Effect of liquid pulses with 6-benzyladenine on the induction of somatic embryogenesis from coffee (Coffea arabica L.) callus cultures

We investigated the effect of the physical state of the nutrient medium on the induction of somatic embryogenesis on cell cultures derived from coffee (Coffea arabica L.). Non-embryogenic callus tissues were pulsed initially with 50 μM 6-benzyladenine (BA) for 6, 24 or 48 h in half-strength liquid Murashige and Skoog (MS) medium. After pretreatment, calli were transferred to agar-solidified half-strength MS medium supplemented with 50 μM BA (‘standard induction medium’). Control callus tissues were incubated directly on the solid standard induction medium. Callus growth was promoted by longer pretreatment periods. Formation of globular somatic embryos was observed on callus tissues pretreated with BA for 24 or 48 h, which developed fully to cotyledonary-stage within only 2 weeks after transfer to agar-solidified medium supplemented with BA. No embryo formation occurred in control cultures. Pretreatment with BA in liquid medium was associated with changes in the redox status of cultured cells, such as alterations of the ascorbate–glutathione redox systems and the accumulation of free radicals and oxidized lipids, as well as the possible reduction of cytochrome c-mediated apoptotic pathways. In particular, the induction of somatic embryogenesis was highly positively correlated (r ² = 0.822) with the accumulation of protein carbonyls. The physiological role of BA as an inducer of both embryonic differentiation and cellular death is discussed.     

CD8+ T lymphocytes protect SCID mice against Encephalitozoon cuniculi infection

Microsporidia are obligate intracellular parasites that cause opportunistic infections in immunocompromised patients. The role of two main T cell subsets in anti-microsporidial immunity has been studied using an Encephalitozoon cuniculi-severe combined immunodeficient (SCID) mouse model. Whereas SCID mice reconstituted with CD4+ T lymphocyte-depleted naive BALB/c splenocytes resolved the infection, adoptive transfer of CD8+ T cell-depleted splenocytes failed to protect the animals against a lethal E. cuniculi infection. Splenocytes from E. cuniculi-immune mice specifically killed syngeneic infected macrophages in a short-term 51Cr-release assay. These results suggest the crucial role of cytotoxic T lymphocytes in the protection against E. cuniculi infection.     

Pulmonary surfactant protein A inhibits the lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria and microsomes

Reactive oxygen species play an important role in several acute lung injuries. The lung tissue contains polyunsaturated fatty acids (PUFAs) that are substrates of lipid peroxidation that may lead to loss of the functional integrity of the cell membranes. In this study, we compare the in vitro protective effect of pulmonary surfactant protein A (SP-A), purified from porcine surfactant, against ascorbate-Fe(2+) lipid peroxidation stimulated by linoleic acid hydroperoxide (LHP) of the mitochondria and microsomes isolated from rat lung; deprived organelles of ascorbate and LHP were utilized as control. The process was measured simultaneously by chemiluminescence as well as by PUFA degradation of the total lipids isolated from these organelles. The addition of LHP to rat lung mitochondria or microsomes produces a marked increase in light emission; the highest value of activation was produced in microsomes (total chemiluminescence: 20.015+/-1.735 x 10(5) cpm). The inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (2.5 to 5.0 microg) of SP-A in rat lung mitochondria and 2.5 to 7.5 microg of SP-A in rat lung microsomes. The inhibitory effect reaches the highest values in the mitochondria, thus, 5.0 microg of SP-A produces a 100% inhibition in this membranes whereas 7.5 microg of SP-A produces a 51.25+/-3.48% inhibition in microsomes. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized membranes was found in the arachidonic acid content; this decreased from 9.68+/-1.60% in the native group to 5.72+/-1.64% in peroxidized mitochondria and from 7.39+/-1.14% to 3.21+/-0.77% in microsomes. These changes were less pronounced in SP-A treated membranes; as an example, in the presence of 5.0 microg of SP-A, we observed a total protection of 20:4 n-6 (9.41+/-3.29%) in mitochondria, whereas 7.5 microg of SP-A produced a 65% protection in microsomes (5.95+/-0.73%). Under these experimental conditions, SP-A produces a smaller inhibitory effect in microsomes than in mitochondria. Additional studies of lipid peroxidation of rat lung mitochondria or microsomes using equal amounts of albumin and even higher compared to SPA were carried out. Our results indicate that under our experimental conditions, BSA was unable to inhibit lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria or microsomes, thus indicating that this effect is specific to SP-A.     

