Gene Expression Profiling of Ampullary Carcinomas Classifies Ampullary Carcinomas into Biliary-Like and Intestinal-Like Subtypes That Are Prognostic of Outcome

Background

Adenocarcinomas of the ampulla of Vater are classified as biliary cancers, though the exact epithelium of origin for these cancers is not known. We sought to molecularly classify ampullary adenocarcinomas in comparison to known adenocarcinomas of the pancreas, bile duct, and duodenum by gene expression analysis.

Methods

We analyzed 32 fresh-frozen resected, untreated periampullary adenocarcinomas (8 pancreatic, 2 extrahepatic biliary, 8 duodenal, and 14 ampullary) using the Affymetrix U133 Plus 2.0 genome array. Unsupervised and supervised hierarchical clustering identified two subtypes of ampullary carcinomas that were molecularly and histologically characterized.

Results

Hierarchical clustering of periampullary carcinomas segregated ampullary carcinomas into two subgroups, which were distinctly different from pancreatic carcinomas. Non-pancreatic periampullary adenocarcinomas were segregated into two subgroups with differing prognoses: 5 year RFS (77% vs. 0%, p = 0.007) and 5 year OS (100% vs. 35%, p = 0.005). Unsupervised clustering analysis of the 14 ampullary samples also identified two subgroups: a good prognosis intestinal-like subgroup and a poor prognosis biliary-like subgroup with 5 year OS of 70% vs. 28%, P = 0.09. Expression of CK7+/CK20- but not CDX-2 correlated with these two subgroups. Activation of the AKT and MAPK pathways were both increased in the poor prognostic biliary-like subgroup. In an independent 80 patient ampullary validation dataset only histological subtype (intestinal vs. pancreaticobiliary) was significantly associated with OS in both univariate (p = 0.006) and multivariate analysis (P = 0.04).

Conclusions

Gene expression analysis discriminated pancreatic adenocarcinomas from other periampullary carcinomas and identified two prognostically relevant subgroups of ampullary adenocarcinomas. Histological subtype was an independent prognostic factor in ampullary adenocarcinomas.     

Maximum glucoamylase production by a temperature-sensitive mutant of Saccharomyces cerevisiae in batch culture

In order to maximize the glucoamylase production by recombinant Saccharomyces cerevisiae in batch culture, first a temperature-controlled expression system for a foreign gene in S. cerevisiae was constructed. A temperature-sensitive pho80 mutant of S. cerevisiae for the PHO regulatory system, YKU131, was used for this purpose. A DNA fragment bearing the promoter of the PHO84 gene, which encodes an inorganic phosphate (P_i) transporter of S. cerevisiae and is derepressed by P_i starvation, was used as promoter. The glucoamylase gene connected with the PHO84 promoter was ligated into a YEp13 vector, designated pKU122. When the temperature-sensitive pho80 ^ts mutant harboring the plasmid pKU122 is cultivated at a lower temperature, the expression of glucoamylase gene is repressed, but at a higher temperature it is expressed. Next the effect of temperature on the specific growth rate, μ, and specific production rate, ρ, was investigated. Maximum values of ρ and ρ at various temperatures were at 30°C and 34°C, respectively. The optimal cultivation temperature strategy for maximum production of glucoamylase by this recombinant strain in batch culture was then determined by the Maximum principle using the relationships of μ and ρ to the cultivation temperature. Finally, the optimal strategy was experimentally realized by changing the cultivation temperature from Tμ (30°C) to Tρ (34°C) at the switching time, t_s. Received 18 September 1997/ Accepted in revised form 07 January 1998     

Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.     

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.     

Directional shape complementarity at the protein-DNA interface

Nature utilizes various styles of architecture for DNA-binding proteins to recognize diverse DNA sequences, a process facilitated by a complementary surface between protein and DNA. However, the extent and ways this 'shape complementarity' occurs at the protein-DNA interface have yet to be characterized. Here, by analyzing a set of diverse protein-DNA complexes of known three-dimensional structures, we investigated whether the normal vectors of a protein surface at the interface exhibited any relationship with DNA conformation. Generally, the normal vectors of a DNA-contacting protein surface distinctly preferred certain angles, enabling them to align with certain axes characterizing the conformation of DNA. Thus, a new geometric property of DNA-binding protein is demonstrated, i.e. the "shape complementarity" of protein-DNA recognition clearly bears the property of "directionality".     

Novel secretory vesicle serpins,endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.     

Potent and selective cathepsin L inhibitors do not inhibit human osteoclast resorption in vitro

Cathepsins K and L are related cysteine proteases that have been proposed to play important roles in osteoclast-mediated bone resorption. To further examine the putative role of cathepsin L in bone resorption, we have evaluated selective and potent inhibitors of human cathepsin L and cathepsin K in an in vitro assay of human osteoclastic resorption and an in situ assay of osteoclast cathepsin activity. The potent selective cathepsin L inhibitors (K(i) = 0.0099, 0.034, and 0.27 nm) were inactive in both the in situ cytochemical assay (IC(50) > 1 micrometer) and the osteoclast-mediated bone resorption assay (IC(50) > 300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC(50) = 63 nm) and resorption (IC(50) = 71 nm) assays. A recently reported dipeptide aldehyde with activity against cathepsins L (K(i) = 0.052 nm) and K (K(i) = 1.57 nm) was also active in both assays (IC(50) = 110 and 115 nm, respectively) These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.     

Expression regulation and function of Pref-1 during adipogenesis of human mesenchymal stem cells (MSCs)

Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARγ (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARγ expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.     

Transgene activity following somatic transgenesis in Nile tilapia Oreochromis niloticus

A detailed study was made of the persistence and expression of a plasmid-enclosed reporter gene construct after intramuscular injection into the somatic muscle tissue of juvenile Nile tilapia Oreochromis niloticus and also the effect of injecting a potentially growth-promoting gene construct. The plasmid-enclosed DNA proved stable at the site of injection, lasting in some cases for up to 6 months, and was, at a very low frequency, detected in gonad tissue, indicating occasional substantial movement from the injected muscle site. It was observed that the reporter gene and regulatory sequences were also functional within the somatic cells. In a comparison of expression levels by direct somatic injection, the 1·6 kb tilapia β-actin regulatory sequence (tiβAP) resulted in c. three-fold higher β-galactosidase activity than the 4·7 kb carp β-actin regulatory sequence (cβAP) when spliced to the lacZ gene. The enhancer element near the end of first intron in the tiβAP, when co-injected with tiβAP/lacZ plasmid at a 3:1 ratio, drove significantly higher reporter activity in somatic cells than the tiβAP/lacZ sequence alone. The introduction of a growth-promoting construct, the Nile tilapia growth hormone gene driven by a tiβAP, yielded no detectable growth enhancement.