Quantification of gene expression in methanotrophs by competitive reverse transcription-polymerase chain reaction

To improve the monitoring of methanotrophic activity, a competitive reverse transcription-polymerase chain reaction (RT-PCR) methodology was developed. Homologous internal RNA standards were created for mmoX and pmoA , genes encoding polypeptides of sMMO and pMMO, respectively. Using specific primer sets, expression of sMMO and pMMO could be quantified by means of competitive RT-PCR and capillary electrophoresis with uncoated bare-fused silica columns and UV detection. Using this technique, it was discovered that the amount of mRNA transcript for both mmoX and pmoA correlated well with whole-cell sMMO and pMMO activity respectively. A method for soil RNA extraction was also developed to utilize this RNA quantification technique for the monitoring of methanotrophic activity in situ . In a model soil slurry system with a background concentration of 2.9 µM copper, it was found that only pmoA was transcribed by cells capable of expressing both forms of MMO. As pMMO and sMMO have very different substrate ranges and kinetics, this methodology may prove useful for optimizing in situ bioremediation by methanotrophs. Provided sufficient sequence information is available to create specific primer sets, these techniques can be applied for monitoring and measuring the activity of other microbial communities in situ .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75134/1/j.1462-2920.2004.00572.x.pd

Guards and Culprits in the Endoplasmic Reticulum: Glucolipotoxicity and β-Cell Failure in Type II Diabetes

The endoplasmic reticulum (ER) is a cellular organelle responsible for multiple important cellular functions including the biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. The ER participates in all branches of metabolism, linking nutrient sensing to cellular signaling. Many pathological and physiological factors perturb ER function and induce ER stress. ER stress triggers an adaptive signaling cascade, called the unfolded protein response (UPR), to relieve the stress. The failure of the UPR to resolve ER stress leads to pathological conditions such as β-cell dysfunction and death, and type II diabetes. However, much less is known about the fine details of the control and regulation of the ER response to hyperglycemia (glucotoxicity), hyperlipidemia (lipotoxicity), and the combination of both (glucolipotoxicity). This paper considers recent insights into how the response is regulated, which may provide clues into the mechanism of ER stress-mediated β-cell dysfunction and death during the progression of glucolipotoxicity-induced type II diabetes

Neuroprotective effect of dexmedetomidine on autophagy in mice administered intracerebroventricular injections of Aβ25–35

Alzheimer’s disease (AD), one of the most prevalent neurodegenerative diseases is associated with pathological autophagy-lysosomal pathway dysfunction. Dexmedetomidine (Dex) has been suggested as an adjuvant to general anesthesia with advantages in reducing the incidence of postoperative cognitive dysfunction in Dex-treated patients with AD and older individuals. Several studies reported that Dex improved memory; however, evidence on the effects of Dex on neuronal autophagy dysfunction in the AD model is lacking. We hypothesized that Dex administration would have neuroprotective effects by improving pathological autophagy dysfunction in mice that received an intracerebroventricular (i.c.v.) injection of amyloid β-protein fragment 25–35 (Aβ25–35) and in an autophagy-deficient cellular model. In the Y-maze test, Dex reversed the decreased activity of Aβ25–35 mice. Additionally, it restored the levels of two memory-related proteins, phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII) and postsynaptic density-95 (PSD-95) in Aβ25–35 mice and organotypic hippocampal slice culture (OHSC) with Aβ25–35. Dex administration also resulted in decreased expression of the autophagy-related microtubule-associated proteins light chain 3-II (LC3-II), p62, lysosome-associated membrane protein2 (LAMP2), and cathepsin D in Aβ25–35 mice and OHSC with Aβ25–35. Increased numbers of co-localized puncta of LC3-LAMP2 or LC3-cathepsin D, along with dissociated LC3-p62 immunoreactivity following Dex treatment, were observed. These findings were consistent with the results of western blots and the transformation of double-membrane autophagosomes into single-membraned autolysosomes in ultrastructures. It was evident that Dex treatment alleviated impaired autolysosome formation in Aβ mice. Our study demonstrated the improvement of memory impairment caused by Dex and its neuroprotective mechanism by investigating the role of the autophagy-lysosomal pathway in a murine Aβ25–35 model. These findings suggest that Dex could be used as a potential neuroprotective adjuvant in general anesthesia to prevent cognitive decline

Bone marrow-derived, alternatively activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model

INTRODUCTION: Tumor-associated macrophages, which are derived from the infiltration of circulating bone marrow-derived monocytes, consist primarily of a polarized M2 macrophage (M2-Mϕ) population and are associated with poor prognosis in various cancers. In the present study, we attempted to assess whether M2-Mϕs derived from bone marrow stimulate the promotion and progression of mammary tumors. METHODS: 4T1 murine mammary carcinoma cells were injected either alone or coupled with M2-Mϕs into the mammary fat pads of syngeneic female Balb/C mice. M2-Mϕs were prepared by treating monocytes isolated from female Balb/C mouse bone marrow with IL-4. Tumor cell growth was determined using an in vivo imaging system and the expression of cell proliferation-related, angiogenesis-related, and lymphangiogenesis-related proteins in tumor tissues was immunohistochemically analyzed. To evaluate the effects of the crosstalk between 4T1 cells and M2-Mϕs on the secretion and mRNA expression of cytokines and the migration of monocytes, 4T1 cells and M2-Mϕs were co-cultured and cytokine antibody array, real-time RT-PCR, and trans-well migration assays were conducted. RESULTS: The co-injection of M2-Mϕs into the mammary fat pads of mice increased solid tumor growth and lung metastasis of 4T1 cells as well as the infiltration of CD45(+ )leukocytes into tumor tissues. The proportions of Ki-67(+ )proliferating cells and the expression of hypoxia inducible factor-1α, vascular endothelial cell growth factor A, CD31, vascular endothelial cell growth factor C, and lymphatic vessel endothelial receptor-1 were increased significantly in the tumor tissues of mice co-injected with 4T1 cells and M2-Mϕs. The in vitro results revealed that the proliferation of 4T1 cells, the migration of monocytes, and the secretion of granulocyte colony-stimulating factor, IFNγ, IL-1α, IL-2, IL-16, IFNγ-induced protein-10, keratinocyte-derived chemokine, macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and RANTES were increased when 4T1 cells were co-cultured with M2-Mϕs, as compared with when the 4T1 cells were cultured alone. CONCLUSION: The crosstalk between 4T1 cells and M2-Mϕs increased the production of cytokines, which may have induced immune cell infiltration into tumor tissues, tumor cell proliferation, angiogenesis, and lymph angiogenesis, thereby increasing solid tumor growth and lung metastasis