Comparative study of immunobiological activities ofPseudomonas aeruginosa andBrucella melitensis lipopolysaccharides

The toxic activity ofBrucella melitensis andPseudomonas aeruginosa lipopolysaccharides as well as their behavior as immunogens, mitogens, and interferon inducers have been studied. Although their toxicities were very similar, the former molecule was incapable of eliciting a primary immune response in mice. Rabbit hyperimmunization gave titers half of those obtained withP. aeruginosa lipopolysaccharide. Optimal mitogenic responses of spleen cell cultures were obtained using 10–50 μg/ml and 50–100 μg/ml ofPseudomonas andBrucella lipopolysaccharide, respectively, giving the latter a lower stimulation of³H-thymidine uptake. Interferon titers induced in chickens byBrucella lipopolysaccharide were three times lower than those obtained withPseudomonas lipopolysaccharide.     

Class III homeodomain-leucine zipper gene family members have overlapping, antagonistic, and distinct roles in Arabidopsis development

The Arabidopsis thaliana genome contains five class III homeodomain-leucine zipper genes. We have isolated loss-of-function alleles for each family member for use in genetic analysis. This gene family regulates apical embryo patterning, embryonic shoot meristem formation, organ polarity, vascular development, and meristem function. Genetic analyses revealed a complex pattern of overlapping functions, some of which are not readily inferred by phylogenetic relationships or by gene expression patterns. The PHABULOSA and PHAVOLUTA genes perform overlapping functions with REVOLUTA, whereas the PHABULOSA, PHAVOLUTA, and CORONA/ATHB15 genes perform overlapping functions distinct from REVOLUTA. Furthermore, ATHB8 and CORONA encode functions that are both antagonistic to those of REVOLUTA within certain tissues and overlapping with REVOLUTA in other tissues. Differences in expression patterns explain some of these genetic interactions, whereas other interactions are likely attributable to differences in protein function as indicated by cross-complementation studies.     

Intrinsically fluorescent cytotoxic cisplatin analogues as DNA marker molecules

Two square planar derivatives of Pt(en)Cl(2) with intrinsic fluorescence in aqueous solution at room temperature, with quantum yields (Phi) 0.11 and 0.10, respectively, have been synthesized and characterized as [Pt(en)(CG)Cl] (Complex 1) and [Pt(en)(CG)(2)] (Complex 2) (en = ethylenediamine, CG = cholylglycinate). Complexes 1 and 2 exchange just one ligand (chloride or cholylglycinate, respectively) when reacted with water or 5'-GMP to give the same chemical species. After reaction with DNA oligonucleotides or DNA plasmids, they show enhanced emission in the visible region, which lasts for long periods of time and makes them potentially useful DNA marker molecules. Incubation with nucleated blood cells followed by microscopic analyses revealed that they enter the cells within minutes of exposure, selectively stain the DNA, and persist after more than 48 h of exposure. Complexes 1 and 2 display cell cycle phase-independent cytotoxic activity against cisplatin-resistant CHO (Chinese hamster ovarian) tumor cells, with an early onset of their effects. Their slightly different biological effects, as compared to cisplatin, are considered to be linked to the bile acids and their vector properties and to the preferential formation of monoadducts.     

Soluble interleukin-2 receptor (sCD25) and interleukin-10 plasma concentrations are associated with severity of primary respiratory syncytial virus (RSV) infection

The role of the immune response in the severity of RSV infection was examined by determining plasma concentrations of interferon-gamma (IFN-gamma), interleukin-10 (IL-10), interleukin-2 receptor (sCD25) and soluble tumor necrosis factor receptor II (sTNFR-II) in 196, previously healthy infants, during acute and convalescence phases of primary RSV infection. The results were analyzed separately for days 1-4 (early) and days 5-7 (late) of symptoms before sample collection and according to disease severity (105 hypoxic, 91 non-hypoxic). Significant associations between plasma levels and severity were found in early samples only. IL-10 and sCD25 concentrations were higher (p=0.01, each) in hypoxic compared with non-hypoxic infants, whereas no differences were observed in IFN-gamma and sTNFR-II levels between the groups. Early sCD25 levels correlated positively with IL-10 concentrations (p= 0.0003; r= 0,401). Amongst the hypoxic infants, the number of days of oxygen supplementation correlated positively with early IL-10 levels (p=0.009; r=0.495) and negatively with the IFN-gamma/IL-10 ratio (p=0.007; r=0.495). IFN-gamma levels were significantly higher in the acute phase than during convalescence for hypoxic and non-hypoxic infants, while IL-10 levels were significantly higher in the acute phase only in hypoxic infants for days 1-4 (early; p=0.0007). sCD25 concentrations were elevated only in hypoxic infants at days 1-4 of the acute phase (p=0.002), whereas sTNFR-II levels did not vary between acute and convalescence phases, independent of severity and time point of sampling. We found no association between plasma levels during the convalescence phase and the severity of the RSV infection.     

