Topology of molecular machines of the endoplasmic reticulum: a compilation of proteomics and cytological data

The endoplasmic reticulum (ER) is a key organelle of the secretion pathway involved in the synthesis of both proteins and lipids destined for multiple sites within and without the cell. The ER functions to both co- and post-translationally modify newly synthesized proteins and lipids and sort them for housekeeping within the ER and for transport to their sites of function away from the ER. In addition, the ER is involved in the metabolism and degradation of specific xenobiotics and endogenous biosynthetic products. A variety of proteomics studies have been reported on different subcompartments of the ER providing an ER protein dictionary with new data being made available on many protein complexes of relevance to the biology of the ER including the ribosome, the translocon, coatomer proteins, cytoskeletal proteins, folding proteins, the antigen-processing machinery, signaling proteins and proteins involved in membrane traffic. This review examines proteomics and cytological data in support of the presence of specific molecular machines at specific sites or subcompartments of the ER

Cigarette smoke worsens lung inflammation and impairs resolution of influenza infection in mice

<p>Abstract</p> <p>Background</p> <p>Cigarette smoke has both pro-inflammatory and immunosuppressive effects. Both active and passive cigarette smoke exposure are linked to an increased incidence and severity of respiratory virus infections, but underlying mechanisms are not well defined. We hypothesized, based on prior gene expression profiling studies, that upregulation of pro-inflammatory mediators by short term smoke exposure would be protective against a subsequent influenza infection.</p> <p>Methods</p> <p>BALB/c mice were subjected to whole body smoke exposure with 9 cigarettes/day for 4 days. Mice were then infected with influenza A (H3N1, Mem71 strain), and analyzed 3 and 10 days later (d3, d10). These time points are the peak and resolution (respectively) of influenza infection.</p> <p>Results</p> <p>Inflammatory cell influx into the bronchoalveolar lavage (BALF), inflammatory mediators, proteases, histopathology, viral titres and T lymphocyte profiles were analyzed. Compared to smoke or influenza alone, mice exposed to smoke and then influenza had more macrophages, neutrophils and total lymphocytes in BALF at d3, more macrophages in BALF at d10, lower net gelatinase activity and increased activity of tissue inhibitor of metalloprotease-1 in BALF at d3, altered profiles of key cytokines and CD4+ and CD8+ T lymphocytes, worse lung pathology and more virus-specific, activated CD8+ T lymphocytes in BALF. Mice smoke exposed before influenza infection had close to 10-fold higher lung virus titres at d3 than influenza alone mice, although all mice had cleared virus by d10, regardless of smoke exposure. Smoke exposure caused temporary weight loss and when smoking ceased after viral infection, smoke and influenza mice regained significantly less weight than smoke alone mice.</p> <p>Conclusion</p> <p>Smoke induced inflammation does not protect against influenza infection.</p> <p>In most respects, smoke exposure worsened the host response to influenza. This animal model may be useful in studying how smoke worsens respiratory viral infections.</p

Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ)

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMD_J) model, a suitable animal model for Duchenne muscular dystrophy.</p> <p>Methods</p> <p>We used surgically biopsied tibialis cranialis muscles of normal male dogs (n = 3) and CXMD_Jdogs (n = 3) at 4, 5 and 6 months of age. Muscle sections were analyzed by conventional morphological methods and <it>in situ </it>zymography to identify the localization of MMP-2 and MMP-9. MMP-2 and MMP-9 activity was examined by gelatin zymography and the levels of the respective mRNAs in addition to those of regulatory molecules, including MT1-MMP, TIMP-1, TIMP-2, and RECK, were analyzed by semi-quantitative RT-PCR.</p> <p>Results</p> <p>In CXMD_Jskeletal muscle, multiple foci of both degenerating and regenerating muscle fibers were associated with gelatinolytic MMP activity derived from MMP-2 and/or MMP-9. In CXMD_Jmuscle, MMP-9 immunoreactivity localized to degenerated fibers with inflammatory cells. Weak and disconnected immunoreactivity of basal lamina components was seen in MMP-9-immunoreactive necrotic fibers of CXMD_Jmuscle. Gelatinolytic MMP activity observed in the endomysium of groups of regenerating fibers in CXMD_Jdid not co-localize with MMP-9 immunoreactivity, suggesting that it was due to the presence of MMP-2. We observed increased activities of pro MMP-2, MMP-2 and pro MMP-9, and levels of the mRNAs encoding MMP-2, MMP-9 and the regulatory molecules, MT1-MMP, TIMP-1, TIMP-2, and RECK in the skeletal muscle of CXMD_Jdogs compared to the levels observed in normal controls.</p> <p>Conclusion</p> <p>MMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle. MMP-9 may be involved predominantly in the inflammatory process during muscle degeneration. In contrast, MMP-2, which was activated in the endomysium of groups of regenerating fibers, may be associated with ECM remodeling during muscle regeneration and fiber growth.</p

Metabolic alterations during the growth of tumour spheroids

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms

Metabolic alterations during the growth of tumour spheroids

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms

Transketolase-Like 1 Expression Is Modulated during Colorectal Cancer Progression and Metastasis Formation

Background Transketolase-like 1 (TKTL1) induces glucose degradation through anaerobic pathways, even in presence of oxygen, favoring the malignant aerobic glycolytic phenotype characteristic of tumor cells. As TKTL1 appears to be a valid biomarker for cancer prognosis, the aim of the current study was to correlate its expression with tumor stage, probability of tumor recurrence and survival, in a series of colorectal cancer patients. Methodolody/Principal Findings Tumor tissues from 63 patients diagnosed with colorectal cancer at different stages of progression were analyzed for TKTL1 by immunohistochemistry. Staining was quantified by computational image analysis, and correlations between enzyme expression, local growth, lymph-node involvement and metastasis were assessed. The highest values for TKTL1 expression were detected in the group of stage III tumors, which showed significant differences from the other groups (Kruskal-Wallis test, P = 0.000008). Deeper analyses of T, N and M classifications revealed a weak correlation between local tumor growth and enzyme expression (Mann-Whitney test, P = 0.029), a significant association of the enzyme expression with lymph-node involvement (Mann-Whitney test, P = 0.0014) and a significant decrease in TKTL1 expression associated with metastasis (Mann-Whitney test, P = 0.0004). Conclusions/Significance To our knowledge, few studies have explored the association between variations in TKTL1 expression in the primary tumor and metastasis formation. Here we report downregulation of enzyme expression when metastasis appears, and a correlation between enzyme expression and regional lymph-node involvement in colon cancer. This finding may improve our understanding of metastasis and lead to new and more efficient therapies against cancer