The response of prion genic variation to selection for scrapie resistance in Hungarian indigenous sheep breeds.

The authors studied the present status of Hungarian indigenous sheep breeds based on the genetic background of scrapie resistance. The aim of this investigation was to estimate the relative frequency of prion haplotypes, genotypes and risk categories, as well as to reveal the efficiency of the scrapie eradication programme achieved over the last decade. A novel approach in the characterisation of prion by using its genic variation was also implemented. The authors established that the proportion of deleterious sites (%) can be a useful indicator of the eradication programme. Based on a large sample size, it was confirmed that the scrapie resistance of the Cikta breed is low, and the classification of this breed according to risk category has not improved. However, the frequent genotype ARQ and risk category 3 can also be considered characteristic of the breed. The careful use of these genotypes is permitted and will contribute to the maintenance of breed diversity. The response of prion genic variation to selection for scrapie resistance in the other breeds (Tsigai, Milking Tsigai, White Racka, Black Racka and Gyimes Racka) was definitely successful.

Evaluation of Maternal Genetic Background of Two Hungarian Autochthonous Sheep Breeds Coming from Different Geographical Directions.

The aim of our research was the evaluation of the maternal genetic background of two Hungarian autochthonous sheep breeds of different geographical origin. A major argument for the preservation of endangered animal breeds is their documented past and historical importance. These also include the registration of pedigree data. This is the first study to evaluate and compare Tsigai and Cikta sheep in Hungary. Our investigation is based on two complete sequences of mitochondrial DNA (cytochrome b gene and control region). Our research was performed on these two sheep breeds with markedly different breed histories and breed characteristics to determine a possible common maternal genetic background, as ultimately the origin of both breeds can be traced back to Asia Minor. Between 2015 and 2017, a total of 203 biological samples were taken using a newly introduced founder sampling method. We found that the prevailing haplogroup B accounted for over 80% of both breeds, strengthening the common ancestral root. However, the pairwise genetic differentiation estimates (KST) calculated using the sequence-based statistics for cytochrome b gene and control region were 0.034 and 0.021, respectively (both at level p < 0.05); thus, revealing genetic differentiation in both sequences between the Tsigai and Cikta. We note that the known different history of the breeds is clearly justified by the currently studied deviations in their maternal genetic background.

Human internal thoracic artery grafts exhibit severe morphological and functional damage and spasmic vasomotion due to oxidative stress.

BACKGROUND: The internal thoracic artery (ITA) is the first choice for myocardial revascularization, but atherosclerotic lesions and perioperative vasospasm may still limit its functionality. Oxidative stress via the peroxynitrite - poly-(ADP-ribose) polymerase (PARP) cascade plays an important role in the pathogenesis of impaired vascular tone via endothelial injury. We aimed to investigate and describe the histology, PARP activation and functionality of ITA grafts and to assess the possible beneficial effect of PARP-inhibition. MATERIAL/METHODS: ITA specimens from 47 patients (26 men, mean age 66.2 ± 1.7 years) who underwent coronary bypass surgery were processed for histological and immunohistochemical studies for oxidative stress and PARP activation, and were functionally tested with acetylcholine (ACh) and sodium nitroprusside (SNP) with or without PARP inhibition. RESULTS: The sections showed atherosclerotic alterations and oxidative and nitrosative stress were evidenced by positive 3-nitrotyrosine, 4-hydroxynonenal and PAR stainings. Functionally, 88.1% reacted to K-Krebs, 68.7% exhibited contraction after 1 µM phenylephrine, 29.9% exhibited relaxation to 30 µM Ach, and all precontracted segments relaxed to 30 µM SNP. High amplitude vasomotion was observed in 47.8% of the segments, which could be abolished by the application of 10 µM SNP.Incubation of the preparations with PJ34 did not improve endothelium-dependent vasodilation. CONCLUSIONS: ITA grafts are severely damaged both morphologically and functionally in patients undergoing coronary artery bypass surgery, but PARP inhibition cannot improve their functional characteristics. The topical use of SNP to the ITA during the operation may improve vascular functions by dilating the vessels and eliminating the eventual spasmic vasomotion.

