is especially visible when searching for information which has not been studied and described in many publications. Therefore, a novel bio-linguistic statistical method is required, capable of 'selecting' true correlations, even when they are low-frequency associations. In this article, we present such statistical approach based on Z-score and implemented in a web-based application 'e-LiSe'.
AVAILABILITY
The software is available at http://miron.ibb.waw.pl/elise
Simple method for plating Escherichia coli bacteriophages forming very small plaques or no plaques under standard conditions
Get PDFThe use of low concentrations (optimally 2.5 to 3.5 microg/ml, depending on top agar thickness) of ampicillin in the bottom agar of the plate allows for formation of highly visible plaques of bacteriophages which otherwise form extremely small plaques or no plaques on Escherichia coli lawns. Using this method, we were able to obtain plaques of newly isolated bacteriophages, propagated after induction of prophages present in six E. coli O157:H(-) strains which did not form plaques when standard plating procedures were employed
Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria
Get PDFDifferent factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600 mM sodium chloride and 40 mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals up to twofold for longer DNA strands (i.e., >300 bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10 min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease of recognition as follows: triple random > quintuple middle > triple middle > single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker
Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)
Get PDFBACKGROUND:
Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days.
RESULTS:
A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2x), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes.
CONCLUSION:
In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems
Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae.
No full textMost replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 3' --> 5' proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 3' --> 5' exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells
Genistin-rich soy isoflavone extract in substrate reduction therapy for Sanfilippo syndrome: An open-label, pilot study in 10 pediatric patients
Get PDFBackground: Mucopolysaccharidoses (MPSs) are a group of severe metabolic disorders caused by deficiencies in enzymes involved in the degradation of glycosaminoglycans (GAGs)—long chains of sugar carbohydrates in cells that help build bone, cartilage, tendons, corneas, skin, and connective tissue. Although enzyme replacement therapy has become available for the treatment of some types of MPS, effective treatment of neurodegenerative forms of MPS has yet to be determined. Recently, genistein (4',5,7-trihydroxyisoflavone), a specific inhibitor of protein tyrosine kinase, has been found to inhibit GAG synthesis and to reduce GAG concentrations in cultures of fibroblasts of MPS patients. Therefore, a potential substrate reduction therapy has been proposed.
Objective: The aim of this study was to examine urinary GAG concentration, hair morphology, and cognitive function in patients receiving genistin treatment for Sanfilippo syndrome (MPS type III).
Methods:Patients aged 3 to 14 years with a biochemically confirmed diagnosis of MPS IIIA or MPS IIIB were eligible to enroll in this open-label, pilot study. Genistin-rich soy isoflavone extract 5 mg/kg/d was administered PO for 12 months. Urinary GAG concentration, hair morphology,and cognitive function (measured using a modified version of the Brief Assessment Examination [BAE] and parent observations)were measured at baseline and after 12 months of treatment.
Results: Ten patients (6 girls, 4 boys; mean age, 8 years [range,3\2-14 years];mean weight, 28 kg [range, 17\2-43 kg]) were included in the study. All patients had Sanfilippo syndrome; 5 patients had MPS IIIA and 5 had MPS IIIB. After 1 year, statistically significant improvement was found in urinary GAG concentration, hair morphology, and cognitive function. Urinary GAG concentration decreased significantly in all 5 patients with MPS IIIA and in 2 patients with MPS IIIB (P = 0.028). Hair morphology improved significantly in all 5 MPS IIIA patients and in 3 MPS IIIB patients (P = 0.012). A significant increase in the BAE score (by 2-6 points) was noted in 8 patients, while the scores of 2 patients did not change after 12 months of treatment (P = 0.012). No adverse events (AEs) considered related to treatment were reported. Moreover, no AEs not related to the treatment (apart from classical symptoms of MPS III) were noted.
Conclusions: This pilot study found some improvements in GAG concentration, hair morphology, and cognitive function in these pediatric patients with Sanfilippo syndrome treated with genistin-rich soy isoflavone extract for 1 year. Clinical trials are needed to evaluate the efficacy and safety of this potential treatment
Fish blood as a chemical signal for Antarctic marine invertebrates
Get PDFChemical cues released from dead or injured organisms constitute important signals informing nearby animals about a feeding possibility. The ability to detect the signal, evaluate its meaning and locate its source can help organisms to exploit food resources efficiently, which is especially important to animals living in environments with limited food supply. Experiments were carried out to study the behavioral responses of several Antarctic benthic invertebrates to fish (Notothenia corriceps) blood. Necrophagous species such as sea stars Odontaster validus and Lysasterias sp., amphipod Waldeckia obesa and nemertean Parborlasia corrugatus responded to fish blood with changes in their behavior. The behavior common to all these species was locomotion directed towards the stimulus source. Behavioral components consistent with food consumption were observed in O. validus and P. corrugatus. The reaction of herbivorous limpets Nacella concinna to fish blood depended on the animal size. Large (>10 mm) limpets showed no behavioral response, whereas small ones (<10 mm) reacted to the stimulus by moving a short distance away. These results indicate that blood released from the tissues of injured or dead animals may be an important chemical signal for organisms belonging to different taxa