The establishment of Neisseria meningitidis serogroup W135 of the clonal complex ET-37/ST-11 as an epidemic clone and the persistence of serogroup A isolates in Burkina Faso

We analyzed 48 invasive isolates of Neisseria meningitidis that were isolated from meningitis cases in Burkina Faso (April 2002 to April 2003). Thirty-nine of these isolates had the phenotype (serogroup:serotype:serosubtype) W135:2a:P1.5,2, eight isolates were A:4:P1.9 and one isolate was nongroupable:nonserotypable:nonserosubtypable. Genotyping of meningococcal isolates showed that W135 isolates belonged to the sequence type (ST)-11. The nongroupable isolate was of genogroup W135 and belonged to ST-192. Isolates of serogroup A belonged to ST-2859 (a member of the subgroup III/ST-5 clonal complex). W135 (ST-11) isolates involved in meningitis outbreaks in Burkina Faso differed from those involved in the Hajj-2000 associated outbreak by their pulsed-field gel electrophoresis profile. These data confirm the changing epidemiology of meningococcal infection in Burkina Faso with the establishment and expansion of serogroup W135 N. meningitidis strains of the ET-37/ST-11 clonal complex, as well as the emergence of a new clone within the subgroup III/ST-5 clonal complex.     

Dynamics of proteins and lipids in the stratum corneum: effects of percutaneous permeation enhancers

We have spin labeled the stratum corneum (SC) with a lysine specific reagent, succinimidyl-2,2,5,5-tetramethyl-3-pirroline-1-oxyl-carboxylate spin label (SSL), to assess the dynamics and hydration degree of SC proteins by electron paramagnetic resonance (EPR) spectroscopy taking measurements directly from the intact tissue. Treating the SC with two percutaneous penetration enhancers, 8 M urea or 20% (v/v) 1-methyl-2-pyrrolidone (1 MP), destabilizes the proteins thus promoting more mobile and solvent-exposed protein conformations. Upon SC lipid depletion the nitroxide side chain becomes more solvent exposed, suggesting that the removal of hygroscopic substances in the extraction process favors more hydrated protein conformations. On the other hand, the treatments with 8 M urea or 40% (v/v) 1 MP did not alter significantly the fluidity in the SC lipid domain as assessed by the probe 5-doxyl stearic acid; these permeation enhancers, specially 1 MP, seem to increase the probe solubility in the solvent leading to a considerable fraction of spin label to be removed from the lipid domain.     

Relevant distance between two different instances of the same potential energy in protein folding

In the context of complex systems and, particularly, of protein folding, a physically meaningful distance is defined which allows to make useful statistical statements about the way in which energy differences are modified when two different instances of the same potential-energy function are used. When the two instances arise from the fact that different algorithms or different approximations are used, the distance herein defined may be used to evaluate the relative accuracy of the two methods. When the difference is due to a change in the free parameters of which the potential depends on, the distance can be used to quantify, in each region of parameter space, the robustness of the modeling to such a change and this, in turn, may be used to assess the significance of a parameters' fit. Both cases are illustrated with a practical example: the study of the Poisson-based solvation energy in the Trp-Cage protein (PDB code 1L2Y).     

Dynamic optimization of bioprocesses: efficient and robust numerical strategies

The dynamic optimization (open loop optimal control) of non-linear bioprocesses is considered in this contribution. These processes can be described by sets of non-linear differential and algebraic equations (DAEs), usually subject to constraints in the state and control variables. A review of the available solution techniques for this class of problems is presented, highlighting the numerical difficulties arising from the non-linear, constrained and often discontinuous nature of these systems. In order to surmount these difficulties, we present several alternative stochastic and hybrid techniques based on the control vector parameterization (CVP) approach. The CVP approach is a direct method which transforms the original problem into a non-linear programming (NLP) problem, which must be solved by a suitable (efficient and robust) solver. In particular, a hybrid technique uses a first global optimization phase followed by a fast second phase based on a local deterministic method, so it can handle the nonconvexity of many of these NLPs. The efficiency and robustness of these techniques is illustrated by solving several challenging case studies regarding the optimal control of fed-batch bioreactors and other bioprocesses. In order to fairly evaluate their advantages, a careful and critical comparison with several other direct approaches is provided. The results indicate that the two-phase hybrid approach presents the best compromise between robustness and efficiency.     

Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports

The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint covalent attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pK(a) of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10.