Comparison of mtDNA control region among descendant breeds of the extinct Zaupel sheep revealed haplogroup C and D in Central Europe.

The consideration of the descendance is indispensable in the preservation of endangered animal breeds. The authors compared mitochondrial DNA (mtDNA) control region sequence in three descendant breeds of the extinct Zaupel sheep, firstly. Their investigation was carried out in order to prove the common origin of Waldschaf (Austria), Bovec sheep (Slovenia) and Cikta (Hungary). A total of 118 biological samples were taken from non-related representatives of the three breeds between 2015 and 2017. A newly designed primer pair was also used to amplify the segment (1180 bp) to be tested. The total number of haplotypes in the whole study population was 49. The majority of which fell into haplogroup B. The significant negative value of the Fu's Fs statistic (Fs statistic = -3.296, p = 0.013) based on haplotype frequencies demonstrated a moderate foreign gene flow. As a novel observation haplogroups C and D appeared in Cikta and Bovec sheep, respectively. The Tajima D-test value in the entire study population was -0.914 (p > 0.10), meaning that the separation of the three descendant breeds did not cause genetic drift, these are collectively in genetic equilibrium. The genetic information confirmed the common origin of the breeds known from the breed history.

Confirmation of a novel siadenovirus species detected in raptors: partial sequence and phylogenetic analysis.

Partial genome characterisation of a novel adenovirus, found recently in organ samples of multiple species of dead birds of prey, was carried out by sequence analysis of PCR-amplified DNA fragments. The virus, named as raptor adenovirus 1 (RAdV-1), has originally been detected by a nested PCR method with consensus primers targeting the adenoviral DNA polymerase gene. Phylogenetic analysis with the deduced amino acid sequence of the small PCR product has implied a new siadenovirus type present in the samples. Since virus isolation attempts remained unsuccessful, further characterisation of this putative novel siadenovirus was carried out with the use of PCR on the infected organ samples. The DNA sequence of the central genome part of RAdV-1, encompassing nine full (pTP, 52K, pIIIa, III, pVII, pX, pVI, hexon, protease) and two partial (DNA polymerase and DBP) genes and exceeding 12 kb pairs in size, was determined. Phylogenetic tree reconstructions, based on several genes, unambiguously confirmed the preliminary classification of RAdV-1 as a new species within the genus Siadenovirus. Further study of RAdV-1 is of interest since it represents a rare adenovirus genus of yet undetermined host origin.

Molecular confirmation of a new herpesvirus from catfish (Ameiurus melas) by testing the performance of a novel PCR method, designed to target the DNA polymerase gene of alloherpesviruses.

A PCR method with consensus degenerate primers was developed for the detection of herpesviruses (HVs) of anamnia. Compared to previously published PCRs, targeting the DNA polymerase gene of fish HVs, the size of PCR products was more than tripled. Although broad applicability of the method could not be proven, approximately 1,600-bp fragments from HVs of white sturgeon (Acipenser transmontanus) and black bullhead (Ameiurus melas) were obtained and sequenced. Phylogenetic tree reconstructions showed both HVs to be monophyletic with the single member (ictalurid HV-1) of the genus Ictalurivirus in the new family Alloherpesviridae.

Human heart mitochondria do not produce physiologically relevant quantities of nitric oxide.

Previous studies raised the possibility that nitric oxide synthase is present in heart mitochondria (mtNOS) and the existence of such an enzyme became generally accepted. However, original experimental evidence is rather scarce and positive identification of the enzyme is lacking. We aimed to detect an NOS protein in human and mouse heart mitochondria and to measure the level of NO released from the organelles. Western blotting with 7 different anti-NOS antibodies failed to detect a NOS-like protein in mitochondria. Immunoprecipitation or substrate-affinity purification of the samples concentrated NOS in control preparations but not in mitochondria. Release of NO from live respiring human mitochondria was below 2 ppb after 45 min of incubation. In a bioassay system, mitochondrial suspension failed to cause vasodilation of human mammary artery segments. These results indicate that mitochondria do not produce physiologically relevant quantities of NO in the heart and are unlikely to have any physiological importance as NO donors, nor do they contain a recognizable mtNOS enzyme.

