Effect of ionic strength on the binding of ascorbate to albumin

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.     

Applications of InterPro in protein annotation and genome analysis

The applications of InterPro span a range of biologically important areas that includes automatic annotation of protein sequences and genome analysis. In automatic annotation of protein sequences InterPro has been utilised to provide reliable characterisation of sequences, identifying them as candidates for functional annotation. Rules based on the InterPro characterisation are stored and operated through a database called RuleBase. RuleBase is used as the main tool in the sequence database group at the EBI to apply automatic annotation to unknown sequences. The annotated sequences are stored and distributed in the TrEMBL protein sequence database. InterPro also provides a means to carry out statistical and comparative analyses of whole genomes. In the Proteome Analysis Database, InterPro analyses have been combined with other analyses based on CluSTr, the Gene Ontology (GO) and structural information on the proteins.     

Hyaluronan content of Wharton's jelly in healthy and Down syndrome fetuses.

The mechanisms by which the excess genetic material of chromosome 21 results in the dysmorphologic features of Down syndrome (DS) are largely unknown. It has been found that the extracellular matrix of nuchal skin of DS fetuses exhibits an higher content of hyaluronan (HA) compared to that of euploid fetuses. Since HA plays a central role in many morphogenetic processes during embryogenesis, an alteration in its metabolism could be involved in the pathogenesis of several structural defects of DS. The extracellular matrix of umbilical cord (UC) is the mammalian tissue with one of the highest content of HA. Therefore we sought to explore the quantitative HA modifications during gestation, tissue distribution and HA metabolism in euploid and DS UCs. Euploid UCs (n=28) and UCs from DS fetuses (n=13) were obtained after termination of pregnancy, spontaneous abortion, or at delivery. Quantitative and molecular size analysis were performed using HPLC and FPLC. Tissue distribution was visualized by immunohistochemistry. Gene expression for HA synthases (HAS) and hyaluronidases (HYAL) were quantified by real-time PCR techniques and HYAL activity was detected by zymography. In euploid UC only HA of a molecular weight of 1700 kDA was present while in DS UC an additional lower weight HA molecule of 1100 kDA was found. Immunohistochemistry showed a larger amount of Wharton's jelly HA in DS UCs than in euploid UC. Real-time PCR analysis showed that HAS 2 and HYAL 2 were expressed at significant levels in all specimens. A higher expression of HAS 2 and a lower expression of HYAL 2 was found in the Wharton's jelly of DS fetuses compared to that of euploid fetuses at 14 weeks of gestation. On the contrary, at term HYAL 2 expression was higher in DS specimens than in those from euploid fetuses. Zymographic studies showed a similar behavior with a lower HYAL activity at early gestation and a higher HYAL activity at term gestation in DS UCs compared to euploid specimens. Therefore we can conclude that HA is more represented in DS UCs than in euploid UCs. A complex alteration of the HA metabolism characterized by an increased synthesis of lower weight HA molecules is a peculiarity of DS UCs.     

Surviving in the High Arctic: dental variation in a casually introduced population of Microtus rossiaemeridionalis (Arvicolinae,Rodentia) on Svalbard

A human‐mediated invasion of a temperate rodent Microtus rossiaemeridionalis on the High Arctic Archipelago of Svalbard in the first half of the 20th century provides an opportunity to explore extent and rate of morphological divergence over decades of isolation. We studied dental size and morphology in 124 voles captured on Svalbard (Spitsbergen) in 1997–2005 and compared the data to mainland conspecific populations across northern Eurasia. Both dental and cranial sizes in the Svalbard population fall within the limits of natural variation of the species. Dental morphology suggests that the population experiences strong effects of isolation as indicated by the significant increase in the frequency of rare dental morphs, possibly caused by inbreeding. No evidence for directional shift towards increased or decreased complexity of the morphotype dental patterns is revealed. Although the population on Svalbard is phenotypically different from the mainland populations (due to increased frequency of rare morphs), those differences are not enough to support the idea of initial rapid evolution related to colonization. The limited spatial extent and environmental homogeneity of suitable habitats on the island allowed the species to initially multiply but not to diversify so that the species exhibits phenotypic conservatism but suffers the consequences of small population size.     

Comparison of interactions of 5′-derivatives of deoxyoctathymidylate with human DNA polymerize α and HIV reverse transcriptase

K_m and V_max values for d(pT₈) and its derivatives containing various 5′-end groups were estimated in the reaction of DNA polymerization α catalyzed by DNA polymerase α and HIV-RT. The effect of 5′-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT₈ is 200-fold higher than that of d(pT₈). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase α the influence of modifiers on primer affinity is much weaker. The effect of 5′-end residues on the V_max values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.     

