Optimization of the Y831C mutation detection in human DNA polymerase gamma by allelic discrimination assay

Many well-defined mutations in the gene for the catalytic subunit of polymerase gamma (POLG1) have been found to be associated with disease, whereas the status of several mutations remains unresolved due to the conflicting reports on their frequencies in populations of healthy individuals. Here, we have developed a highly sensitive, real-time allelic discrimination assay enabling detection of the Y831C mutation in the POLG1 gene. The Y831C mutation is present in the Polish population at a frequency of 2.25%. The new assay is well suited to both extensive population studies and molecular diagnostics of POLG1.     

Molecular analysis of plasmid pAP4 from Acetobacter pasteurianus

The complete nucleotide sequence of plasmid pAP4 isolated from Acetobacter pasteurianus 2374^T has been determined. Plasmid pAP4 was analysed and found to be 3,870 bp in size with a G+C content of 50.1%. Computer assisted analysis of sequence data revealed 2 possible ORFs with typical promoter regions. ORF1 codes for a protein responsible for kanamycin resistance similar with Tn5 transposone, ORF2 encodes a resistance to ampicillin identical with Tn3 transposone. Plasmid has in A. pasteurianus five copies and in E. coli DH1 about 30 copies per chromosome and it segregation stability in both strains is very high. Based on the data on replication region, plasmid does not code for a replication protein and origin region is similar with ColE1-like plasmid.     

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (E _m) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 μM to 1 mM) for several hours to one week were studied. The E _m values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (E _D) when the Cd concentration exceeded 100 μM. The values of E _D ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of E _m was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 μM) or complete repolarization (Cd < 100 μM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of E _m a continuous concentration-dependent decrease of E _m followed at all Cd concentrations. Depolarization of E _m was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 μM to 1 mM Cd, with the exception of root cells treated with 1 and 10 μM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 μM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h. All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 μM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H⁺ ATPase because fusicoccin, an H⁺ ATPase activator diminished the depolarizing effect of Cd on the E _m. The depolarization of E _m in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H⁺ ATPase.     

A new colorimetric assay for methionyl aminopeptidases: examination of the binding of a new class of pseudopeptide analog inhibitors

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.     

Confined and Chemically Flexible Grain Boundaries in Polycrystalline Compound Semiconductors

Grain boundaries (GBs) in polycrystalline Cu(In,Ga)Se₂ thin films exhibit only slightly enhanced recombination, as compared with the grain interiors, allowing for very high power‐conversion efficiencies of more than 20% in the corresponding solar‐cell devices. This work highlights the specific compositional and electrical properties of Cu(In,Ga)Se₂ GBs by application of appropriate subnanometer characterisation techniques: inline electron holography, electron energy‐loss spectroscopy, and atom‐probe tomography. It is found that changes of composition at the GBs are confined to regions of only about 1 nm in width. Therefore, these compositional changes are not due to secondary phases but atomic or ionic redistribution within the atomic planes close to the GBs. For different GBs in the Cu(In,Ga)Se₂ thin film investigated, different atomic or ionic redistributions are also found. This chemical flexibility makes polycrystalline Cu(In,Ga)Se₂ thin films particularly suitable for photovoltaic applications.     

Postconditioning and Anticonditioning: Possibilities to Interfere to Evoked Apoptosis

The aim of this study was to validate the ability of postconditioning, used 2 days after kainate intoxication, to protect selectively vulnerable hippocampal CA1 neurons against delayed neuronal death. Kainic acid (8 mg/kg, i.p.) was used to induce neurodegeneration of pyramidal CA1 neurons in rat hippocampus. Fluoro Jade B, the specific marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 days after intoxication without and with delayed postconditioning (norepinephrine, 3.1 μmol/kg i.p., 2 days after kainate administration) and anticonditioning (Extract of Ginkgo biloba, 40 mg/kg p.o used simultaneously with kainate). Morris water maze was used on 6th and 7th day after kainate to test learning and memory capabilities of animals. Our results confirm that postconditioning if used at right time and with optimal intensity is able to prevent delayed neuronal death initiated not only by ischemia but kainate intoxication, too. The protective effect of repeated stress–postconditioning was suppressed if extract of Ginkgo biloba (EGb 761, 40 mg/kg p.o.) has been administered together with kainic acid. It seems that combination of lethal stress and antioxidant treatment blocks the activation of endogenous protecting mechanism known as ischemic tolerance, aggravates neurodegeneration and, after repeated stress is able to cause cumulative damage. This observation could be very valuable in situation when the aim of treatment is elimination of unwanted cell population from the organism.     