Influence of modulated structural dynamics on the kinetics of alpha-chymotrypsin catalysis. Insights through chemical glycosylation, molecular dynamics and domain motion analysis

Although the chemical nature of the catalytic mechanism of the serine protease alpha-chymotrypsin (alpha-CT) is largely understood, the influence of the enzyme's structural dynamics on its catalysis remains uncertain. Here we investigate whether alpha-CT's structural dynamics directly influence the kinetics of enzyme catalysis. Chemical glycosylation [Solá RJ & Griebenow K (2006) FEBS Lett 580, 1685-1690] was used to generate a series of glycosylated alpha-CT conjugates with reduced structural dynamics, as determined from amide hydrogen/deuterium exchange kinetics (k(HX)). Determination of their catalytic behavior (K(S), k(2), and k(3)) for the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) revealed decreased kinetics for the catalytic steps (k(2) and k(3)) without affecting substrate binding (K(S)) at increasing glycosylation levels. Statistical correlation analysis between the catalytic (DeltaG( not equal)k(i)) and structurally dynamic (DeltaG(HX)) parameters determined revealed that the enzyme acylation and deacylation steps are directly influenced by the changes in protein structural dynamics. Molecular modelling of the alpha-CT glycoconjugates coupled with molecular dynamics simulations and domain motion analysis employing the Gaussian network model revealed structural insights into the relation between the protein's surface glycosylation, the resulting structural dynamic changes, and the influence of these on the enzyme's collective dynamics and catalytic residues. The experimental and theoretical results presented here not only provide fundamental insights concerning the influence of glycosylation on the protein biophysical properties but also support the hypothesis that for alpha-CT the global structural dynamics directly influence the kinetics of enzyme catalysis via mechanochemical coupling between domain motions and active site chemical groups.     

A new surface plasmon resonance sensor for high-throughput screening applications

We report a new high-throughput surface plasmon resonance (SPR) sensor based on combination of SPR imaging with polarization contrast and a spatially patterned multilayer SPR structure. We demonstrate that this approach offers numerous advantageous features including high-contrast SPR images suitable for automated computer analysis, minimum crosstalk between neighboring sensing channels and inherent compensation for light level fluctuations. Applications of a laboratory prototype of the high-throughput SPR sensor with 108 sensing channels for refractometry and biosensing are described. In refractometric experiments, the noise-limited refractive index resolution of the system has been established to be 3 x 10(-6) refractive index unit (RIU). Experimental data on detection of human choriogonadotropin (hCG) suggest that in conjunction with monoclonal antibodies against hCG, the reported SPR imaging sensor is capable of detecting hCG at concentrations lower than 500 ng/ml.     

Relics of the Europe's warm past: phylogeography of the Aesculapian snake

Understanding how species responded to past climate change can provide information about how they may respond to the current global warming. Here we show how a European reptile species responded to the last natural global warming event at the Pleistocene-Holocene transition that led to the Holocene climatic optimum approximately 5000-8000 years ago. The Aesculapian snake, Zamenis longissimus, is a thermophilous species whose present-day distribution in the southern half of Europe is a remnant of much wider range during the Holocene climatic optimum when populations occurred as far north as Denmark. These northern populations went extinct as the climate cooled, and presently the species is extinct from all central Europe, except few relic populations in locally suitable microhabitats in Germany and the Czech Republic. Our phylogenetic and demographic analyses identified two major clades that expanded from their respective western and eastern refugia after the last glacial maximum (18,000-23,000 years ago) and contributed approximately equally to the present range. Snakes from the relic northern populations carried the Eastern clade, showing that it was primarily the snakes from the eastern, probably Balkan, refugium that occupied the central and northern Europe during the Holocene climatic optimum. Two small, deep-branching clades were identified in near the Black Sea and in Greece. These clades provide evidence for two additional refugia, which did not successfully contribute to the colonization of Europe. If, as our results suggest, some populations responded to the mid-Holocene global warming by shifting their ranges further north than other populations of the same species, knowing what populations were able to expand in different species may provide information about what populations will be important for the species' ability to cope with the current global warming.     

