Identification and functionality of proteomes secreted by rat cardiac stem cells and neonatal cardiomyocytes

In the heart, the proteomes secreted by both cardiac stem cells (CSCs) and cardiac myocytes could act synergistically, but the identification and functionality of the proteins comprising the individual secretomes have not yet been described. In this study, we have identified proteins present in the media obtained from cultured rat CSCs and from cultured neonatal rat ventricular myocytes (NRVMs) and compared them with proteins identified in the media alone. Briefly, 83 unique proteins were identified after analysis by RPLC and MS. In total 49 and 23% were NRVM‐specific or CSC‐specific proteins, respectively, and 63% of total 83 proteins were integral plasma membrane and/or known secreted proteins. Fifteen proteins met our criteria for paracrine/autocrine factors: (i) robust protein identification, (ii) cell specific and (iii) known to be secreted. Most of these proteins have not been previously linked to stem cells. NRVM‐specific proteins atrial natriuretic factor (ANP) and connective tissue growth factor, and CSC‐specific protein interleukin‐1 receptor‐like 1 (ST2) were found to affect rat CSC proliferation. These findings suggest that relative concentration of each protein may be crucial for cellular intertalk and for the final outcome of cardiac cell therapy.     

Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica

The freshwater filamentous green oxyphotobacterium Prochlorothrix hollandica is an unusual oxygenic photoautotrophic cyanobacterium differing from most of the others by the presence of light-harvesting Pcb antenna binding both chlorophylls a and b and by the absence of phycobilins. The pigment-protein complexes of P. hollandica SAG 10.89 (CCAP 1490/1) were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose density gradient and characterized by biochemical, spectroscopic and immunoblotting methods. The Pcb antennae production is suppressed by high light conditions (> 200 μmol photons m⁻² s⁻¹) in P. hollandica. PcbC protein was found either in higher oligomeric states or coupled to PS I (forming antenna rings around PS I). PcbA and PcbB are most probably only very loosely bound to photosystems; we assume that these pigment-protein complexes function as low light-induced mobile antennae. Further, we have detected α-carotene in substantial quantities in P. hollandica thylakoid membranes, indicating the presence of chloroplast-like carotenoid synthetic pathway which is not present in common cyanobacteria.     

Natural hybridization in tropical spikerushes of Eleocharis subgenus Limnochloa (Cyperaceae): Evidence from morphology and DNA markers

? Premise of the study: Natural hybridization represents an important force driving plant evolution and affecting community structure and functioning. Hybridization may be overlooked, however, among morphologically highly uniform congeners. An excellent example of such a group is Eleocharis subgenus Limnochloa, which has no reliably proven hybrids. Does this reflect biological barriers to interspecific crosses or difficulties in detecting the hybrids? We tested the hypothesis that hybridization occurs among sympatric Eleocharis cellulosa, E. interstincta, and E. mutata in northern Belize, Central America. ? Methods: Morphometric study (407 plants) was followed by examination of inter-simple sequence repeat (ISSR) polymorphisms (44 plants) and ITS sequence variation (33 plants). ? Key results: Two putatively hybrid morphotypes were discerned-E. cellulosa-resembling and E. interstincta-resembling. DNA markers of E. cellulosa and E. interstincta displayed additive constitution in plants from one E. cellulosa-resembling population only. The other putatively hybrid populations contained ISSR and ITS markers of the species they resembled morphologically, several unique ISSR markers, and ITS sequences of an undescribed South American Limnochloa entity. DNA markers of E. mutata were absent in the putative hybrids. ? Conclusions: Simultaneous use of various types of molecular markers can overcome many pitfalls of investigations concerning hybridization among closely related and morphologically similar species. Northern Belize represents a hybrid zone of E. cellulosa and E. interstincta. A third participant in the hybridization events occurring in this zone is an unknown Limnochloa lineage but is not E. mutata. Interspecific hybridization may play a significant role in the diversification of Eleocharis.     

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.     

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.     

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.     

Mitochondrial DNA variability in the Czech population, with application to the ethnic history of Slavs

Mitochondrial DNA (mtDNA) variability was studied in a sample of 179 individuals representing the Czech population of Western Bohemia. Sequencing of two hypervariable segments, HVS I and HVS II, in combination with screening of coding-region haplogroup-specific RFLP markers revealed that most Czech mtDNAs belong to the common West Eurasian mitochondrial haplogroups (H, pre-V HV*, J, T, U, N1, W, and X). However, about 3% of Czech mtDNAs encompass East Eurasian lineages (A, N9a, D4, M*). A comparative analysis with published data showed that different Slavonic populations in Central and Eastern Europe contain small but marked amounts of East Eurasian mtDNAs. We suggest that the presence of East Eurasian mtDNA haplotypes is not an original feature of the gene pool of the proto-Slavs but rather may be mostly a consequence of admixture with Central Asian nomadic tribes, who migrated into Central and Eastern Europe in the early Middle Ages.     

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.     

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.     

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.