Specific patterns of Cdc42 activity are related to distinct elements of T cell polarization

T cell polarization toward and within the cellular interface with an APC is critical for effective T cell activation. The Rho family GTPase Cdc42 is a central regulator of cellular polarization. Using live-cell imaging, we characterized the spatiotemporal patterns of Cdc42 activity and their physiological regulation. Using three independent means of experimental manipulation of Cdc42 activity, we established that Cdc42 is a critical regulator of T cell actin dynamics, TCR clustering, and cell cycle entry. Using quantification of three-dimensional data, we could relate distinct spatiotemporal patterns of Cdc42 activity to specific elements of T cell activation. This result suggests that Cdc42 activity in specific locations at specific times is most critical for its function in T cell activation.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

The angiogenic factor midkine is regulated by dexamethasone and retinoic acid during alveolarization and in alveolar epithelial cells

Background

A precise balance exists between the actions of endogenous glucocorticoids (GC) and retinoids to promote normal lung development, in particular during alveolarization. The mechanisms controlling this balance are largely unknown, but recent evidence suggests that midkine (MK), a retinoic acid-regulated, pro-angiogenic growth factor, may function as a critical regulator. The purpose of this study was to examine regulation of MK by GC and RA during postnatal alveolar formation in rats.

Methods

Newborn rats were treated with dexamethasone (DEX) and/or all-trans-retinoic acid (RA) during the first two weeks of life. Lung morphology was assessed by light microscopy and radial alveolar counts. MK mRNA and protein expression in response to different treatment were determined by Northern and Western blots. In addition, MK protein expression in cultured human alveolar type 2-like cells treated with DEX and RA was also determined.

Results

Lung histology confirmed that DEX treatment inhibited and RA treatment stimulated alveolar formation, whereas concurrent administration of RA with DEX prevented the DEX effects. During normal development, MK expression was maximal during the period of alveolarization from postnatal day 5 (PN5) to PN15. DEX treatment of rat pups decreased, and RA treatment increased lung MK expression, whereas concurrent DEX+RA treatment prevented the DEX-induced decrease in MK expression. Using human alveolar type 2 (AT2)-like cells differentiated in culture, we confirmed that DEX and cAMP decreased, and RA increased MK expression.

Conclusion

We conclude that MK is expressed by AT2 cells, and is differentially regulated by corticosteroid and retinoid treatment in a manner consistent with hormonal effects on alveolarization during postnatal lung development.     

Design and optimization of a series of novel 2-cyano-pyrimidines as cathepsin K inhibitors

Morphing structural features of HTS-derived chemotypes led to the discovery of novel 2-cyano-pyrimidine inhibitors of cathepsin K with good pharmacokinetic profiles, for example, compound 20 showed high catK potency (IC₅₀ = 4 nM), >580-fold selectivity over catL and catB, and oral bioavailability in the rat of 52%.     

Mathematical modelling and simulation of aqueous two-phase continuous protein extraction.

A mathematical model has been developed to describe the continuous, steady-state operation of an aqueous two-phase system for protein extraction. The model is based on steady-state mass balances of the main components and phase equilibrium data. Experimental data on the separation of thaumatin from contaminant proteins of an homogenate of E. coli in a PEG4000/Phosphate system was used. The data shows the effect of the presence and absence of NaCl which was used to carry out the extraction of thaumatin into the PEG phase and back into the PO4(-3) phase. Simulation results showing the sensitivity to key process parameters, and the effect of process variables on performance are presented and discussed. The model can be used to predict performance and thus 'robustness' of process conditions as well as predict protein recovery yield and purity. This model can also be used to implement a suitable control strategy to maintain process stability.     

Crystallographic analysis of triclosan bound to enoyl reductase

Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses correlated with inhibition data, on the complexes of E. coli and Brassica napus ENR with triclosan and NAD(+) which reveal how triclosan acts as a site-directed, picomolar inhibitor of the enzyme by mimicking its natural substrate. Elements of both the protein and the nucleotide cofactor play important roles in triclosan recognition, providing an explanation for the factors controlling its tight binding to the enzyme and for the emergence of triclosan resistance.     

Selective inhibition of low affinity IgE receptor (CD23) processing: P1' bicyclomethyl substituents

Using a variety of alpha-hydroxy hydroxamic acid derivatives, the size and shape of the S1' pocket for the CD23 processing metalloprotease has been explored. It has been demonstrated that a P1' 2-naphthylmethyl group occupies most of the available space and gives excellent selectivity against fibroblast collagenase (matrix metalloproteinase-1, MMP-1) and other MMPs.     

Deoxycytidine glyoxal: lesion induction and evidence of repair following vitamin C supplementation in vivo

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p =.001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p =.0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected.