Organ transplantation in Bulgaria

The transplantation program in Bulgaria started in 1968 with renal transplantations to a child and adult woman. In 1986 the first heart transplantation was performed. To date a total of 10 heart transplants have been performed, including one combined heart/lung. A liver transplantation program was launched in 2005 with a total number of 16 transplantations—7 from living donors and 9 from deceased donors. The highest transplantation activity is registered in the field of renal transplantation. During the period 1980–2006, 462 Bulgarian recipients of kidney were transplanted in Bulgaria. The ratio between transplantations from deceased and living related donors is approximately 1:0.9. Annual transplantation activity varies among the years from 1 to 12 renal transplantations p.m.p./per year. The 1- (80.7% vs. 63.1%), 5- (57.86% vs. 39.0%) and 10-year (42.65% vs. 23.62%) graft survival rates are higher for recipients of living donor kidneys compared to those of deceased donor. In 1983 a National kidney waiting list was established. Currently the number of the registered patients eligible for renal transplantation is 885. The proportion of sensitized patients in the waiting list is 20.45% and 4.34% of them are hyperimmunized. Recently HLAMatchmaker program has been implemented not only for sensitized patients but also for those with rare alleles and haplotypes. Post-transplant immunological monitoring showed a strong association between alloantibody presence and delayed graft function (Chi-square = 10.73, P < 0.001), acute rejection (Chi-square = 14.504, P < 0.001), chronic rejection (Chi-square = 12.84, P < 0.001) and graft loss (Chi-square = 20.283, P < 0.001). Based on the experience in our transplant center a strategy for improvement of long-term renal graft survival was developed and implemented.     

The DNA binding and bending activities of truncated tail-less HMGB1 protein are differentially affected by Lys-2 and Lys-81 residues and their acetylation

The role of lysines 2 and 81 as target sites for acetylation in full-length HMGB1 and truncated tail-less protein, respectively, has been studied by mutation analysis for the abilities of these proteins to bind and bend DNA. The DNA bending ability of truncated tail-less HMGB1 containing Lys-2 mutated to alanine does not differ from that of the wild-type protein, while the same mutation of Lys-81 reduced the bending capacity of the mutant protein. These data demonstrate that Lys-81 is critical for the DNA bending ability of truncated HMGB1. Such a conclusion is further confirmed by the experiments carried out with CBP-acetylated proteins: acetylation of Lys-2 in mutant protein K81/A81 alleviated DNA bending and induced DNA end-joining. On the contrary, the acetylation of Lys-81 in the mutant K2/A2 enhanced the bending potential of HMGB1∆C. Regarding the ability of HMGB1 to specifically bind bent DNA, the individual mutations of either K2 or K81 as well as the double mutation of both residues to alanine were found to completely abolish binding of truncated tail-less HMGB1 to cisplatin-modified DNA. We conclude that unlike the case with the bending ability of truncated HMGB1, where Lys-81 has a primary function, Lys-2 and Lys-81 are both critical for the protein''s binding to cisplatin-modified DNA. The mutation K2/A2 in full-length HMGB1 and acidic tail removal induce the same conformational changes. Any further substitutions at the acetylable lysines in the truncated form of HMGB1 do not have an additional effect.     

Excitation transfer and trapping kinetics in plant photosystem I probed by two-dimensional electronic spectroscopy

Photosystem I is a robust and highly efficient biological solar engine. Its capacity to utilize virtually every absorbed photon’s energy in a photochemical reaction generates great interest in the kinetics and mechanisms of excitation energy transfer and charge separation. In this work, we have employed room-temperature coherent two-dimensional electronic spectroscopy and time-resolved fluorescence spectroscopy to follow exciton equilibration and excitation trapping in intact Photosystem I complexes as well as core complexes isolated from Pisum sativum. We performed two-dimensional electronic spectroscopy measurements with low excitation pulse energies to record excited-state kinetics free from singlet–singlet annihilation. Global lifetime analysis resolved energy transfer and trapping lifetimes closely matches the time-correlated single-photon counting data. Exciton energy equilibration in the core antenna occurred on a timescale of 0.5 ps. We further observed spectral equilibration component in the core complex with a 3–4 ps lifetime between the bulk Chl states and a state absorbing at 700 nm. Trapping in the core complex occurred with a 20 ps lifetime, which in the supercomplex split into two lifetimes, 16 ps and 67–75 ps. The experimental data could be modelled with two alternative models resulting in equally good fits—a transfer-to-trap-limited model and a trap-limited model. However, the former model is only possible if the 3–4 ps component is ascribed to equilibration with a “red” core antenna pool absorbing at 700 nm. Conversely, if these low-energy states are identified with the P₇₀₀ reaction centre, the transfer-to-trap-model is ruled out in favour of a trap-limited model.

