Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies: Src kinase as a model system

High-throughput screening (HTS), a major component of lead identification, often utilizes fluorescence-based assay technologies. For example, HTS kinase assays are formatted using a variety of fluorescence-based assay technologies including, but not limited to, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP). These assays offer tremendous advantages such as a nonradioactive format, ease of automation, and excellent reproducibility. Fluorescence-based assays frequently used for lead identification can also be useful for structure activity relationship (SAR) studies during lead optimization. An important issue when assessing an assay to be used for SAR is the ability of the assay to discriminate high-affinity small molecule inhibitors (pM-nM) from low-affinity inhibitors (microM-mM). The purpose of this study was to utilize HTS-friendly assay formats for SAR by developing TR-FRET, FP, and DELFIAassays measuring Src kinase activity and to define the theoretical lower limit of small molecule inhibitor detection achievable with these assay formats. The authors show that 2 homogeneous assay formats, TR-FRET and FP, allowed for the development of Src kinase assays with a lower limit of detection of K(i) = 0.01 nM. This study indicates that assay technologies typically used for HTS can be used during lead optimization by providing quantitative measurements of compound activity critical to driving SAR studies.     

Still Heart Encodes a Structural HMT,SMYD1b,with Chaperone-Like Function during Fast Muscle Sarcomere Assembly

The vertebrate sarcomere is a complex and highly organized contractile structure whose assembly and function requires the coordination of hundreds of proteins. Proteins require proper folding and incorporation into the sarcomere by assembly factors, and they must also be maintained and replaced due to the constant physical stress of muscle contraction. Zebrafish mutants affecting muscle assembly and maintenance have proven to be an ideal tool for identification and analysis of factors necessary for these processes. The still heart mutant was identified due to motility defects and a nonfunctional heart. The cognate gene for the mutant was shown to be smyd1b and the still heart mutation results in an early nonsense codon. SMYD1 mutants show a lack of heart looping and chamber definition due to a lack of expression of heart morphogenesis factors gata4, gata5 and hand2. On a cellular level, fast muscle fibers in homozygous mutants do not form mature sarcomeres due to the lack of fast muscle myosin incorporation by SMYD1b when sarcomeres are first being assembled (19hpf), supporting SMYD1b as an assembly protein during sarcomere formation.     

An Algorithm that Predicts the Viability and the Yield of Human Hepatocytes Isolated from Remnant Liver Pieces Obtained from Liver Resections

Isolated human primary hepatocytes are an essential in vitro model for basic and clinical research. For successful application as a model, isolated hepatocytes need to have a good viability and be available in sufficient yield. Therefore, this study aims to identify donor characteristics, intra-operative factors, tissue processing and cell isolation parameters that affect the viability and yield of human hepatocytes. Remnant liver pieces from tissue designated as surgical waste were collected from 1034 donors with informed consent. Human hepatocytes were isolated by a two-step collagenase perfusion technique with modifications and hepatocyte yield and viability were subsequently determined. The accompanying patient data was collected and entered into a database. Univariate analyses found that the viability and the yield of hepatocytes were affected by many of the variables examined. Multivariate analyses were then carried out to confirm the factors that have a significant relationship with the viability and the yield. It was found that the viability of hepatocytes was significantly decreased by the presence of fibrosis, liver fat and with increasing gamma-glutamyltranspeptidase activity and bilirubin content. Yield was significantly decreased by the presence of liver fat, septal fibrosis, with increasing aspartate aminotransferase activity, cold ischemia times and weight of perfused liver. However, yield was significantly increased by chemotherapy treatment. In conclusion, this study determined the variables that have a significant effect on the viability and the yield of isolated human hepatocytes. These variables have been used to generate an algorithm that can calculate projected viability and yield of isolated human hepatocytes. In this way, projected viability can be determined even before isolation of hepatocytes, so that donors that result in high viability and yield can be identified. Further, if the viability and yield of the isolated hepatocytes is lower than expected, this will highlight a methodological problem that can be addressed.     

Amyloid Beta Precursor Protein and Prion Protein Have a Conserved Interaction Affecting Cell Adhesion and CNS Development

Genetic and biochemical mechanisms linking onset or progression of Alzheimer Disease and prion diseases have been lacking and/or controversial, and their etiologies are often considered independent. Here we document a novel, conserved and specific genetic interaction between the proteins that underlie these diseases, amyloid-β precursor protein and prion protein, APP and PRP, respectively. Knockdown of APP and/or PRNP homologs in the zebrafish (appa, appb, prp1, and prp2) produces a dose-dependent phenotype characterized by systemic morphological defects, reduced cell adhesion and CNS cell death. This genetic interaction is surprisingly exclusive in that prp1 genetically interacts with zebrafish appa, but not with appb, and the zebrafish paralog prp2 fails to interact with appa. Intriguingly, appa & appb are largely redundant in early zebrafish development yet their abilities to rescue CNS cell death are differentially contingent on prp1 abundance. Delivery of human APP or mouse Prnp mRNAs rescue the phenotypes observed in app-prp-depleted zebrafish, highlighting the conserved nature of this interaction. Immunoprecipitation revealed that human APP and PrP^C proteins can have a physical interaction. Our study reports a unique in vivo interdependence between APP and PRP loss-of-function, detailing a biochemical interaction that considerably expands the hypothesized roles of PRP in Alzheimer Disease.     

