Cell Kinetic Evidence Suggests Elevated Oxidative Stress in Cultured Cells of Bloom's Syndrome

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse disturbed cell proliferation of fibroblasts and lymphoblastoid cells from Bloom's syndrome (BS). Fibroblasts show poor activation, arrest in the G2 phase of the cell cycle along with a prolongation of the Gl phase. This pattern of perturbed cells proliferation is akin to that elicited in normal fibroblasts by 4-hydroxy-nonenal, a breakdown product of lipid peroxides. Treatment with vitamin E improved growth of BS fibroblasts more strongly than growth of normal fibroblasts. Lymphoblastoid cells from BS, to the contrary, experience only a minor arrest in the G2 phase after one round of bromodeoxyuridine incorporation, but are strongly inhibited during and after the second S phase. Thus, their cell cycle arrest is dependent upon BrdU incorporation, as has been found previously in normal cells exposed to elevated concentrations of oxygen or paraquat, a superoxide generating compound. These results suggest that BS cells may suffer from an elevated, endogenous generation of oxygen free radicals.     

The measurement of chemically-induced DNA repair synthesis in human cells by BND-cellulose chromatography.

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.     

The role of AdipoR1 and AdipoR2 in liver fibrosis

Activation of the adiponectin (APN) signaling axis retards liver fibrosis. However, understanding of the role of AdipoR1 and AdipoR2 in mediating this response is still rudimentary. Here, we sought to elucidate the APN receptor responsible for limiting liver fibrosis by employing AdipoR1 and AdipoR2 knock-out mice in the carbon tetrachloride (CCl₄) model of liver fibrosis. In addition, we knocked down receptor function in primary hepatic stellate cells (HSCs) in vitro. Following the development of fibrosis, AdipoR1 and AdipoR2 KO mice had no quantitative difference in fibrosis by Sirius red staining. However, AdipoR2 KO mice had an enhanced fibrotic signature with increased Col1-α1, TGFß-1, TIMP-1, IL-10, MMP-2 and MMP-9. Knockdown of AdipoR1 or AdipoR2 in HSCs followed by APN treatment demonstrated that AdipoR1 and AdipoR2 did not affect proliferation or TIMP-1 gene expression, while AdipoR2 modulated Col1-α1 and α-SMA gene expression, HSC migration, and AMPK activity. These finding suggest that AdipoR2 is the major APN receptor on HSCs responsible for mediating its anti-fibrotic effects.     

Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures

Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.     

The Escherichia coli protein YjjG is a house-cleaning nucleotidase in vivo

House-cleaning enzymes protect cells from the adverse effects of noncanonical metabolic chemical compounds. The Escherichia coli nucleotide phosphatase YjjG (B4374, JW4336) functions as a house-cleaning phosphatase in vivo. YjjG protects the cell against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine (5-FdUridine), 5-fluorouridine, 5-fluoroorotic acid (5-FOA), 5-fluorouracil, and 5-aza-2'-deoxycytidine. YjjG prevents the incorporation of potentially mutagenic nucleotides into DNA as shown for 5-bromo-2'-deoxyuridine (BrdU). Its enzymatic activity in vitro towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP) is higher than towards canonical thymidine monophosphate (dTMP). The closest homolog in humans, HDHD4, does not show a protective effect against noncanonical nucleotides, excluding an involvement of HDHD4 in resistance against noncanonical nucleotides used for cancer chemotherapy. The substrate spectrum of YjjG suggests that its in vivo substrates are noncanonical pyrimidine derivatives, which might also include oxidized nucleobases such as 5-formyluracil and 5-hydroxyuracil.     

Hedgehog-mediated paracrine interaction between hepatic stellate cells and marrow-derived mesenchymal stem cells

During liver injury, bone marrow-derived mesenchymal stem cells (MSCs) can migrate and differentiate into hepatocytes. Hepatic stellate cell (SC) activation is a pivotal event in the development of liver fibrosis. Therefore, we hypothesized that SCs may play an important role in regulating MSC proliferation and differentiation through the paracrine signaling pathway. We demonstrate that MSCs and SCs both express hedgehog (Hh) pathway components, including its ligands, receptors, and target genes. Transwell co-cultures of SCs and MSCs showed that the SCs produced sonic hedgehog (Shh), which enhanced the proliferation and differentiation of MSCs. These findings demonstrate that SCs indirectly modulate the activity of MSCs in vitro via the Hh pathway, and provide a plausible explanation for the mechanisms of transplanted MSCs in the treatment of liver fibrosis.     

Taenia taeniaeformis: colonic hyperplasia in heavily infected rats

Only one study previously mentioned the involvement of colon during Taenia taeniaeformis larvae infection in rats with inconsistent occurrence of lesions. Present study aimed to determine the consistency of histopathologic changes in colonic epithelia, and the proliferation of mucosal cells through BrdU and PCNA immunohistochemistry. Results demonstrated that crypt hyperplasia of the colon was found in all infected rats, although variable in degree even in a single tissue section. Cystic cavities were frequently seen in severely hyperplastic mucosa. Proliferative zone lengths were significantly increased and PCNA positive cells were observed throughout the colonic crypt lengths at 9 but not at 6 weeks post infection. Cell proliferation involving the major types of cells in the epithelial colon was also increased in infected rats at 9 weeks post infection, with labeling indices significantly greater than the control rats throughout the BrdU time course labeling. Findings suggested that massive increases in epithelial cells and depth of colonic crypts were due to a remarkable increase in cell proliferation. The study concluded that enteropathy in the colon during T. taeniaeformis infection could be consistently observed in heavily infected rats.     

Taenia taeniaeformis: fate and proliferation of mucosal cells during gastric hyperplasia in larvae infected rats

Fate and proliferation of gastric mucosal cells during hyperplasia of Taenia taeniaeformis eggs inoculated Wistar rats were investigated using PCNA immunohistochemistry, BrdU labeling and other histopathologic staining techniques. Results revealed marked cell proliferation in gastric corpus and antral mucosa of infected rats as evidenced by increased lengths of proliferative zones and indices of BrdU labeling. The gastropathy in corpus was characterized by massive accumulation of precursors, neck and intermediate cells following significant decreases in numbers of parietal and zymogenic cells. Gastropathy in antrum was described with significant increases in precursors and mucous cells. Our results suggested that T. taeniaeformis-induced gastric hyperplasia was initiated by depletion of parietal cells presumably due to the cestode's ES products. As a result, there was inhibition of zymogenic cell differentiation due to the disruption of normal development pathways of gastric mucosal lineages. These sequences of events were considered to cause the increase in cell proliferation and accumulation of intermediate cells resulting to the hyperplastic lesions.