Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line <Emphasis Type="Italic">in vitro</Emphasis>

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellullar carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.     

Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons

Using neurotoxicity and inositol phosphate release as criteria for receptor expression, we report the differential expression of excitatory amino acid receptor subtypes in cerebellar granule cells grown in serum-free media containing either high (25 mM) or low (5 mM) KCl. NMDA receptors are expressed in neurons grown in high, but not low, KCl. In contrast, ionotropic quisqualate receptors are expressed in neurons grown in low KCl, but not in those grown in high KCl. Addition of NMDA to cultures containing low KCl appears to mimic high KCl conditions: NMDA receptors are expressed, but ionotropic quisqualate receptors are not. Glutamate and kainate are toxic to cells grown in either condition.     

Altered ion transporter expression in bronchial epithelium in mountaineers with high-altitude pulmonary edema.

Hypoxia inhibits activity and expression of transport proteins of cultured lung alveolar epithelial cells. Here we tested whether hypoxia at high altitude affected the expression of ion transport proteins in tissues obtained from controls and mountaineers with high-altitude pulmonary edema (HAPE) at the Capanna Margherita (4,559 m). Expression was determined by RT-PCR and Western blots from brush biopsies of bronchial epithelium and from leukocytes obtained before and during the stay at high altitude. At low altitude, amounts of mRNAs were not different between control and HAPE-susceptible subjects. At high altitude, the amount of mRNA of Na-K-ATPase, CFTR, and beta-actin of brush biopsies did not change in controls but decreased significantly (-60%) in HAPE-susceptible subjects. There was no change in Na channel mRNAs at high altitude in controls and HAPE. No statistically significant correlation was found between the expression of Na transporters and PO2 and O2 saturation. In leukocytes, 28S-rRNA and Na-K-ATPase decreased at altitude in control and HAPE-susceptible subjects, but no significant change in Na-K-ATPase protein was found. Hypoxia-inducible factor-1alpha mRNA and GAPDH mRNA tended to increase in leukocytes obtained from HAPE-susceptible subjects at high altitude but did not change in controls. These results show that hypoxia induces differences in mRNA expression of ion transport-related proteins between HAPE-susceptible and control subjects but that these changes may not necessarily predict differences in protein concentration or activity. It is therefore unclear whether these differences are related to the pathophysiology of HAPE.     

Cutting edge: distal regulatory elements are required to achieve selective expression of IFN-gamma in Th1/Tc1 effector cells

Using a transgenic approach, we analyzed the contribution of introns located within the IFN-gamma gene and distal regulatory regions to IFN-gamma gene expression. Intron 1 and 3 from the IFN-gamma gene displayed strong enhancer activity. This activity appeared to be dependent upon integration into the genome but resulted in a loss of Th1 selectivity. We also found that distal regulatory elements are not required for high level expression of the human IFN-gamma gene, but rather for cell lineage-specific expression. An 8.6-kb human IFN-gamma transgene was sufficient to yield high level expression but a 191-kb IFN-gamma transgene with approximately 90 kb of flanking 5' and 3' sequence was necessary to achieve both high level and Th1 selective expression of human IFN-gamma.     

Does expression of Bt toxin matter in farmer's pesticide use?

Despite the widespread adoption of Bt cotton, farmers still spray excessive pesticides in their cotton fields. In contrast to scientists who always use high quality seeds in the laboratory and/or experimental fields, farmers may plant low quality seeds with a low expression of Bt toxin. How does the expression of Bt toxin influence farmers' pesticide use? On the basis of a plot‐level survey and laboratory test data, this study shows that pesticide use on one cotton plot is influenced not only by the expression of Bt crops in this plot, but also by the average expression in the village in the early stage of the cotton growing season. In other words, high expression of Bt toxin benefits not only the farmers who plant the varieties but also all the other villagers.     

Differential expression of the chitin synthase genes of Aspergillus nidulans, chsA, chsB, and chsC, in response to developmental status and environmental factors

To understand the role of the chitin synthase genes of Aspergillus nidulans, we analyzed the expression of chsA, chsB, and chsC both by Northern blotting and by a vital reporter system with sgfp encoding a modified version of green fluorescent protein, sGFP. chsA was expressed specifically during asexual differentiation, but not during either vegetative growth or sexual differentiation. The expression of chsB was ubiquitous throughout the fungal body and relatively independent of the change in developmental status of the cells. chsC was expressed moderately during sexual development as well as during the early phase of vegetative growth, but was expressed weakly in old vegetative mycelia and in asexual structures. Furthermore its expression was spatially differentiated, i.e., relatively strong in young cleistothecia and in mature ascospores, but negligible in Hülle cells. Osmostress caused by high concentrations (up to 1.2M) of KCl or NaCl stimulated the expression of chsA and chsC, but not that of chsB. Sodium acetate, especially at high concentration (3%), strongly enhanced the expression of all the three genes. Neither heat shock nor the sugar carbon sources tested (glucose, sucrose, or lactose) affected the expression of any of the three chitin synthase genes.     

Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants

GUS (uidA) reporter gene expression for two sugarcane polyubiquitin promoters, ubi4 and ubi9, was compared to expression from the maize Ubi-1 promoter in stable transgenic rice (only ubi9) and sugarcane (ubi4 and ubi9). Ubi9 drove high-level GUS expression, comparable to the maize Ubi-1 promoter, in both callus and regenerated plants of rice transformed by Agrobacterium. This high level expression was inherited in R1 plants. Expression from ubi4 and ubi9 was quite high in sugarcane callus transformed via particle bombardment. Expression dropped to very low or undetectable levels in the resulting plants; this drop in expression resulted from PTGS. PTGS in regenerated sugarcane plants also occurred with the maize Ubi-1 promoter. In sugarcane callus, ubi4 was HS inducible, but ubi9 was not. This physiological difference corresponds to a MITE insertion that is present in the putative HSEs of ubi9 but not present in ubi4.     

