Cathepsin D expression in colorectal adenocarcinomas and adenomas

The aim of this study was to investigate the role of cathepsin D in colorectal cancer. For this purpose cathepsin D expression was evaluated by means of immunohistochemistry in stromal and tumor cells of 31 colorectal carcinomas and 29 adenomas. Cytoplasmic cathepsin D expression of tumor cells was present in 90.3% of the carcinoma cases and various degrees of stromal cell cathepsin D expression were present in all cases. In the adenomas, the epithelial cells and stromal cells expressed cathepsin D in 68.96% and 96.55% of cases, respectively. The staining intensity was always weaker in the adenomas. When the stromal and tumor cell cathepsin D expression in the adenocarcinoma and adenoma cases were compared, a statistically significant difference was observed in the staining of stromal cells. Furthermore, stromal cathepsin D expression in the adenocarcinomas was related to tumor stage when the carcinomas were divided into low and high stage. Cathepsin D expression in stromal cells may be an important indicator of poor prognosis in colorectal adenocarcinomas.     

Reduction of atherosclerosis by the peroxisome proliferator-activated receptor alpha agonist fenofibrate in mice

Several clinical and angiographic intervention trials have shown that fibrate treatment leads to a reduction of the coronary events associated to atherosclerosis. Fibrates are ligands for peroxisome proliferator-activated receptor alpha (PPARalpha) that modulate risk factors related to atherosclerosis by acting at both systemic and vascular levels. Here, we investigated the effect of treatment with the PPARalpha agonist fenofibrate (FF) on the development of atherosclerotic lesions in apolipoprotein (apo) E-deficient mice and human apoA-I transgenic apoE-deficient (hapoA-I Tg x apoE-deficient) mice fed a Western diet. In apoE-deficient mice, plasma lipid levels were increased by FF treatment with no alteration in the cholesterol distribution profile. FF treatment did not reduce atherosclerotic lesion surface area in the aortic sinus of 5-month-old apoE-deficient mice. By contrast, FF treatment decreased total cholesterol and esterified cholesterol contents in descending aortas of these mice, an effect that was more pronounced in older mice exhibiting more advanced lesions. Furthermore, FF treatment reduced MCP-1 mRNA levels in the descending aortas of apoE-deficient mice, whereas ABCA-1 expression levels were maintained despite a significant reduction of aortic cholesterol content. In apoE-deficient mice expressing a human apoA-I transgene, FF increased human apoA-I plasma and hepatic mRNA levels without affecting plasma lipid levels. This increase in human apoA-I expression was accompanied by a significant reduction in the lesion surface area in the aortic sinus. These data indicate that the PPARalpha agonist fenofibrate reduces atherosclerosis in these animal models of atherosclerosis.     

Thermodynamics of the pyruvate kinase reaction and the reversal of glycolysis in heart and skeletal muscle

The effect of temperature, pH, and free [Mg(2+)] on the apparent equilibrium constant of pyruvate kinase (phosphoenol transphosphorylase) (EC ) was investigated. The apparent equilibrium constant, K', for the biochemical reaction P-enolpyruvate + ADP = ATP + Pyr was defined as K' = [ATP][Pyr]/[ADP][P-enolpyruvate], where each reactant represents the sum of all the ionic and metal complexed species in M. The K' at pH 7.0, 1.0 mm free Mg(2+) and I of 0.25 m was 3.89 x 10(4) (n = 8) at 25 degrees C. The standard apparent enthalpy (DeltaH' degrees ) for the biochemical reaction was -4.31 kJmol(-1) in the direction of ATP formation. The corresponding standard apparent entropy (DeltaS' degrees ) was +73.4 J K(-1) mol(-1). The DeltaH degrees and DeltaS degrees values for the reference reaction, P-enolpyruvate(3-) + ADP(3-) + H(+) = ATP(4-) + Pyr(1-), were -6.43 kJmol(-1) and +180 J K(-1) mol(-1), respectively (5 to 38 degrees C). We examined further the mass action ratio in rat heart and skeletal muscle at rest and found that the pyruvate kinase reaction in vivo was close to equilibrium i.e. within a factor of about 3 to 6 of K' in the direction of ATP at the same pH, free [Mg(2+)], and T. We conclude that the pyruvate kinase reaction may be reversed under some conditions in vivo, a finding that challenges the long held dogma that the reaction is displaced far from equilibrium.     