     

Stone-dwelling actinobacteria Blastococcus saxobsidens,Modestobacter marinus and Geodermatophilus obscurus proteogenomes

The Geodermatophilaceae are unique model systems to study the ability to thrive on or within stones and their proteogenomes (referring to the whole protein arsenal encoded by the genome) could provide important insight into their adaptation mechanisms. Here we report the detailed comparative genome analysis of Blastococcus saxobsidens (Bs), Modestobacter marinus (Mm) and Geodermatophilus obscurus (Go) isolated respectively from the interior and the surface of calcarenite stones and from desert sandy soils. The genome-scale analysis of Bs, Mm and Go illustrates how adaptation to these niches can be achieved through various strategies including ‘molecular tinkering/opportunism'' as shown by the high proportion of lost, duplicated or horizontally transferred genes and ORFans. Using high-throughput discovery proteomics, the three proteomes under unstressed conditions were analyzed, highlighting the most abundant biomarkers and the main protein factors. Proteomic data corroborated previously demonstrated stone-related ecological distribution. For instance, these data showed starvation-inducible, biofilm-related and DNA-protection proteins as signatures of the microbes associated with the interior, surface and outside of stones, respectively.     

Terpenes as Useful Markers in Differentiation of Natural Populations of Relict Pines Pinus heldreichii,P. nigra,and P. peuce

Comparative analysis of terpene diversity and differentiation of relict pines Pinus heldreichii, P. nigra, and P. peuce from the central Balkans was performed at the population level. Multivariate statistical analyses showed that the composition of needle terpenes reflects clear divergence among the pine species from different subgenera: P. peuce (subgenus Strobus) vs. P. nigra and P. heldreichii (subgenus Pinus). In addition, despite the described morphological similarities and the fact that P. nigra and P. heldreichii may spontaneously hybridize, our results indicated differentiation of their populations naturally growing in the same area. In accordance with recently proposed concept of ‘flavonic evolution’ in the genus Pinus, we assumed that the terpene profile of soft pine P. peuce, defined by high amounts of six monoterpenes, is more basal than those of hard pines P. nigra and P. heldreichii, which were characterized by high content levels of mainly sesquiterpenes. In order to establish precise positions of P. heldreichii, P. nigra and P. peuce within the taxonomic and phylogenetic tree, as well as develop suitable conservation strategies and future breeding efforts, it is necessary to perform additional morphological, biochemical, and genetic studies.     

The role of human tissue kallikreins 7 and 8 in intracranial malignancies

Recent evidence suggests that many tissue kallikreins are implicated in carcinogenesis. Kallikrein 8 (KLK8) plays a role in the physiology of the central nervous system. Kallikrein 7 (KLK7) takes part in skin desquamation. Both show altered expression in ovarian and breast cancer. In this study, we examined the level of mRNA expression of the KLK7 and KLK8 genes in 73 intracranial tumors using qualitative RT-PCR. The results were correlated with clinical and histomorphological variables and patient outcome. The expression of both genes was also examined in the brain cancer cell lines U-251 MG, D54 and SH-SY5Y and the invasive capacity of glioblastoma cells U-251 MG overexpressing hK7 or hK8 was also investigated in an in vitro Matrigel assay. Follow-up analysis revealed that expression of KLK7 mRNA was associated with shorter overall survival (OS) compared to patients with no KLK7 expression, as determined by Cox proportional hazard regression analysis. Overexpression of hK7 protein by cultivated brain tumor cells significantly enhanced the invasive potential in the Matrigel invasion assay, in contrast to cells overexpressing hK8 protein. Our data suggest that hK7 protein overexpression is associated with a more aggressive phenotype in brain cancer cells.     

Light-adapted charge-separated state of photosystem II: structural and functional dynamics of the closed reaction center

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSII_O), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSII_C). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSII_C into a light-adapted charge-separated state (PSII_L). The PSII_C-to-PSII_L transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (F_v) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSII_L requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events—pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, F_m, is associated with PSII_L rather than with PSII_C, and thus the F_v/F_m parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

The closed-state of photosystem II possesses a hitherto unrecognized structural and functional plasticity and upon illumination assumes a light-adapted charge-separated state.     

Essential‐Oil Variability of Juniperus deltoides R.P.Adams along the East Adriatic Coast – How Many Chemotypes Are There?

The composition of the essential oils isolated from twigs of ten Juniperus deltoides R.P . Adams populations from the east Adriatic coast was determined by GC‐FID and GC/MS analyses. Altogether, 169 compounds were identified, representing 95.6–98.4% of the total oil composition. The oils were dominated by monoterpenes (average content of 61.6%), which are characteristic oil components of species of the Juniperus section. Two monoterpenes, α‐pinene and limonene, were the dominant constituents, comprising on average 46.78% of the essential oils. Statistical methods were deployed to determine the diversity of the terpene classes and the common terpenes between the investigated populations. These statistical analyses revealed the existence of three chemotypes within all populations, i.e., a α‐pinene, limonene, and limonene/α‐pinene type.