The Phosphate-Binding Protein of Escherichia coli Is Not Essential for Pi-Regulated Expression of the pho Regulon

Disruption of pstS encoding the P(i)-binding protein in Escherichia coli generally leads to the constitutive expression of the pho regulon. We demonstrate that P(i)-controlled expression is restored when the activity of the P(i) transporter PitA or PitB is increased. Apparently, PstS is not an essential component of the signal transduction pathway.     

Nerve fiber formation and catecholamine content in adult rat adrenal medullary transplants after treatment with NGF, NT-3, NT-4/5, bFGF, CNTF, and GDNF

Adrenal chromaffin cells have been characterized by the ability to change the phenotype in response to neurotrophic factor stimulation. The adrenal gland expresses numerous trophic factors endogenously, but there is still a lack of knowledge as to how the adrenal medullary cells respond to these factors. Accordingly, we evaluated nerve fiber outgrowth and cell morphology, and measured catecholamine content in adult rat adrenal medullary tissue transplanted to the anterior chamber of the eye after exposure to neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), or glial cell line-derived neurotrophic factor (GDNF) compared with the effects after exposure to recombinant human nerve growth factor (rhNGF). The results show that rhNGF was the most potent factor in inducing neurite outgrowth from the grafted chromaffin cells. CNTF was also a powerful inducer of nerve fiber formation, while NT-4/5, GDNF, and bFGF were less potent. NT-3 did not produce neurite outgrowth above that seen in vehicle-treated eyes. Combining two neurotrophins, rhNGF and NT-3, reduced nerve fiber formation. Tyrosine hydroxylase (TH) immunohistochemistry revealed good cell survival in all grafts, and no morphological differences were detected with the different treatments. The adrenaline: noradrenaline: dopamine ratio was approximately 49%: 49%: 2%, independent of treatment, and the catecholamine content was equal irrespective of treatment. In conclusion, all neurotrophic factors used, except for NT-3, promoted neurite outgrowth from adult rat chromaffin transplants. Differences in outgrowth induced by the various trophic factors did not, however, change the catecholamine content in grafts when analyzed together with the graft-derived nerve plexus.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.     

Level of daily physical activity in individuals with COPD compared with healthy controls

Background

Persons with Chronic Obstructive Pulmonary Disease (COPD), performing some level of regular physical activity, have a lower risk of both COPD-related hospital admissions and mortality. COPD patients of all stages seem to benefit from exercise training programs, thereby improving with respect to both exercise tolerance and symptoms of dyspnea and fatigue. Physical inactivity, which becomes more severe with increasing age, is a point of concern in healthy older adults. COPD might worsen this scenario, but it is unclear to what degree. This literature review aims to present the extent of the impact of COPD on objectively-measured daily physical activity (DPA). The focus is on the extent of the impact that COPD has on duration, intensity, and counts of DPA, as well as whether the severity of the disease has an additional influence on DPA.

Results

A literature review was performed in the databases PubMed [MEDLINE], Picarta, PEDRO, ISI Web of Knowledge and Google scholar. After screening, 11 studies were identified as being relevant for comparison between COPD patients and healthy controls with respect to duration, intensity, and counts of DPA. Four more studies were found to be relevant to address the subject of the influence the severity of the disease may have on DPA. The average percentage of DPA of COPD patients vs. healthy control subjects for duration was 57%, for intensity 75%, and for activity counts 56%. Correlations of DPA and severity of the disease were low and/or not significant.

Conclusions

From the results of this review, it appears that patients with COPD have a significantly reduced duration, intensity, and counts of DPA when compared to healthy control subjects. The intensity of DPA seems to be less affected by COPD than duration and counts. Judging from the results, it seems that severity of COPD is not strongly correlated with level of DPA. Future research should focus in more detail on the relation between COPD and duration, intensity, and counts of DPA, as well as the effect of disease severity on DPA, so that these relations become more understandable.