Overexpression of P-glycoprotein in L1210/VCR cells is associated with changes in several endoplasmic reticulum proteins that may be partially responsible for the lack of thapsigargin sensitivity

L1210/VCR cells, which express an abundant amount of P-glycoprotein (P-gp), were found to be resistant to thapsigargin--an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). In the current paper, we have studied the possible differences among L1210 and L1210/VCR cells in expression of endoplasmic reticulum proteins involved in the regulation of calcium homeostasis and calcium-dependent processes. Amounts of mRNA encoding both calcium release channels (ryanodine receptor channels--RyR and IP3-receptor channels--IP3R) were found to be at similar levels in sensitive and resistant cells. However, mRNAs encoding IP3R1 or 2 were decreased in resistant cells cultivated in the presence of VCR (1.08 micromol/l), while mRNA encoding RyR remained unchanged. The amount of mRNA for SERCA2 was decreased in resistant cells when compared with sensitive cells. This decrease was more pronounced when resistant cells were cultivated in the presence of vincristine (VCR). Calnexin was found to be less expressed at the protein level in resistant as in sensitive cells. The level of mRNA encoding calnexin was decreased only when resistant cells were cultivated in the presence of VCR. Calnexin was found to be associated with immature P-gp in resistant cells. Thus, differences exist between sensitive and resistant cells in the expression of endoplasmic reticulum proteins involved in the control of intracellular calcium homeostasis or calcium-dependent processes. These changes may be at least partially responsible for the lack of sensitivity of resistant cells to thapsigargin.     

Macleya cordata and Prunella vulgaris in oral hygiene products - their efficacy in the control of gingivitis

A double-blind, placebo-controlled clinical trial was performed to investigate the effectiveness of a herbal-based dentifrice in the control of gingivitis. Forty volunteers completed the 84-day study. All subjects were balanced for parameters measured - plaque index (PI), community periodontal index of treatment needs (CPITN) and papillary bleeding index (PBI). The dentifrice was effective in reducing symptoms of gingivitis as evaluated by the CPITN and PBI indexes.     

Lectin-like binding and antibiotic sensitivity of enterococci from wild herbivores

Fifty eight enterococcal isolates from wild herbivores were tested for their antibiotic sensitivity pattern and lectin-like binding of extracellular matrix (ECM) and serum proteins. Kanamycin resistance was very frequent; many multiresistant strains were also isolated. All isolates were sensitive to rifampicin. Resistance to gentamicin, novobiocin, and tetracycline was widely distributed in the microflora of wild herbivores breeded in zoological garden in Košice.

No autoaggregating strains were detected among these 58 enterococcal isolates. Various degrees of binding of mucins, fetuin, heparin, fibrinogen, and fibronectin were observed in individual strains. However, bovine lactoferrin binding by enterococci from deers and chamoises was either negative (0) or strongly positive (3).

With regard to influence of growth media, TH agar was found to be better for the expression of lectin-like binding than blood agar, TH broth and Nutrient broth.

A significant effect (P < 0.001 or P < 0.05) of proteolytic treatment was observed in six selected strains. However, there is a difference between the effect of trypsin and pronase P.Pronase treatment more effectively decreased binding of some strains (1H, 6A, EF 1111, EC 1292), while trypsin treatment decreased more binding of other enterococcal strains (EF953 and 1E). Significant (P < 0.001) influence of metaperiodate, which cleaves the C-C bond between vicinal groups of sugars, on collagen I binding by three selected strains (1E, 1H, 6A) and bovine lactoferrin binding (by EF 1111, EC 1292, EF 953) was also observed. However, its influence was very different. In two strains (1H and EC 1292), ECM binding was decreased, while in four other strains (1E, 6A, EF 1111, EF 953) it was increased.