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By Daniel C. Liebler Totowa, NJ:Humana Press, Inc., 2002. 198 pp. ISBN: 0 89603-991-9, $49.50 cloth. ISBN: 0-89603-992-7, $29.50 paper The so-called New Biology is really a fresh approach to [...]

Introduction: The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUPTFII- interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer. Results: FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by at RA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC- 1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade. Conclusions: Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII. [ABSTRACT FROM AUTHOR]

The article presents a case study of how the University of Louisville (UofL) became a leader in technology transfer. It mentions that the university became indulged with the industry through its start-up pharmaceutical company Pradama Inc. which was rooted in the research done by the faculty. It also cites the beginning of the university's research profile which started in the 1980. The issue on financial capability of the university's research center is also presented.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive decline in multiple cognitive domains. Its pathological hallmarks include senile plaques and neurofibrillary tangles. Mild cognitive impairment (MCI) is the earliest detectable stage of AD with limited symptomology and no dementia. The yearly conversion rate of patients from MCI to AD is 10–15%, although conversion back to normal is possible in a small percentage. Early diagnosis of AD is important in an attempt to intervene or slow the advancement of the disease. Early AD (EAD) is a stage following MCI and characterized by full-blown dementia; however, information involving EAD is limited. Oxidative stress is well-established in MCI and AD, including protein oxidation. Protein nitration also is an important oxidative modification observed in MCI and AD, and proteomic analysis from our laboratory identified nitrated proteins in both MCI and AD. Therefore, in the current study, a proteomics approach was used to identify nitrated brain proteins in the inferior parietal lobule from four subjects with EAD. Eight proteins were found to be significantly nitrated in EAD: peroxiredoxin 2, triose phosphate isomerase, glutamate dehydrogenase, neuropolypeptide h3, phosphoglycerate mutase1, H+– transporting ATPase, α-enolase and fructose-1,6-bisphosphate aldolase. Many of these proteins are also nitrated in MCI and late-stage AD, making this study the first to our knowledge to link nitrated proteins in all stages of AD. These results are discussed in terms of potential involvement in the progression of this dementing disorder. [ABSTRACT FROM AUTHOR]

The article discusses a study which assessed that 2-Amino-3,8-dimethylmidazo-[4,5-f]quinoxaline-induced DNA damage and subsequent mutagenesis would be faster than NAT2 acetylators using nucleotide excision repair-deficient Chinese hamster ovary cells expressing human alleles, p4501Aa1 and either NAT2*4 and NAT2*5B. Through the measurement of catalytic activity, transfection of human alleles were confirmed. High performance liquid chromatography was used to assay acetyltransferases.

Diabetic cardiomyopathy is a common complication leading to heightened risk of heart failure and death. In the present report, we performed proteomic analysis on total cardiac proteins from the OVE26 mouse model of type 1 diabetes to identify protein changes that may contribute to diabetic cardiomyopathy. This analysis revealed that a surprising high proportion (12 of 20) of the altered proteins that could be identified by mass spectrometry were of mitochondrial origin. All but one of these proteins were upregulated by diabetes. Quantitative RT-PCR, performed for two of these proteins, indicated that part of the upregulation was attributed to increased messenger RNA levels. Morphological study of diabetic hearts showed significantly increased mitochondrial area and number as well as focal regions with severe damage to mitochondria. Diabetic mitochondria also showed reduced respiratory control ratio (9.63 ± 0.20 vs. 6.13 ± 0.41, P < 0.0001), apparently due to reduced state 3 rate, and diminished GSH level (5.5 ± 0.9 vs. 8.2 ± 2.5 µmol/mg protein, P < 0.05), indicating impaired mitochondrial function and increased oxidative stress. Further examination revealed increased mitochondrial DNA (1.03 ± 0.18 vs. 0.69 ± 0.13 relative copy number, P < 0.001) and a tendency to higher protein yield in OVE26 cardiac mitochondria, as well as increased mRNA level for mitochondrial transcription factor A and two mitochondrial encoded proteins. Taken together, these results show that mitochondria are a primary target in the diabetic heart, probably due to oxidative stress, and that this damage coincides with and may stimulate mitochondrial biogenesis. [ABSTRACT FROM AUTHOR]

Humans are exposed to arylamine carcinogens such as 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MelQx) 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) and 4-aminobiphenyl (ABP) in diet and cigarette smoke. Bioactivation includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in MelQx-, IQ-, and ABP-induced mutagenesis and DNA damage, nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. MelQx, IQ, and ABP each caused significantly (p<0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the CYP1A2/NAT2*4 acetylator cell line compared to the CYP1A2, CYP1A2/NAT2*5B, and untransfected cell lines. For all three carcinogens, induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs, dG-C8-MelQx, dG-C8-ABP, and dG-C8-IQ adduct levels were identified and quantified by liquid chromatography-mass spectrometry and were each significantly greater in the rapid NAT2*4 acetylator cell line following treatment with MelQx, ABP, and IQ as compared to all other cell lines. Partially supported by USPHS grant CA034627 and a grant from Philip Morris, USA. [ABSTRACT FROM AUTHOR]

To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris. The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene. A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification. In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins. Radioligand binding assays demonstrated that the CB2 receptors expressed in P. pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems. Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor. ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P. pastoris for purification. [Copyright &y& Elsevier]