Quantitative proteomic analysis of Myc oncoprotein function

This study applies a new quantitative proteomics technology to the analysis of the function of the Myc oncoprotein in mammalian cells. Employing isotope-coded affinity tag (ICAT) reagent labeling and tandem mass spectrometry, the global pattern of protein expression in rat myc-null cells was compared with that of myc-plus cells (myc-null cells in which myc has been introduced) to generate a differential protein expression catalog. Expression differences among many functionally related proteins were identified, including reduction of proteases, induction of protein synthesis pathways and upregulation of anabolic enzymes in myc-plus cells, which are predicted to lead to increased cell mass (cell growth). In addition, reduction in the levels of adhesion molecules, actin network proteins and Rho pathway proteins were observed in myc-plus cells, leading to reduced focal adhesions and actin stress fibers as well as altered morphology. These effects are dependent on the highly conserved Myc Box II region. Our results reveal a novel cytoskeletal function for Myc and indicate the feasibility of quantitative whole-proteome analysis in mammalian cells.     

CXCR5-transduced bone marrow-derived dendritic cells traffic to B cell zones of lymph nodes and modify antigen-specific immune responses

Skin-derived migratory dendritic cells (DC), in contrast to bone marrow-derived DC (BMDC), express CXCR5, respond to the chemokine CXC ligand 13 (CXCL13) in vitro, and are capable of migrating to B cell zones (BCZ) in lymph nodes (LN) in vivo. Herein, we analyzed the surface phenotype of skin-derived migratory DC and found that 15-35% of MHC class II(high) cells showed high levels of expression of CXCR5 but expressed low levels of DEC205, a suggested characteristic of dermal-type DC in mice. To study the effects of CXCR5 on the trafficking dynamics of DC, we stably expressed CXCR5 in BMDC by retroviral gene transduction. CXCR5 was detected by flow cytometry on transduced cells, which responded to CXCL13 in vitro in chemotaxis assays (3-fold over nontransduced BMDC, p < 0.01). When injected into the footpads of mice, approximately 40% of injected CXCR5-BMDC were observed in BCZ of draining LN. Mice were vaccinated with CXCR5- and vector-BMDC that were pulsed with keyhole limpet hemocyanin (KLH) to induce Ag-specific cellular and humoral immune responses. Mice injected with CXCR5-BMDC (vs vector-BMDC) demonstrated marginally less footpad swelling in response to intradermal injection of KLH. Interestingly, significantly higher levels of KLH-specific IgG (p < 0.05) and IgM (p < 0.01) were found in the serum of mice injected with CXCR5-BMDC compared with mice immunized with vector-transduced BMDC. Thus, CXCR5 is predominantly expressed by dermal-type DC. Moreover, CXCR5 directs BMDC to BCZ of LN in vivo and modifies Ag-specific immune responses induced by BMDC vaccination.     

Production of helper-dependent adenovirus vector relies on helper virus structure and complementing

Background

The helper‐dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD‐Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD‐Ad vector is generally lower than that of an E1‐deleted first‐generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD‐Ad vector.

Methods

To study this question and improve HD‐Ad vector production, we have generated four different helper viruses with a wild‐type or deleted E3 region, and with a relocated loxP. We have also constructed a first‐generation vector with a wild‐type E3 region and without the loxP site. We compared the replication of these viruses in Cre‐positive and ‐negative cells and studied their complementing for HD‐Ad vector production.

Results

Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD‐Ad vector was obtained when a helper virus with wild‐type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability.

Conclusions

Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD‐Ad vector can be further improved by modifications in helper virus structure. Copyright © 2002 John Wiley & Sons, Ltd.