High level expression of human proteins in murine hybridoma cells: induction by methotrexate in the absence of gene amplification.

We have developed an inducible system for high level expression of heterologous genes in murine hybridoma cells. The rapid induction by methotrexate (MTX) does not involve gene amplification and is controlled at the level of mRNA accumulation. Transfection was achieved by protoplast fusion with an expression vector containing the cDNA of interest and a marker gene encoding dihydrofolate reductase. The initial clones, selected at 100 nM MTX, produced high levels of the protein of interest and contained about 100-400 copies of the integrated plasmid DNA. They could adapt to a 100- to 1000-fold stepwise increase in MTX concentration in a few weeks, during which the expression of the gene of interest but not its copy number, increased several-fold. Furthermore, the induction is freely reversible. If cells were propagated in MTX-free media, the expression level decreased, but the cells could be reinduced to their original high level of expression by adding MTX back to the media. A several-fold increase in the mRNA levels of the dihydrofolate reductase and the gene of interest could be detected after induction for 18 h.     

Directed evolution of single-chain Fv for cytoplasmic expression using the beta-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation

BACKGROUND: Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli. RESULTS: We used the beta-galactosidase alpha-complementation system to monitor and evolve two antibody fragments for high expression levels in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>107 clones), clones exhibiting high levels of beta-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the beta-galactosidase activity present in the cells. CONCLUSION: This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free alpha-peptide released from the protein fusion by the host proteases. This means that the alpha-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released alpha-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein.     

Expression and Prognostic Significance of Human Epidermal Growth Factor Receptors 1, 2 and 3 in Periampullary Adenocarcinoma

Periampullary adenocarcinoma, including pancreatic cancer, is a heterogeneous group of tumours with dismal prognosis, for which there is an urgent need to identify novel treatment strategies. The human epithelial growth factor receptors EGFR, HER2 and HER3 have been studied in several tumour types, and HER-targeting drugs have a beneficial effect on survival in selected types of cancer. However, these effects have not been evident in pancreatic cancer, and remain unexplored in other types of periampullary cancer. The prognostic impact of HER-expression in these cancers also remains unclear. The aim of this study was therefore to examine the expression and prognostic value of EGFR, HER2 and HER3 in periampullary cancer, with particular reference to histological subtype. To this end, protein expression of EGFR, HER2 and HER3, and HER2 gene amplification was assessed by immunohistochemistry and silver in situ hybridization, respectively, on tissue microarrays with tumours from 175 periampullary adenocarcinomas, with follow-up data on recurrence-free survival (RFS) and overall survival (OS) for up to 5 years. EGFR expression was similar in pancreatobiliary (PB) and intestinal (I) type tumours, but high HER2 and HER3 expression was significantly more common in I-type tumours. In PB-type cases receiving adjuvant gemcitabine, but not in untreated cases, high EGFR expression was significantly associated with a shorter OS and RFS, with a significant treatment interaction in relation to OS (p_{interaction =} 0.042). In I-type cases, high EGFR expression was associated with a shorter OS and RFS in univariable, but not in multivariable, analysis. High HER3 expression was associated with a prolonged RFS in univariable, but not in multivariable, analysis. Neither HER2 protein expression nor gene amplification was prognostic. The finding of a potential interaction between the expression of EGFR and response to adjuvant chemotherapy in PB-type tumours needs validation, and merits further study.     

The role of hepatic, renal and intestinal gluconeogenic enzymes in glucose homeostasis of juvenile rainbow trout

Rainbow trout is unable to utilize high levels of dietary carbohydrates and experiences hyperglycemia after consumption of carbohydrate-rich meals. Carbohydrates stimulate hepatic glycolytic activity, but gene expression of the rate-limiting gluconeogenic enzymes glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) remains high. Although there is significant mRNA expression and activity of gluconeogenic enzymes in trout intestine and kidney, the regulation of these enzymes by diet is not known. We tested the hypothesis that dietary carbohydrate modulates intestinal and renal G6Pase, FBPase and PEPCK. Fish were either fasted or fed isocaloric carbohydrate-free (CF) or high carbohydrate (HC) diets for 14 days. As expected, fish fed HC exhibited postprandial hyperglycemia and enhanced levels of hepatic glucokinase mRNA and activity. Dietary carbohydrates had no significant effect on the expression and activity of PEPCK, FBPase and G6Pase in all three organs. In contrast, fasting enhanced the activity, but not the mRNA expression of both hepatic and intestinal PEPCK, as well as intestinal FBPase. Therefore, the activity of rate-limiting gluconeogenic enzymes in trout can be modified by fasting, but not by the carbohydrate content of the diet, potentially causing hyperglycemia when fed high levels of dietary carbohydrates. In this species consuming low carbohydrate diets at infrequent intervals in the wild, fasting-induced increases in hepatic and intestinal gluconeogenic enzyme activities may be a key adaptation to prevent perturbations in blood glucose during food deprivation. Presented in part at Experimental Biology, April 2006, San Francisco, CA [Kirchner S., Panserat S., Kaushik S. and Ferraris R. FASEB-IUPS-2006 A667.6].