Noduler, a novel immune up-regulated protein mediates nodulation response in insects

Insect immune system comprises of both humoral and cellular defenses. Nodulation is one of the major, yet very poorly understood cellular responses against microbial infections in insects. Through screening for novel immune genes from an Indian saturniid silkmoth Antheraea mylitta, we identified a protein up-regulated in hemolymph within minutes upon bacterial challenge. We have shown here, for first time, the involvement of this novel protein in mediating nodulation response against bacteria and hence designated it as Noduler. Noduler possessed a characteristic reeler domain found in several extracellular matrix vertebrate proteins. Noduler was shown in vitro to bind a wide range of bacteria, yeast, and also insect hemocytes. Furthermore, Noduler specifically bound LPS, lipotechoic acid, and beta-1, 3 glucan components of microbial cell walls. RNA-interference mediated knock-down of the Noduler resulted in significant reduction in the number of nodules and consequent increase in bacterial load in larval hemolymph. The results suggest that the Noduler is widely conserved and is involved in very early clearance of bacteria by forming nodules of hemocytes and bacterial complexes in insects. The results would promote further studies for understanding of the crucial but hitherto overlooked nodulation mechanism in insects and also provide cues for the study of similar mammalian proteins whose function is not understood.     

Serum Selenium and Lead Levels: a Possible Link with Diabetes and Associated Proteinuria

Biological Trace Element Research - The study assessed trace element selenium (Se) and a heavy metal lead (Pb) in patients with type II diabetes mellitus (T2DM) and its associated proteinuria....     

2-Aminoimidazoles inhibitors of TGF-β receptor 1

The 4-(5-fluoro-6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-1H-imidazol-2-ylamine 3 is a potent and selective inhibitor of TGF-βR1. Substitution of the amino group of 3 typically led to a slight decrease in the affinity for the receptor and in TGF-β-inducted PAI-luciferase reporter activity. However, 2-acetamidoimidazoles were identified as attractive candidates for further optimization as a result of their significant activity combined to their superior pharmacokinetic profile.     

Ornithine-urea cycle and urea synthesis in African lungfishes, Protopterus aethiopicus and Protopterus annectens, exposed to terrestrial conditions for six days

The objectives of this study were (1) to determine the type of carbamoyl phosphate synthetase (CPS) present, and the compartmentalization of arginase, in the livers of the African lungfishes, Protopterus aethiopicus and Protopterus annectens, and (2) to elucidate if these two lungfishes were capable of increasing the rates of urea synthesis and capacities of the ornithine-urea cycle (OUC) during 6 days of aerial exposure without undergoing aestivation. Like another African lungfish, Protopterus dolloi, reported elsewhere, the CPS activities from the livers of P. aethiopicus and P. annectens had properties similar to that of the marine ray (Taeniura lymma), but dissimilar to that of the mouse (Mus musculus). Hence, they possessed CPS III, and not CPS I as reported previously. CPS III was present exclusively in the liver mitochondria of both lungfishes, but the majority of the arginase activities were present in the cytosolic fractions of their livers. Glutamine synthetase (GS) activity was also detected in the hepatic mitochondria of both specimens. Therefore, our results suggest that the evolution of CPS III to CPS I might not have occurred before the evolution of extant lungfishes as suggested previously, prompting an examination of the current view on the evolution of CPS and OUC in vertebrates. Aerial exposure led to significant decreases in rates of ammonia excretion in P. aethiopicus and P. annectens, but there were no accumulations of ammonia in their tissues. However, urea contents in their tissues increased significantly after 6 days of aerial exposure. The estimated rates of urea synthesis in P. aethiopicus and P. annectens increased 1.2- and 1.47-fold, respectively, which were smaller than that in P. dolloi (8.6-fold) reported elsewhere. In addition, unlike P. dolloi, 6 days of aerial exposure had no significant effects on the hepatic CPS III activities of P. aethiopicus and P. annectens. In contrast, aerial exposure induced relatively greater degrees of reductions in ammonia production in P. aethiopicus (34%) and P. annectens (37%) than P. dolloi (28%) as previously reported. Thus, our results suggest that various species of African lungfishes respond to aerial exposure differently with respect to nitrogen metabolism and excretion, and it can be concluded that P. aethiopicus and P. annectens depended more on reductions in ammonia production than on increases in urea synthesis to ameliorate ammonia toxicity when exposed to terrestrial conditions.