Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

Deficiency of functional mannose-binding lectin is not associated with infections in patients with systemic lupus erythematosus

Infection imposes a serious burden on patients with systemic lupus erythematosus (SLE). The increased infection rate in SLE patients has been attributed in part to defects of immune defence. Recently, the lectin pathway of complement activation has also been suggested to play a role in the occurrence of infections in SLE. In previous studies, SLE patients homozygous for mannose-binding lectin (MBL) variant alleles were at an increased risk of acquiring serious infections in comparison with patients who were heterozygous or homozygous for the normal allele. This association suggests a correlation between functional MBL level and occurrence of infections in SLE patients. We therefore investigated the biological activity of MBL and its relationship with the occurrence of infections in patients with SLE. Demographic and clinical data were collected in 103 patients with SLE. Functional MBL serum levels and MBL-induced C4 deposition were measured by enzyme-linked immunosorbent assay using mannan as coat and an MBL- or C4b-specific monoclonal antibody. The complete MBL-dependent pathway activity was determined by using an assay that measures the complete MBL pathway activity in serum, starting with binding of MBL to mannan, and was detected with a specific monoclonal antibody against C5b-9. Charts were systematically reviewed to obtain information on documented infections since diagnosis of SLE. Major infections were defined as infections requiring hospital admission and intravenous administration of antibiotics. In total, 115 infections since diagnosis of lupus, including 42 major infections, were documented in the 103 SLE patients (mean age 41 ± 13 years, mean disease duration 7 ± 4 years). The percentage of SLE patients with severe MBL deficiency was similar to that in 100 healthy controls: 13% versus 14%, respectively. Although deposition of C4 to mannan and MBL pathway activity were reduced in 21% and 43% of 103 SLE patients, respectively, neither functional MBL serum levels nor MBL pathway activity was associated with infections or major infections in regression analyses. In conclusion, SLE patients frequently suffer from infections, but deficiency of functional MBL does not confer additional risk.     

Evaluation of stability and size distribution of sunflower oil-coated micro bubbles for localized drug delivery

Background

In recent years magnesium alloys have been intensively investigated as potential resorbable materials with appropriate mechanical and corrosion properties. Particularly in orthopedic research magnesium is interesting because of its mechanical properties close to those of natural bone, the prevention of both stress shielding and removal of the implant after surgery.

Methods

ZEK100 plates were examined in this in vitro study with Hank's Balanced Salt Solution under physiological conditions with a constant laminar flow rate. After 14, 28 and 42 days of immersion the ZEK100 plates were mechanically tested via four point bending test. The surfaces of the immersed specimens were characterized by SEM, EDX and XRD.

Results

The four point bending test displayed an increased bending strength after 6 weeks immersion compared to the 2 week group and 4 week group. The characterization of the surface revealed the presence of high amounts of O, P and Ca on the surface and small Mg content. This indicates the precipitation of calcium phosphates with low solubility on the surface of the ZEK100 plates.

Conclusions

The results of the present in vitro study indicate that ZEK100 is a potential candidate for degradable orthopedic implants. Further investigations are needed to examine the degradation behavior.     

Abatacept decreases disease activity in a absence of CD4+ T cells in a collagen-induced arthritis model

IntroductionAbatacept is a fusion protein of human cytotoxic T-lymphocyte–associated protein (CTLA)-4 and the Fc portion of human immunoglobulin G1 (IgG1). It is believed to be effective in the treatment of rheumatoid arthritis by inhibiting costimulation of T cells via blocking CD28–B7 interactions as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 molecules is crucial for activation of naive cells, whereas it is unclear whether the action of already activated CD4⁺ T cells, which are readily present in established disease, also depends on this interaction. The aim of this study was to determine whether the mode of action of abatacept depends solely on its ability to halt T cell activation in established disease.MethodsArthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4⁺ T cells. Abatacept or control treatment was started when 80 % of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws, and anti-collagen antibody levels over time were determined by enzyme-linked immunosorbent assay.ResultsTreatment with abatacept in the absence of CD4⁺ T cells resulted in lower disease activity. This was associated with decreasing levels of collagen-specific IgG1 and IgG2a antibodies, whereas the antibody levels in control or CD4⁺ T cell–depleted mice increased over time.ConclusionsThese results show that abatacept decreased disease activity in the absence of CD4⁺ T cells, indicating that the mode of action of abatacept in established arthritis does not depend entirely on its effects on CD4⁺ T cell activation.     

Glutaraldehyde and glyoxal fixation of ribonucleoproteins for their analysis by cesium chloride equilibrium gradient centrifugation]

Conditions for fixation of different RNP (ribosomes, poliribosomes, informosomes) by glutaraldehyde and glyoxal for their subsequent analysis in CsCl density-gradient has been developed. Higher dialdehyde concentration and longer incubation time should be used for fixation of ribosomes and polyribosomes than for that of informosomes. For the fixation of all RNP studied their incubation with 0.01 M (0.1%) glutaraldehyde for several minutes is sufficient. Much higher concentration of the fixating agent (about 0.2-0.5 M i. e. 1-3%) and more prolonged time of incubation (in order of several 10 hours) are needed for the fixation of the RNP in the case of glyoxal. Conditions for selective aldehyde fixation of informosomes in the presence of ribosomes and polyribosomes has been developed.     

Complex type of kinetics of fructose-1,6-diphosphate from the rat and rabbit liver

Studies on rat and rabbit liver fructose 1.6-bisphosphatase inhibition by AMP showed that with an increase in EDTA concentration the hyperbolic AMP inhibition curve is transformed into a sigmoidal one. At intermediate EDTA concentrations, the kinetic curves have a plateau. The appearance of the intermediate plateau may be due to the superposition of kinetic curves corresponding to two enzyme forms simultaneously present in the assay mixture. One of these forms deprived of endogenous Me2+ (presumably Zn2+) is inhibited by AMP in a cooperative manner, while the other one retains Me2+ which prevents the cooperative response of the enzyme to AMP.