Differential Galois integrability obstructions for nonlinear three-dimensional differential systems.

In this short communication, we deal with an integrability analysis of nonlinear three-dimensional differential systems. Right-hand sides of these systems are linear in one variable, which enables one to find explicitly a particular solution and to calculate variational equations along this solution. The conditions for the complete integrability with two functionally independent rational first integrals for B-integrability and the partial integrability are obtained from an analysis of properties of the differential Galois group of variational equations. They have a very simple form of numbers, which is necessary to check whether they are appropriate integers. An application of the obtained conditions to some exemplary nonlinear three-dimensional differential systems is shown.

Preliminary crystal structure analysis of an Fab specific for a Salmonella O-polysaccharide antigen.

The Fab from an IgG1, lambda murine monoclonal antibody with specificity for the O-polysaccharide antigen of Salmonella typhimurium has been crystallized in the absence and presence of hapten. The conditions for crystal growth were vapor diffusion equilibration with 16 to 23% polyethylene glycol 8000 solutions. Data have been collected from crystals of the complex in space group P212121, a = 60.6 A, b = 111.3 A, c = 61.1 A, and refinement of a molecular replacement solution is underway.

Structure of oncomodulin refined at 1.85 A resolution. An example of extensive molecular aggregation via Ca2+.

The crystal structure of oncomodulin, a 12,000 Mr protein isolated from rat tumours, has been determined by molecular replacement using the carp parvalbumin structure as a starting model. Refinement was performed by cycles of molecular fitting and restrained least-squares, using area-detector intensity data to 1.85 A resolution. For the 5770 reflections in the range 6.0 to 1.85 A, which were used in the refinement, the crystallographic R-factor is 0.166. The refined model includes residues 2 to 108, three Ca2+ and 87 water molecules per oncomodulin molecule. The oncomodulin backbone is closely related to that of parvalbumin; however, some differences are found after a least-squares fit of the two backbones, with root-mean-square (r.m.s.) deviations of 1 to 2 A in residues 2 to 6, 59 to 61 of the CD loop, 87, 90 and 108. The overall r.m.s. deviation of the backbone residues 5 to 108 is 0.62 A. Each of the two Ca2+ atoms that are bound to the CD and EF loops is co-ordinated to seven oxygen atoms, including one water molecule. The third Ca2+ is also seven-co-ordinated, to five oxygen atoms belonging to three different oncomodulin molecules and to two water molecules which form hydrogen bonds to a fourth oncomodulin; thus, this intermolecular Ca2+ and its equivalents interlink the molecules into zigzag layers normal to the b axis with a spacing of b/2 or 32.14 A. No such extensive molecular aggregation has been reported for any of the related Ca-binding regulatory proteins of the troponin-C family studied thus far. The Ca-O distances in all three polyhedra are in the range 2.07 A to 2.64 A, indicating tightly bound Ca polyhedra.

Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes.

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with SssI-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible cause- and-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.

Increased duration of transgene expression in the lung with plasmid DNA vectors harboring adenovirus E4 open reading frame 3.

For gene therapy to be effective in the treatment of chronic diseases, plasmid DNA (pDNA) vectors that provide persistent expression of therapeutic levels of the transgene product are desirable. Studies in the lung with adenovirus vectors showed that products of the adenovirus E4 region can act both in cis and in trans to increase the duration of expression when transcription of the transgene was under the control of the human cytomegalovirus (CMV) promoter. To determine if these E4-encoded proteins could also effect greater persistence of expression from a nonviral vector, a complex composed of cationic lipid GL-67, a CMV promoter plasmid (pCF1-CAT), and an E4-containing adenovirus vector (Ad2/betagal-4) was instilled into the lungs of BALB/c nu/nu mice. Significant increases in the duration of transgene expression were observed for up to 10 weeks postinstillation compared with expression from mice instilled with control complexes containing an adenovirus vector deleted of most of E4 (Ad2/betagal-2). This effect could also be observed in immunodeficient NIH-rnu rats as well as in immunocompetent BALB/c mice. Studies with CMV promoter mutants indicated that a region proximal to the promoter was necessary for the E4-mediated increase in longevity of expression. In addition to the CMV promoter, a CMV enhancer-human mucin I (MUC-I) hybrid promoter also responded to these E4-encoded proteins with increased persistence of transgene expression, but a human interleukin 8 (IL-8) promoter did not. Ad2/betagal-4 could be replaced by a pDNA vector expressing only the E4 region, indicating that products of the E4 region alone were sufficient in the absence of expression from the rest of the adenovirus genome. Further analysis indicated that the protein encoded by open reading frame 3 (ORF3) alone was sufficient for conferring the increase in persistence of expression. These data indicate that expression of a single protein from the adenovirus genome can significantly improve the duration of transgene expression from pDNA vectors, and increases the feasibility of using nonviral vectors for the treatment of chronic diseases.

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.

Morphological changes of human erythrocytes induced by cholesterol sulphate.

OBJECTIVES: Morphological alterations of human erythrocytes induced by cholesterol sulphate (5-cholesten-3 beta-ol sulphate, CS) were studied. DESIGN AND METHODS: Influence of CS on red blood cell stability (in isotonic conditions) by simultaneous application of flow cytometry and scanning electron microscopy was studied. RESULTS: In isotonic medium CS induces erythrocyte size and shape changes in dose-and time-dependent manner. Incubation (in vitro) of erythrocytes with CS concentrations from 4 x 10(-5) mol/dm3 to 8 x 10(-5) mol/dm3 led to a progressive sphero-echinocitic shape transformation accompanied by a cell size decrease. In contrast to this, for CS content equal to 1 x 10(-5) mol/dm3 the maintenance of the normal biconcave shape of red blood cells was observed. CONCLUSIONS: The results suggest that CS, similarly to numerous evaginating amphiphilic agents, induces a transformation of the erythrocyte normal discoid shape to echinocytic form. This effect may be caused, at least partly, by an asymmetric expansion of the membrane lipid bilayer due to asymmetric distribution of CS incorporated into the membrane. The echinocytic shape transformation of erythrocytes indicated that CS intercalates in the outer hemileaflet of the lipid bilayer leading to membrane externalization.

