Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports

A new protocol for the stabilization of the quaternary structure of multimeric enzymes has been attempted using as model enzyme (tetrameric) L-asparaginase from Escherichia coli. Such strategy is based upon multisubunit covalent immobilization of the enzyme onto activated supports (agarose-glutaraldehyde). Supports activated with different densities of reactive groups were used; the higher the density of groups, the higher the stabilization attained. However, because of the complexity of that enzyme, even the use of the highest densities of reactive groups was not enough to encompass all four subunits in the immobilization process. Therefore, a further chemical intersubunit cross-linking with aldehyde-dextran was pursued; these derivatives displayed a fully stabilized multimeric structure. In fact, boiling the modified enzyme derivative in the presence of sodium dodecyl sulfate and beta-mercaptoethanol did not lead to release of any enzyme subunit into the medium. Such a derivative, prepared under optimal conditions, retained ca. 40% of the intrinsic activity of the free enzyme and was also functionally stabilized, with thermostabilization enhancements of ca. 3 orders of magnitude when compared with its soluble counterpart. This type of derivative may be appropriate for extracorporeal devices in the clinical treatment of acute leukemia and might thus bring about inherent advantages in that all subunits are covalently bound to the support, with a longer half-life and a virtually nil risk of subunit release into the circulating blood stream.     

Membrane-associated phosphoinositides-specific phospholipase C forms from <Emphasis Type="Italic">Catharanthus roseus</Emphasis> transformed roots

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membrane-associated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.     

Inactivation of tyrosinase photoinduced by pterin

Tyrosinase catalyzes in mammals the first and rate-limiting step in the biosynthesis of the melanin, the main pigment of the skin. Pterins, heterocyclic compounds able to photoinduce oxidation of DNA and its components, accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder in which the protection against UV radiation fails due to the lack of melanin. Aqueous solutions of tyrosinase were exposed to UV-A irradiation (350nm) in the presence of pterin, the parent compound of oxidized pterins, under different experimental conditions. The enzyme activity in the irradiated solutions was determined by spectrophotometry and HPLC. In this work, we present data that demonstrate unequivocally that the enzyme is photoinactivated by pterin. The mechanism of the photosensitized process involves an electron transfer from tyrosinase to the triplet excited state of pterin, formed after UV-A excitation of pterin. The biological implications of the results are discussed.     

Photosynthetic Traits in Wheat Grown under Decreased and Increased CO2 Concentration, and after Transfer to Natural CO2 concentration

Wheat plants were grown from sowing to day 18 in 26-dm³ chambers at three different CO₂ concentrations: 150 (-CO₂), 350 (C, control), 800 (+CO₂) mol mol^-1. Afterwards, plants of the three variants were grown at the same natural CO₂ concentration. Plant characteristics were measured just before the transfer (0 days after CO₂ treatment, DAT), and at 5 – 8 DAT on the 1^st leaf, and at 12 – 22 DAT on the 4^th leaf. Decreased or increased CO₂ concentrations caused acclimations which persisted after transplantation to natural CO₂ concentration. At 5 – 8 DAT, stomatal density, stomatal conductance (g_s), CO₂ saturated net photosynthetic rate (P_Nsat0), radiation saturated net photosynthetic rate (P_Nsat1), and carboxylation efficiency () were higher in -CO₂ plants and lower in +CO₂ plants than in C plants. As compared with C plants, the photochemical efficiency () was lower in -CO₂ and higher in -CO₂ plants, however, chlorophyll (Chl) a, Chl b, Chl a–b and carotenoid contents were lower in both -CO₂ and +CO₂ plants. On the 4^th leaf, which emerged on plant after finishing CO₂ treatments, at 12 – 22 DAT, no differences in stomatal density and g, between treatments were observed. In -CO₂ plants, pigment content and P_Nsat0 were higher, was lower, and P_Nsat1 and were not different from C plants. In contrast, in +CO₂ plants, pigment content, P_Nsat1 and were lower, and P_Nsat0 and were unchanged. Leaf area, dry mass, and tiller development increased in +CO₂ plants and decreased in -CO₂ plants. In the interval between 8 and 22 DAT, lower net assimilation rate in +CO₂ than in -CO₂ plants was observed.     

Sex-sorted bovine spermatozoa and DNA damage: II. Dynamic features

This study examined the dynamic response of Spermatozoa DNA Fragmentation after sex selection in bulls using a MoFlo^® SX (Beckman Coulter, Miami FL) spermatozoa sorter. The dynamic response of spermatozoa DNA fragmentation refers to the changing values of SDF, i.e., rate of SDF (rSDF), when analyzed periodically over a set incubation time at 37 °C. A dynamic assessment of SDF using non-sorted and sex-sorted spermatozoa samples during 72 h of incubation at 37 °C was performed. Results showed a reduced DNA longevity in sex-sorted frozen-thawed spermatozoa, with spermatozoa DNA damage appearing between 24 h and 48 h. The baseline SDF level was higher in conventional frozen-thawed than in sex-sorted frozen-thawed spermatozoa samples; while the reverse occurred for the rSDF. The afore-mentioned result produced a crossover point between both dynamic tendencies of SDF for sex-sorted versus conventional samples. We defined this crossover point as the Crossover Positioning Time (CPT) or the time (in hours) where both curves crossover after a period of spermatozoa incubation at 37 °C. The point at which the CPT occurs could be used as an indicator of the rSDF for individual bulls after X- and Y-chromosome bearing spermatozoa selection. CPT values produced a window of SDF ranging between 24 h and 48 h in the present experiment. It is proposed that higher values for CPT are indicative of bulls presenting chromatin that is more resistant to the external stressors affecting spermatozoa DNA after spermatozoa sorting.     

RNA binding is more critical to the suppression of silencing function of Cucumber mosaic virus 2b protein than nuclear localization

Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios ~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.     

Cyclic expression of HLA class I and II molecules on the surface of purified human spermatozoa and their control by serum inhibin B levels

HLA class I and class II expression was analyzed weekly by cytofluorometry on spermatozoa samples from four donors during a 15-wk trial. On the same day that semen samples were studied, and to analyze whether this expression was hormone-controlled, serum levels of testosterone, LH, FSH, inhibin B, activin, and pro-alphaC on the one hand, and seminal plasma levels of inhibin B, activin, and alpha-inhibin on the other, were also measured. Inhibin B and related peptides were quantitated using a novel two-site assay with monoclonal antibodies to the alpha and beta subunits of inhibin. Our results showed that HLA class I and class II molecules were expressed on the spermatozoa's surface, following a cyclic pattern, and that there was a simultaneous and coordinated expression of both types of molecules (r = 0.801, P < 0.0001). Furthermore, when the expression of these molecules was plotted against the different hormone levels, serum inhibin B showed a clear inverse correlation with HLA class I (r = -0.612, P < 0.0001) and class II (r = -0.534, P < 0.0001). This finding reveals unexpected functions of inhibin B, which may be relevant in the fertilization process and on male fertility control.     

Screening of a Leptospira biflexa Mutant Library To Identify Genes Involved in Ethidium Bromide Tolerance

Leptospira spp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyte L. biflexa is a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants in L. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduce L. biflexa susceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n = 11), transport (n = 6), membrane structure (n = 5), stress response (n = 2), DNA damage repair (n = 1), and other processes (n = 3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth of L. biflexa in the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenic L. interrogans.