Two glucuronoyl esterases of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The white-rot fungus Phanerochaete chrysosporium produces glucuronoyl esterase, a recently discovered carbohydrate esterase, during growth on sugar beet pulp. Two putative genes encoding this enzyme, ge1 and ge2, were isolated and cloned. Heterologous expression in Aspergillus vadensis, Pycnoporus cinnabarinus and Schizophyllum commune resulted in extracellular glucuronoyl esterase activity, demonstrating that these genes encode this enzymatic function. The amino acid sequence of GE1 was used to identify homologous genes in the genomes of twenty-four fungi. Approximately half of the genomes, both from ascomycetes and basidiomycetes, contained putative orthologues, but their presence could not be assigned to any of fungal class or subclass. Comparison of the amino acid sequences of identified and putative glucuronoyl esterases to other types of carbohydrate esterases (CE) confirmed that they form a separate family of CEs. These enzymes are interesting candidates for biotechnological applications such as the separation of lignin and hemicellulose.     

The Changes in Endogenous Antioxidant Enzyme Activity After Postconditioning

1. The aim of this work was to study potential mechanisms participating in postischemic protection of selectively vulnerable CA1 neurons in the hippocampus. Experiments were focused on measuring changes in endogenous antioxidant enzyme activity.2. Forebrain cerebral ischemia was induced in a rat by four-vessel occlusion. Ten minutes of ischemia induces so-called delayed neuronal death in selectively vulnerable CA1 region 3 days later. After 7 days of reperfusion, 71.6% of neurons succumb to neurodegeneration. When 5 min of ischemia was used as postconditioning, 2 days after 10 min of cerebral ischemia, delayed neuronal death in CA1 was almost completely (89.9%) prevented.3. Searching for mechanisms of protection, we measured the activity of endogenous antioxidant enzymes. Activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were measured in the hippocampus, striatum and cortex by spectrophotometric methods after 10 min of ischemia used as the preconditioning. Two days after the preconditioning or the sham operation, second ischemia was induced for 5 min. We observed significant increase of total SOD activity in all studied regions of the brain 5 h after postconditioning (5 min of ischemia). SOD activity decreased to control values after 24 h.4. In some experiments, we used intraperitoneal injections of norepinephrine (3.1 μM/kg) or 3-nitropropionic acid (20 mg/kg) as postconditioning, instead of ischemia. All three treatments resulted in significant increase of SOD activity, but norepinephrine was the most effective. The same effect as was seen for total SOD activity could be observed for CuZn-SOD as well as Mn-SOD activity. Similarly, considerable increase in the activity of catalase was detected 5 h after postconditioning (5 min of ischemia). It is interesting that the greatest changes were established in selectively vulnerable hippocampus and striatum. As in the case of SOD, the highest levels of CAT activity were induced by norepinephrine, while lower but significant increase in CAT activity was induced by 3-nitropropionic acid.5. Our results suggest that endogenous antioxidants SOD and CAT could play considerable neuroprotective role after postconditioning.     

Application of potential probiotic Lactobacillus fermentum AD1 strain in healthy dogs

Probiotic utilization is becoming increasingly popular in veterinary medicine. However, only few probiotic products are available commercially for use in dogs in our market. Therefore, the aim of our study was to determine the properties of new potential probiotic Lactobacillus fermentum AD1 strain-own canine isolate and to investigate its effect on several microbiological and biochemical parameters in healthy dogs. The strain expressed in vitro survival by pH 3.0 after 3h (86.8%) and in the presence of 1% bile (75.4%). The AD1 strain adhered to the canine and human intestinal mucus. It was sensitive to commonly used antimicrobials. Fifteen healthy dogs were supplemented with 10(9)L. fermentum AD1 for 7 days. At the end of AD1 strain application, numbers of faecal lactobacilli and enterococci increased significantly in the canine faeces. Significant increase of total protein and total lipid and significant reduction of glucose in serum of dogs were noted. These data indicate that L. fermentum AD1 survive transit through the canine gastrointestinal tract, and populate the colon and probably increased absorption of some nutrients. Whether longer time of its application lead to the same results as well as its potential to improve immune function in dogs remains to be determined.