Impact of hypoxia conditions on the Mnemiopsis leidyi A. Agassiz, 1865 bioluminescence

The findings of the study on the impact of hypoxia on the glow of the Black Sea ctenophore Mnemiopsis leidyi A. Agassiz, 1865 of three size groups (20–30, 30–45, and 45–60 mm) were obtained under experimental conditions. Peculiarities of ctenophore bioluminescence were studied during mechanical and chemical stimulation under the conditions of normoxia (at an oxygen concentration of 5.6–6.7 mg O₂ L⁻¹), moderate hypoxia (2.5–2.8 mg O₂ L⁻¹), and acute hypoxia (1.2–1.5 mg O₂ L⁻¹). An increase in the amplitude and energy of luminescence of the ctenophores mechanically and chemically stimulated was observed at an oxygen concentration of 1.2–1.5 mg O₂ L⁻¹ (acute hypoxia) in two size groups in the lobate form (30–45 and 45–60 mm). The inhibition of amplitude, energy, and duration of the signal was registered in M. leidyi ctenophores at the transitional stage from larva to the lobate form under conditions of acute hypoxia. It was noted that in normoxia, the values of the amplitude and energy of the bioluminescent signal of M. leidyi increase along with a size growth of an individual. This phenomenon was observed both during mechanical and chemical stimulations. Under conditions of acute hypoxia, this trend was mainly preserved. The universality of the relation between the bioluminescence of the organisms and their bioenergetics is obvious. The bioluminescent system of ctenophores has the role of an antioxidant system and is engaged in the neutralization of reactive oxygen species (ROS), that is the process during which photons are emitted. The response of the bioluminescent system to a decrease in oxygen concentration can be associated with an increase in the production of ROS that provides high values of the ctenophore luminescence under hypoxic conditions.     

Vitrification of human laser treated blastocysts within cut standard straws (CSS): novel aseptic packaging and reduced concentrations of cryoprotectants

Vitrification of laser treated human blastocysts using reduced concentrations of permeable cryoprotectants was carried out by submerging cut standard straws (CSS) into liquid nitrogen. The CSS were made by cutting a standard 0.25 ml straw at an angle of approximately 45 degrees . After laser assisted hatching, 6 day blastocysts (n=250) were loaded into droplets of approximately 0.75 microl in the CSS and were either plunged directly into liquid nitrogen or first encased in a standard 0.5 ml straw (aseptic technique) before being vitrified. Permeable cryoprotectants (ethylene glycol+Me(2)SO) at concentrations of 15% and 20% v:v were tested for their effect on post warming re-expansion and post transfer pregnancy rates. Our results indicate that the use of reduced concentrations of cryoprotectants and aseptic packaging of blastocysts did not have any statistically significant impact on the study outcomes.     

Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

Biosurfactant Production by Antarctic Facultative Anaerobe <Emphasis Type="Italic">Pantoea</Emphasis> sp. During Growth on Hydrocarbons

The facultative anaerobe Pantoea sp. strain A-13, isolated from ornithogenic soil of Dewart Island (Frazier Islands), Antarctica, produced glycolipid biosurfactants when grown on n-paraffins or kerosene as the sole source of carbon and energy. Hemolysis of erythrocytes, growth inhibition of Bacillus subtilis, and thin-layer chromatography studies have suggested that the secreted glycolipids are rhamnolipids. Glycolipids produced by kerosene-grown cells decreased the surface tension at the air–water interface to 30 mN/m and possessed a low critical micelle concentration value of 40 mg/l, which indicated high surface activity. They efficiently emulsified aromatic hydrocarbons, kerosene, and n-paraffins. Biosurfactant production contributed to an increase in cell hydrophobicity, which correlated with increased growth of the strain on tested hydrocarbons. According to the results, the Antarctic biosurfactant-producing strain Pantoea sp. A-13 appears to be valuable source for application in accelerated environmental bioremediation.     

Metal particle-enhanced fluorescent immunoassays on metal mirrors

We present fluoroimmunoassays on plain metal-coated surfaces (metal mirrors) enhanced by metal nanoparticles (silver island films [SIFs]). Metal mirrors (aluminum, gold, or silver protected with a thin silica layer) were coated with SIFs, and an immunoassay (model assay for rabbit immunoglobulin G or myoglobin immunoassay) was performed on this surface using fluorescently labeled antibodies. Our results showed that SIFs alone (on glass surface not coated with metal) enhance the immunoassay signal approximately 3- to 10-fold. Using a metal mirror instead of glass as support for SIFs results in up to 50-fold signal enhancement.