Neurogliaform neurons form a novel inhibitory network in the hippocampal CA1 area.

We studied neurogliaform neurons in the stratum lacunosum moleculare of the CA1 hippocampal area. These interneurons have short stellate dendrites and an extensive axonal arbor mainly located in the stratum lacunosum moleculare. Single-cell reverse transcription-PCR showed that these neurons were GABAergic and that the majority expressed mRNA for neuropeptide Y. Most neurogliaform neurons tested were immunoreactive for alpha-actinin-2, and many stratum lacunosum moleculare interneurons coexpressed alpha-actinin-2 and neuropeptide Y. Neurogliaform neurons received monosynaptic, DNQX-sensitive excitatory input from the perforant path, and 40 Hz stimulation of this input evoked EPSCs displaying either depression or initial facilitation, followed by depression. Paired recordings performed between neurogliaform neurons showed that 85% of pairs were electrically connected and 70% were also connected via GABAergic synapses. Injection of sine waveforms into neurons during paired recordings resulted in transmission of the waveforms through the electrical synapse. Unitary IPSCs recorded from neurogliaform pairs readily fatigued, had a slow decay, and had a strong depression of the synaptic response at a 5 Hz stimulation frequency that was antagonized by the GABA(B) antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP55845). The amplitude of the first IPSC during the 5 Hz stimulation was also increased by CGP55845, suggesting a tonic inhibition of synaptic transmission. A small unitary GABA(B)-mediated IPSC could also be detected, providing the first evidence for such a component between GABAergic interneurons. Electron microscopic localization of the GABA(B1) subunit at neurogliaform synapses revealed the protein in both presynaptic and postsynaptic membranes. Our data disclose a novel interneuronal network well suited for modulating the flow of information between the entorhinal cortex and CA1 hippocampus.

Organization of dye-coupled cerebellar granule cells labeled from afferent vestibular and dorsal root fibers in the frog Rana esculenta.

Application of neurobiotin to the nerves of individual labyrinthine organs and dorsal root fibers of limb-innervating segments of the frog resulted in labeling of granule cells in the cerebellum showing a significant overlap with a partial segregation in the related areas of termination. In different parts of the cerebellum, various combinations of different canal and otolith organ-related granule cells have been discerned. The difference in the extension of territories of vertical canals vs. horizontal canals may reflect their different involvement in the vestibuloocular and vestibulospinal reflex. Dye-coupled cells related to the lagenar and saccular neurons were localized in more rostral parts of the cerebellum, whereas cells of the utricle were represented only in its caudal half. This separation is supportive of the dual function of the lagena and the saccule. The territories of granule cells related to the cervical and lumbar segments of the spinal cord were almost completely separated along the rostrocaudal axis of cerebellum, whereas their territories were almost entirely overlapping in the mediolateral and ventrodorsal directions. The partial overlap of labyrinthine organ-related and dorsal root fiber-related granule cells are suggestive of a convergence of sensory modalities involved in the sense of balance. We propose that the afferent input of vestibular and proprioceptive fibers mediated by gap junctions to the cerebellar granule cells subserve one of the possible morphological correlates of a very rapid modification of the motor activity in the vestibulocerebellospinal neuronal circuit.

[Do we use digitalis properly in the management of elderly patients suffering from the signs of chronic heart failure?]. / Helyesen alkalmazzuk-e a digitáliszt idos, krónikus szivelégtelenség tüneteit mutató betegeinkben?

