Cellulolytic enzymes for the hydrolysis of leached beet cosette

Summary Cellulolytic fungi were isolated from rotting leaves and tested for extra-cellular cellulase activities (CMCase, avicelase, cellobiase and xylanase). The effect of the proportion of the enzyme activities on the rate of degradation of leached beet cosette was observed using a range of supernatant fluids in appropriate combinations. At low cosette concentrations (1.5–3.0 g/l), avicelase and cellobiase were the rate limiting enzymes; avicelase in the initial stages of reaction and cellobiase after 6–8 hours, when cellobiose inhibition becomes important. A ratio of celiobiase to avicelase of approx 2.0 was established as appropriate. At higher substrate concentrations (10 g/l, 40 g/l) the best cellobiase to avicelase ratio was maintained and up to 40% hydrolysis was obtained in the 10 g/l incubation with 10 Uav/l and 20 Ucellob/l. At 100 g/l cosette concentration, substrate inhibition was observed.     

Cellular Source and Mechanisms of High Transcriptome Complexity in the Mammalian Testis

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Five batches representative of Ontario-grown American ginseng root produce comparable reductions of postprandial glycemia in healthy individuals

Evidence indicates that the glycemia-lowering effect of American ginseng root may be batch dependent. We therefore evaluated the effect of 5 root batches, representative of Ontario-grown American ginseng, on postprandial glucose and insulin indices. Twelve healthy subjects (5 male, 7 female), mean +/- SE age 26.5 +/- 2 years, body mass index 23.96 +/- 3.41 kg/m2, fasting blood glucose 4.77 +/- 0.04 mmol/L, were assigned to consume 9 g of American ginseng from 5 farms (A-E), administered in randomized sequence on 5 separate visits, and a water-control during the 6th and last visit. Treatments were consumed 40 min before a 2-hour 75-gram oral glucose tolerance test. Plasma glucose and insulin were measured at baseline, before, and during the test. Compared with control, batches A and C reduced glucose incremental area under the curve (IAUC) by 35.2% (156 vs. 240 mmol.min/L) and 32.6% (162 vs. 240 mmol.min/L), respectively. Batches A, C, and E reduced incremental peak glucose by 1.3, 1.2, and 1.1 mmol/L, respectively. Batch C reduced the insulin IAUC by 27.7% (15.8 vs. 21.8 nmol.min/L). Effects on glucose and insulin parameters were not different across ginseng treatments. The mean of the 5 ginseng treatments reduced peak postprandial glucose by 1.0 mmol/L, glucose IAUC by 27.7% (173 vs. 240 mmol.min/L), and insulin IAUC by 23.8% (16.6 vs. 21.8 nmol.min/L) relative to control. (All results statistically significant at p < 0.05.) American ginseng decreased postprandial glycemia and insulinemia; however, 40% of the batches did not reduce glycemia with the anticipated magnitude, irrespective of their saponin composition.     

Closure of rRNA related gaps in the Chromobacterium violaceum genome with the PCR-assisted contig extension (PACE) protocol

In the finishing phase of the Chromobacterium violaceum genome project, the shotgun sequences were assembled into 57 contigs that were then organized into 19 scaffolds, using the information from shotgun and cosmid clones. Among the 38 ends resulting from the 19 scaffolds, 10 ended with sequences corresponding to rRNA genes (seven ended with the 5S rRNA gene and three ended with the 16S rRNA gene). The 28 non-ribosomal ends were extended using the PCR-assisted contig extension (PACE) methodology, which immediately closed 15 real gaps. We then applied PACE to the 16S rRNA gene containing ends, resulting in eight different sequences that were correctly assembled within the C. violaceum genome by combinatory PCR strategy, with primers derived from the non-repetitive genomic region flanking the 16S and 5S rRNA gene. An oriented combinatory PCR was used to correctly position the two versions (copy A and copy B, which differ by the presence or absence of a 100-bp insert); it revealed six copies corresponding to copy A, and two to copy B. We estimate that the use of PACE, followed by combinatory PCR, accelerated the finishing phase of the C. violaceum genome project by at least 40%.     

Nodal/Activin signaling drives self-renewal and tumorigenicity of pancreatic cancer stem cells and provides a target for combined drug therapy

Nodal and Activin belong to the TGF-β superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and Activin regulate self-renewal of pancreatic cancer stem cells. Nodal and Activin were hardly detectable in more differentiated pancreatic cancer cells, while cancer stem cells and stroma-derived pancreatic stellate cells markedly overexpressed Nodal and Activin, but not TGF-β. Knockdown or pharmacological inhibition of the Nodal/Activin receptor Alk4/7 in cancer stem cells virtually abrogated their self-renewal capacity and in vivo tumorigenicity, and reversed the resistance of orthotopically engrafted cancer stem cells to gemcitabine. However, engrafted primary human pancreatic cancer tissue with a substantial stroma showed no response due to limited drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor enhanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival. Therefore, inhibition of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a therapeutic strategy for targeting cancer stem cells.     

Targeting MAGE-C1/CT7 expression increases cell sensitivity to the proteasome inhibitor bortezomib in multiple myeloma cell lines

The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26-27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70-80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p<0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p<0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p<0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be a strategy for future therapies in MM, in particular in combination with proteasome inhibitors.     

Stem cells as the root of pancreatic ductal adenocarcinoma

Emerging evidence suggests that stem cells play a crucial role not only in the generation and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.