A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage

CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called "dynamic mutation" diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.     

Accuracy of diagnostic registers and management of chronic obstructive pulmonary disease: the Devon primary care audit

Background

Guidelines on COPD diagnosis and management encourage primary care physicians to detect the disease at an early stage and to treat patients according to their condition and needs. Problems in guideline implementation include difficulties in diagnosis, using spirometry and the disputed role of reversibility testing. These lead to inaccurate diagnostic registers and inadequacy of administered treatments. This study represents an audit of COPD diagnosis and management in primary care practices in Devon.

Methods

Six hundred and thirty two patients on COPD registers in primary care practices were seen by a visiting Respiratory Specialist Nurse. Diagnoses were made according to the NICE guidelines. Reversibility testing was carried out either routinely or based on clinical indication in two sub-samples. Dyspnoea was assessed. Data were entered into a novel IT-based software which computed guideline-based treatment recommendations. Current and recommended treatments were compared.

Results

Five hundred and eighty patients had spirometry. Diagnoses of COPD were confirmed in 422 patients (73%). Thirty nine patients were identified as asthma only, 94 had normal spirometry, 23 were restrictive and 2 had a cardiac disorder. Reversibility testing changed diagnosis of 11% of patients with airflow obstruction, and severity grading in 18%. Three quarters of patients with COPD had been offered practical help with smoking cessation. Short and long-acting anticholinergics and long acting beta-2 agonists had been under-prescribed; in 15–18% of patients they were indicated but not received. Inhaled steroids had been over-prescribed (recommended in 17%; taken by 60%), whereas only 4% of patients with a chronic productive cough were receiving mucolytics. Pulmonary rehabilitation was not available in some areas and was under-used in other areas.

Conclusion

Diagnostic registers of COPD in primary care contain mistakes leading to inaccurate prevalence estimates and inappropriate treatment decisions. Use of pre-bronchodilator readings for diagnosis overestimates the prevalence and severity in a significant minority, thus post bronchodilator readings should be used. Management of stable COPD does often not correspond to guidelines. The IT system used in this study has the potential to improve diagnosis and management of COPD in primary care.     

Polyclonal carcinoembryonic antigen staining in the cytologic differential diagnosis of primary and metastatic hepatic malignancies.

In order to assess the utility of immunocytochemical staining of bile canaliculi with a polyclonal antiserum to carcinoembryonic antigen (pCEA) in the differentiation of primary hepatocellular carcinomas from metastatic malignancies, pCEA staining was performed on fine needle aspiration specimens from hepatic lesions in 60 patients. The original cytologic diagnoses were hepatocellular carcinoma in 22 patients, metastatic neoplasm or cholangiocarcinoma in 27 patients and benign hepatocytes in 11 cases. The cytologic diagnoses of malignancy were confirmed by surgical excision, autopsy or clinical investigations in 82% of the patients. Follow-up data, supported by pCEA staining, reversed the original cytologic diagnosis in three cases. Bile canalicular pCEA staining was identified in 18 of 22 cases of hepatocellular carcinoma and in all 11 benign hepatocellular aspirates. All 27 cases of metastatic malignancy or cholangiocarcinoma were negative for canalicular pCEA staining, although 11 cases exhibited cytoplasmic staining. Interpretation of pCEA staining was not affected by the intermingling of malignant cells and benign hepatocytes. Predictive values were 100% for a positive test and 87% for a negative test. These findings indicate that staining with pCEA antiserum is a useful adjunct in the differential cytologic diagnosis of malignant hepatic lesions.     

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.

     

Epithelial expression of TLR4 is modulated in COPD and by steroids,salmeterol and cigarette smoke

The toll-like receptors (TLRs) are a key component of host defense in the respiratory epithelium. Cigarette smoking is associated with increased susceptibility to infection, while COPD is characterised by bacterial colonisation and infective exacerbations. We found reduced TLR4 gene expression in the nasal epithelium of smokers compared with non-smoking controls, while TLR2 expression was unchanged. Severe COPD was associated with reduced TLR4 expression compared to less severe disease, with good correlation between nasal and tracheal expression. We went on to examine the effect of potential modulators of TLR4 expression in respiratory epithelium pertinent to airways disease. Using an airway epithelial cell line, we found a dose-dependent downregulation in TLR4 mRNA and protein expression by stimulation with cigarette smoke extracts. Treatment with the corticosteroids fluticasone and dexamethasone resulted in a dose-dependent reduction in TLR4 mRNA and protein. The functional significance of this effect was demonstrated by impaired IL-8 and HBD2 induction in response to LPS. Stimulation with salmeterol (10^-6 M) caused upregulation of TLR4 membrane protein presentation with no upregulation of mRNA, suggesting a post-translational effect. The effect of dexamethasone and salmeterol in combination was additive, with downregulation of TLR4 gene expression, and no change in membrane receptor expression. Modulation of TLR4 in respiratory epithelium may have important implications for airway inflammation and infection in response to inhaled pathogens.     

Selective modulation of MAP kinase in embryonic palate cells

Murine embryonic palate mesenchyme (MEPM) cells are responsive to a number of endogenous factors found in the local embryonic tissue environment. Recently, it was shown that activation of the cyclic AMP (cAMP) or the transforming growth factor β (TGFβ) signal transduction pathways modulates the proliferative response of MEPM cells to epidermal growth factor (EGF). Since the mitogen-activated protein kinase (MAPK) cascade is a signal transduction pathway that mediates cellular responsiveness to EGF, we examined the possibility that several signaling pathways which abrogate EGF-stimulated proliferation do so via the p42/p44 MAPK signaling pathway. We demonstrate that EGF stimulates MAPK phosphorylation and activity in MEPM cells maximally at 5 minutes. Tyrosine phosphorylation and activation of MAPK was unaffected by treatment of MEPM cells with TGFβ or cholera toxin. Similarly, TGFβ altered neither EGF-induced MAPK tyrosine phosphorylation nor activity. However, the calcium ionophore, A23187, significantly increased MAPK phosphorylation which was further increased in the presence of EGF, although calcium mobilization reduced EGF-induced proliferation. Despite the increase in phosphorylation, we could not demonstrate induction of MAPK activity by A23187. Like EGF, phorbol ester, under conditions which activate PKC isozymes in MEPM cells, increased MAPK phosphorylation and activity but was also growth inhibitory to MEPM cells. The MEK inhibitor, PD098059, only partially abrogated EGF-induced phosphorylation. Likewise, depletion of PKC isozymes partially abrogated EGF-induced MAPK phosphorylation. Inhibition of both MEK and PKC isozymes resulted in a marked decrease in MAPK activity, confirming that EGF uses multiple pathways to stimulate MAPK activity. These data indicate that the MAPK cascade does not mediate signal transduction of several agents that inhibit growth in MEPM cells, and that there is a dissociation of the proliferative response and MAP kinase activation. Furthermore, other signaling pathways known to play significant roles in differentiation of palatal tissue converge with the MAPK cascade and may use this pathway in the regulation of alternative cellular processes. J. Cell. Physiol. 176:266–280, 1998. © 1998 Wiley-Liss, Inc.     

Thermal torsion of F-actin

The equipartition theorem is used to calculate the mode amplitudes for F-actin thermal torsion. The amplitudes phi n are found to scale as n-1, (EI)-1/2, and L1/2, where n is mode number, EI is actin bending stiffness, and L is filament length. Depending on conditions, the amplitude can be as large as 15 degrees; this is discussed briefly in terms of skeletal muscle mechanics.