INTRODUCTION: The indications of digoxin therapy has been significantly narrowed and also the effective target therapeutic blood level has been decreased (0.9 micromol/L) compared to the previously desired one. OBJECTIVE: In this retrospective trial the data of 60 consecutive patients over 65 years (25 male, 35 female, mean age 77.3 +/- 5.0 y), hospitalized between 01. 01. 2002 and 31. 12. 2003 with a diagnosis of chronic heart failure and elevated (> 1.2 microg/I) serum level of digoxin, were analyzed. METHODS: Beside the analysis of the age, sex, serum level of digoxin and potassium, creatinine clearance value, symptoms and ECG-signs of digitalis intoxication, presence of atrial fibrillation, concomitant diseases and left ventricular ejection fraction value, the reasonability of digitalis treatment and therapy applied at the time of discharge (considering actual treatment guidelines) were also reviewed. RESULTS: At the admission mean serum level of digoxin was 2.1 +/- 0.9 microg/l. 20 patient's value (33.3%) was found above 2.2 microg/l. Symptoms characteristic for digitalis intoxication were observed in 28 patients. On the ECG performed at admission signs of digitalis effect/overdose were observed in 54 cases ("bigemin" ventricular extrasystoles, bradycardia, characteristic down-sloping ST-depressions). The mean left ventricular ejection fraction of the patients (51.5 +/- 12.7%) did not suggest to a significant left ventricular systolic dysfunction. For the elevated serum level of digoxin the impaired renal function (mean creatinine clearance 42.9 +/- 21.3 mL/min) was responsible in most cases. In patients with the highest serum level of digoxin (n = 20, 3.2 +/- 0.7 microg/L) the creatinine clearance was even lower, 30.4 +/- 13.7 mL/min. During hospital treatment the administration of digitalis was found to be unnecessary and thus terminated in 44 patients. At the discharge only 16 patients were receiving digitalis, 14 of them digoxin and 2 patients digitoxin. CONCLUSIONS: The authors emphasize, that in case of elderly patients the indication and control of digitalis therapy requires greater precaution and tight doctor-patient cooperation.

[Analysis of vascular complications of IABP therapy in open-heart surgery patients 1999-2004]. / Szívmuitött betegek intraaortikus ballonpumpa kezelése során kialakult vaszkuláris szövodményeinek elemzése (1999-2004).

Intraaortic balloon pump (IABP) is being used in cardiac surgery in an increased ratio. IABP therapy involves considerable risk, mainly vascular complications, postoperative bleeding and infection can represent danger. Between 1999 and 2004 out of 4443 open heart surgery operations we have performed intraaortic balloon pump treatment in case of 75 patients. The mean age was 64 years, 23 patients had diabetes mellitus, 47 patients had hypertension, 20 patients had peripheral vascular disease as well. We performed IABP therapy most frequently during isolated coronary bypass operations (42 cases), but also combined operations (implantation of valve prosthesis + coronary bypass) represent a significant part (implantation of aortic valve prosthesis + CABG: 5 cases, implantation of mitral valve prosthesis + CABG: 8 cases). Vascular complications occurred in 10 cases--13.3%--out of 75 patients, including 7 fatal ones. Three cases are due to the IABP treatment itself: Crush syndrome was developed leading to the loss of the patient. Applying the multiple logistic regression model we have examined the effect of the following factors on the occurrence of vascular complications: gender, age, body surface, accompanying diseases (hypertension, diabetes, peripheral vascular disease), the method and timing of insertion. Peripheral vascular disease (p < 0.005) and hypertension (p = 0.01) represent independent risk factors regarding the occurrence of complications. Having performed chi-square test we have not identified significant correlations between mortality and vascular complications. In case of prevailing peripheral vascular disease, the application of alternative insertion techniques--via the ascending aorta, the axillary artery--are recommended.

Effects of amniotic epithelial cell transplantation in endothelial injury.

