Characteristics of protection by MgADP and MgATP of α3β3γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis

MgADP and MgATP binding to catalytic sites of βY341W-α₃β₃Γ subcomplex of F₁-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α₃β₃Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K _d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α₃β₃Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F₁-ATPases.     

Thyroid disease is a favorable prognostic factor in achieving sustained virologic response in chronic hepatitis C undergoing combination therapy: A nested case control study

Background

Interferon-α in combination with ribavirin is the current gold standard for treatment of chronic hepatitis C. It is unknown if the development of autoimmune thyroid disease (TD) during treatment confers an improved chance of achieving sustained virologic response. The aim of this study is to assess the chance of achieving sustained virologic response (SVR) in patients who developed TD during treatment when compared with those who did not.

Methods

We performed a tertiary hospital-based retrospective nested case-control analysis of 19 patients treated for hepatitis C who developed thyroid disease, and 76 controls (matched for age, weight, gender, cirrhosis and aminotransferase levels) who did not develop TD during treatment. Multivariate logistic-regression models were used to compare cases and controls.

Results

The development of TD was associated with a high likelihood of achieving SVR (odds ratio, 6.0; 95% confidence interval, 1.5 to 24.6) for the pooled group containing all genotypes. The likelihood of achieving SVR was increased in individuals with genotype 1 HCV infection who developed TD (odds ratio, 5.2; 95% confidence interval, 1.2 to 22.3), and all genotype 3 patients who developed TD achieved SVR.

Conclusions

Development of TD during treatment for hepatitis C infection is associated with a significantly increased chance of achieving SVR. The pathophysiogical mechanisms for this observation remain to be determined.

Trial Registration

The Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRB12610000830099     

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.     

Induction of long-term B-cell depletion in refractory rheumatoid arthritis patients preferentially affects autoreactive more than protective humoral immunity

Introduction

B-cell depletion has become a common treatment strategy in anti-TNF-refractory rheumatoid arthritis (RA). Although the exact mechanism of how B-cell depletion leads to clinical amelioration in RA remains to be elucidated, repetitive treatment with B-cell-depleting agents leading to long-term B-cell depletion has been reported to be beneficial. The latter has led to the hypothesis that the beneficial effects of B-cell depletion might act through their influence on pathogenic autoreactive plasma cells.

Methods

In this study, we investigated the effects of a fixed retreatment regimen with anti-CD20 mAbs on the humoral (auto)immune system in a cohort of therapy-refractory RA patients.

Results

Fixed retreatment led to long-term B-cell depletion in peripheral blood, bone marrow and, to a lesser extent, synovium. Also, pathologic autoantibody secretion (that is, anticitrullinated peptide antibodies (ACPAs)) was more profoundly affected by long-term depletion than by physiological protective antibody secretion (that is, against measles, mumps and rubella). This was further illustrated by a significantly shorter estimated life span of ACPA-IgG secretion compared to total IgG secretion as well as protective antibody secretion.

Conclusion

By studying plasma cell function during an extensive 2-year period of B-cell depletion, autoantibody secretion was significantly shorter-lived than physiologically protective antibody secretion. This suggests that the longevity of autoreactive plasma cells is different from protective long-lived plasma cells and might indicate a therapeutic window for therapies that target plasma cells.     

Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA

A lambda gt10 library containing DNAs complementary to messenger RNAs from human breast cancer T47-D cells was constructed and screened with a cDNA probe encoding the rabbit progesterone receptor. Four overlapping clones have been sequenced. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da. The cysteine rich basic region supposed to be involved in DNA binding is completely homologous in the human and rabbit receptors, whereas the C-terminal end, where hormone binding is thought to take place, differs by a single amino acid change. The human progesterone receptor is characterized, as is the rabbit receptor, by the very high proline content of its N-terminal region. When mRNAs from either human breast cancer cell lines T47-D and MCF-7 or from normal human uterus tissue were blotted and probed with the cloned cDNA, four main bands were observed (5100, 4300, 3700, and 2900 nucleotides).     

Interaction of glucocorticoid · receptor complexes with rat liver nuclei

Some previous reports on acellular binding of glucocorticoid · receptor complexes to rat liver nuclei have pointed to the conclusion that there exists a small number of high affinity nuclear “receptor” sites. Various investigations lead us to the opposite conclusion and suggest that these results were actually due to the presence, in the cytosol, of one or several macromolecules which inhibited the binding to nuclei of steroid · receptor complexes. The mechanism of this inhibition was examined. It appeared to be due not to a competition between both molecules for the same nuclear acceptor site but to an interaction in the cytosol between teh inhibitor and the steroid · receptor complex which prevented the binding of the latter to the nuclei. The search for high affinity specific acceptor sites was also negative for physiological saline conditions and for the non-salt-extractable fraction of the nuclear receptor. When 940-fold purified receptor · steroid complexes were used, very high concentrations of complexes could be achieved and saturation of nuclei was then observed, but only under physiological ionic strength conditions. However, the interaction was of relatively low affinity (K_A = 3.8 · 10⁷ M^?1) and to a great number of acceptor sites (N = 26.2 pmol/mg DNA), largely exceeding the cellular concentration of receptor (5.8 pmol/mg DNA).These results suggested that saturation of nuclei by steroid · receptor complexes should not occur in the intact liver cell. They were confirmed by studies on the distribution of steroid · receptor complexes in liver slices incubated with various concentrations of [³H]dexamethasone. For all hormone concentrations a constant proportion (90%) of the complexes was found in the nuclei, thus showing no saturation of the nuclear acceptor sites.