Estimation of cholesterol sulphate in blood plasma and in erythrocyte membranes from individuals with Down's syndrome or diabetes mellitus type I.

OBJECTIVES: Plasma and erythrocyte membrane cholesterol sulphate (CS) were measured in patients suffering from diabetes and Down's syndrome. DESIGN AND METHODS: The procedure for separation and determination of CS comprised HPTLC (high-performance thin-layer chromatography) and densitometry. RESULTS: The mean plasma and RBC membranes CS concentrations (+/- SD) of the control group (n = 16) was 188 +/- 47 micrograms/dL and 343 +/- 57 micrograms/10(12) RBC, respectively. In 15 patients with diabetes and 12 Down's syndrome patients substantially higher CS levels were found (diabetes: plasma-348 +/- 60 micrograms/dL; RBC membranes-646 +/- 113 micrograms/10(12) RBC; Down's syndrome: plasma-245 +/- 54 micrograms/dL; RBC membranes 427 +/- 74 micrograms/10(12) RBC). Analysis of variance and multiple comparison (Newman-Keuls test) show statistically significant differences between all samples both for erythrocytes, F(2.41) = 52.24, p < 0.05, and plasma, F(2.41) = 34.92, p < 0.05. CONCLUSIONS: It is postulated that differences in CS levels may contribute to changes of erythrocyte properties in these pathological states.

Thermal properties and fluidity of human erythrocyte membranes in diabetes mellitus.

Exposure of human erythrocytes to elevated temperatures induces a decrease in stability of the cell membrane. Thermally induced haemolysis of erythrocytes from patients with type 1 diabetes and from healthy control individuals was measured as a function of duration of exposure to heat between 48.0 and 54.0 degrees C. Results indicate that the thermosensitivity of erythrocytes from patients with type 1 diabetes is lower than for control individuals. Activation energies for lysis were similar for both control and 'diabetic' erythrocytes, being 298.3 and 287.7 kJ/mol, respectively. The steady-state fluorescence anisotropy measurement of TMA-DPH for each step of haemolysis was employed as a parameter characterizing membrane fluidity. We found that 'diabetic' erythrocyte membranes had significantly decreased fluidity. The relationship between fluidity and rate of haemolysis indicates that the rate-limiting step in the haemolysis reaction involves the rupturing of the membrane bilayer.

Thermosensitivity of red blood cells from Down's syndrome individuals.

Biochemical disturbances of the reactive oxygen species metabolism revealed in subjects with Down's syndrome (DS), and the findings indicating that heat-induced cell alterations have been, at least, partly mediated by reactive oxygen species, made the elucidation of the response of trisomic cells to elevated temperatures of special interest. Kinetic analysis of cell-survival curves, accompanied by the flow cytometry and the scanning electron microscopy (SEM) examinations, and their relationship with the cell membrane fluidity, were undertaken. At each temperature (48-54 degrees C), Dq parameters, representing the ability to accumulate sublethal damages, were similar for both cell groups. D0 parameters (inverse leakage rates; D0 = 1/k) were greater for DS cells at each temperature below 54 degrees C. The haemolysis sensitivity ratio (HSR) showed that DS erythrocytes were, in average, 1.60 times more resistant to heat injury than those from normal subjects. Activation energies of haemolysis, calculated according to the Arrhenius equation, were similar both for normal (290.8 +/- 6.5 [kJ/mol]) and DS erythrocytes (288.0 +/- 5.5 [kJ/mol]). Flow cytometry studies showed that the scattering properties of intact DS erythrocytes (reflecting size, volume, shape and cell membrane surface morphology) were different than those of normal cells. Scanning electron micrographs and scattering diagrams obtained for cells submitted to heat stress (51 degrees C) confirmed that DS erythrocytes were more resistant, to a certain extent, to heat-induced disruption than normal cells. The steady-state fluorescence anisotropy of TMA-DPH (1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene) showed that untreated DS erythrocytes had substantially lower fluidity (r = 0.356 +/- 0.008) of the outer monolayer of cell membranes as compared to normal cells (r = 0.324 +/- 0.011). The increase of the cell membrane fluidity during exposure to heat was observed. The greatest elevation of cell membrane fluidity occurred during the preleakage period, immediately upon the heat treatment and was considered as a rate-limiting step of heat-induced haemolysis.

[Evaluation of side effects and complications during the treatment of affective psychoses with thymoleptics]. / Ocena dzialan ubocznych i powiklan podczas leczenia psychoz afektywnych tymoleptykami.

Evaluation of effects of amitriptyline, imipramine, maprotiline, mianserin , clomipramine and citalopram was performed in 84 patients (49 females and 35 males) age on average 40 years with diagnosis of affective psychosis treated in the Department of Psychiatry Medical School of Szczecin. Antidepressants independently from their pharmacological profile cause in above 50% of patients side effects mainly from autonomous nervous system. Tricyclic antidepressants caused some cardiotoxic effects which were not observed during administration of antidepressant drugs of different chemical structure, especially of citalopram. No effects on the haemopoietic system and on parenchymatous organs were observed. Neither were affected hypothalamic mechanisms for basal secretion of thyrotropic hormone, prolactin , cortisol and so ACTH. Multifactorial analysis of positive and untoward effects observed during the treatment shows comparable clinical value of all six evaluated drugs. A choice of a drug for an individual should depend on particular clinical contraindications.