PURPOSE: Human amniotic epithelial cells (hAECs) are promising tools for endothelial repair in vascular regenerative medicine. We hypothesized that these epithelial cells are capable of repairing the damaged endothelial layer following balloon injury of the carotid artery in adult male rats. RESULTS: Two days after injury, the transplanted hAECs were observed at the luminal side of the arterial wall. Then, 4 weeks after the injury, significant intimal thickening was observed in both untreated and cell implanted vessels. Constriction was decreased in both implanted and control animals. Immunohistochemical analysis showed a few surviving cells in the intact arterial wall, but no cells were observed at the site of injury. Interestingly, acetylcholine-induced dilation was preserved in the intact side and the sham-transplanted injured arteries, but it was a trend toward decreased vasodilation in the hAECs' transplanted vessels. CONCLUSION: We conclude that hAECs were able to incorporate into the arterial wall without immunosuppression, but failed to improve vascular function, highlighting that morphological implantation does not necessarily result in functional benefits and underscoring the need to understand other mechanisms of endothelial regeneration.

[Modified Jatene operation for the repair of left ventricle aneurysm]. / Tapasztalataink a bal kamra aneurysma sebészetében--módosított Jatene mútét alkalmazásával.

INTRODUCTION: 134 left ventricle aneurysm operations were performed at our institute between 1993 and 2000. The original Jatene operation was modified by our workgroup: running sutures and abandoning Teflon pledges for faster operation; an additional linear closure of the remnant aneurysm above the patch to eliminate bleeding complications. OBJECT: The aim of this study was to describe our surgical technique, its advantages against other surgical methods in left ventricle aneurysms. METHOD: We divided our patients into three subgroups: linear techniques, such as plication (PL) n = 54, resection (RE) n = 45, and circular technique modified Jatene operation (MJ) n = 35. We performed a I. retrospective study in these subgroup, and a II. long-term clinical follow-up in MJ group. RESULTS: We did not find any technical difficulties in modified Jatene operations (there was no significant difference in operating times and cross clamping times in linear versus circular techniques). On the other hand, we found significantly better left ventricle geometry after circular method with echocardiography. There was no short-term (< 30 days) mortality in RM group. II. In the RM group long-term clinical follow-up we found further improvement in left ventricle geometry. We lost 1 patient (2.94%) because of cardiac death, and 2 patients (5.88%) had heart transplantation. All patients were in NYHA class I or II. CONCLUSIONS: In left ventricle aneurysm surgery circular techniques should be preferred because the better results in left ventricle geometry. We think that our improvements in Jatene's operation significantly decreased surgical difficulties, and through decreasing operating time short and long term outcome can be positively affected.

Designing magnetic superlattices that are composed of single domain nanomagnets.

BACKGROUND: The complex nature of the magnetic interactions between any number of nanosized elements of a magnetic superlattice can be described by the generic behavior that is presented here. The hysteresis characteristics of interacting elliptical nanomagnets are described by a quasi-static method that identifies the critical boundaries between magnetic phases. A full dynamical analysis is conducted in complement to this and the deviations from the quasi-static analysis are highlighted. Each phase is defined by the configuration of the magnetic moments of the chain of single domain nanomagnets and correspondingly the existence of parallel, anti-parallel and canting average magnetization states. RESULTS: We give examples of the phase diagrams in terms of anisotropy and coupling strength for two, three and four magnetic layers. Each phase diagrams character is defined by the shape of the magnetic hysteresis profile for a system in an applied magnetic field. We present the analytical solutions that enable one to define the "phase" boundaries between the emergence of spin-flop, anti-parallel and parallel configurations. The shape of the hysteresis profile is a function of the coupling strength between the nanomagnets and examples are given of how it dictates a systems magnetic response. Many different paths between metastable states can exist and this can lead to instabilities and fluctuations in the magnetization. CONCLUSION: With these phase diagrams one can find the most stable magnetic configurations against perturbations so as to create magnetic devices. On the other hand, one may require a magnetic system that can easily be switched between phases, and so one can use the information herein to design superlattices of the required shape and character by choosing parameters close to the phase boundaries. This work will be useful when designing future spintronic devices, especially those manipulating the properties of CoFeB compounds.

Partial characterization of a new adenovirus lineage discovered in testudinoid turtles.

In the USA and in Hungary, almost simultaneously, adenoviruses of a putative novel lineage were detected by PCR and sequencing in turtles belonging to four different species (including two subspecies) of the superfamily Testudinoidea. In the USA, partial sequence of the adenoviral DNA-dependent DNA polymerase was obtained from samples of a captive pancake tortoise (Malacochersus tornieri), four eastern box turtles (Terrapene carolina carolina) and two red-eared sliders (Trachemys scripta elegans). In Hungary, several individuals of the latter subspecies as well as some yellow-bellied sliders (T. scripta scripta) were found to harbor identical, or closely related, putative new adenoviruses. From numerous attempts to amplify any other genomic fragment by PCR, only a nested method was successful, in which a 476-bp fragment of the hexon gene could be obtained from several samples. In phylogeny reconstructions, based on either DNA polymerase or hexon partial sequences, the putative new adenoviruses formed a clade distinct from the five accepted genera of the family Adenoviridae. Three viral sub-clades corresponding to the three host genera (Malacochersus, Terrapene, Trachemys) were observed. Attempts to isolate the new adenoviruses on turtle heart (TH-1) cells were unsuccessful. Targeted PCR screening of live and dead specimens revealed a prevalence of approximately 25% in small shelter colonies of red-eared and yellow-bellied sliders in Hungary. The potential pathology of these viruses needs further investigation; clinically healthy sliders were found to shed the viral DNA in detectable amounts. Based on the phylogenetic distance, the new adenovirus lineage seems to merit the rank of a novel genus.

Complete sequence of raptor adenovirus 1 confirms the characteristic genome organization of siadenoviruses.

Currently, the family Adenoviridae contains five genera, out of which Siadenovirus is one of the two least densely populated ones. A new member representing a new species in this genus has been detected in various birds of prey. The virus, named raptor adenovirus 1 (RAdV-1), could not be isolated, probably because no appropriate permissive cell-line was available. Partial genomic sequences, obtained by PCR and suggesting that the virus is a new siadenovirus species, have been published earlier. In the present paper, determination and analysis of the complete RAdV-1 genome are reported. This is the first complete genome sequence acquired from a non-isolated adenovirus (AdV). The sole source was a mixture of the internal organs of the diseased and dead birds. Until now, the genomic organization considered characteristic to siadenoviruses had been deduced from the detailed study of only two virus species, one of which originated from birds and the other from a frog. The present analysis of RAdV-1 confirmed the genus-specific genetic content and genomic features of siadenoviruses, and a putative novel gene was found as well. In general, AdVs and most of the AdV genera are thought to be strictly host specific. In the genus Siadenovirus, however, two virus species of rather divergent (avian and amphibian) host origin were present when the genus was found. Although by now the greatest number of known siadenoviruses infect birds, the original hosts of the genus remain unknown.

Recognition and partial genome characterization by non-specific DNA amplification and PCR of a new siadenovirus species in a sample originating from Parus major, a great tit.

A seemingly novel siadenovirus species was detected by PCR and sequencing in the sample of a great tit (Parus major) found dead in Hungary. Since the genus Siadenovirus has very few known members so far, further study of the virus was intriguing not only from epizootiological but also from taxonomical aspects. The sample, which had been tested in another PCR survey previously, consisted of less than 50 microl of extracted nucleic acid. To ensure sufficient target DNA for an extended study, the viral genome had to be preserved. To this end, the sample was subjected to a novel method of non-specific DNA amplification. Using the amplified DNA as target, different PCR and sequencing strategies were applied with consensus or specific primers for the study of the central genome part of the putative tit adenovirus. The sequence of supposedly one half (13,628 bp) of the genome was determined including eight full genes between the genes of the IVa2 and hexon proteins. The gene content of the viral genome fragment as well as the results of the phylogenetic analyses with different proteins confirmed the discovery of a new species in the genus Siadenovirus. This is the first report on the detection of an adenovirus in great tits. The methods, described in this work, proved suitable for the recovery of nucleic acid samples that contain irreplaceable microbial genomic DNA but are only available in limited quantities.