ARH is a modular adaptor protein that interacts with the LDL receptor,clathrin, and AP-2

Mutations in the phosphotyrosine binding domain protein ARH cause autosomal recessive hypercholesterolemia, a disorder caused by defective internalization of low density lipoprotein receptors (LDLR) in the liver. To examine the function of ARH, we used pull-down experiments to test for interactions between ARH, the LDLR, and proteins involved in clathrin-mediated endocytosis. The phosphotyrosine binding domain of ARH interacted with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR in a sequence-specific manner. Mutations in the NPVY sequence that were previously shown to decrease LDLR internalization abolished in vitro binding to ARH. Recombinant ARH bound purified bovine clathrin with high affinity (K(D), approximately 44 nm). The interaction between ARH and clathrin was mapped to a canonical clathrin box sequence (LLDLE) in ARH and to the N-terminal domain of the clathrin heavy chain. A highly conserved 20-amino acid sequence in the C-terminal region of ARH bound the beta(2)-adaptin subunit of AP-2. Mutation of a glutamic acid residue in the appendage domain of beta(2)-adaptin that is required for interaction with the adapter protein beta-arrestin markedly reduced binding to ARH. These data are consistent with the hypothesis that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery.     

Identification of a Human Cancer Related Organ-Specific Neoantigen

Human cancers express organ-specific neoantigens (OSNs) which elicit specific cellular immune responses in the cancer patient, as demonstrated by leukocyte adherence inhibition (LAI), an in vitro immune response assay. A purified protein of MW 40,000 (p40) exhibiting OSN (colon specific) activity was cleaved into specific peptide fragments and their partial amino acid sequences determined. This information was used in the polymerase chain reaction (PCR) to obtain a 992 bp cDNA clone (PCR-992) from a human colon adenocarcinoma cell line (LS-180). By comparison of the predicted amino acid sequence of PCR-992 with the known sequence of p40 peptides, PCR-992 was shown to correspond to almost the entire coding region of p40. Nucleotide sequence analysis suggested that the protein was mycoplasmal in origin due to its high A+T content (76%) and the presence of five in frame TGA termination codons; at least two of the latter are actually read as tryptophan, a known feature of mycoplasma translation. We have confirmed this origin by direct isolation of a contaminating mycoplasma species from the LS-180 cell line and demonstration that it could be hybridized with the PCR-992 probe. Northern and PCR analysis of RNA preparations from the contaminated LS-180 cell line showed that p40 was part of the high affinity transport system operon of Mycoplasma hyorhinis (Dudler et al, EMBO J., 7: 3963-3970, 1988). Total protein lysates of Mycoplasma hyorhinis cultivated without animal cells could elicit positive LAI responses when incubated with cancer patient leukocytes but not with normal patient leukocytes. The organ-specific nature of the response was, however, not observed indicating that host cell-mycoplasmal interactions may play a role in determining the organ-specific nature of p40 seen with the LAI. The significance of these findings will be discussed in the context of previous thinking regarding the origin of OSNs.     

Molecular docking analysis of penta galloyl glucose with the bcl-2 family of anti-apoptotic targets

Apoptosis requires cellular proteins from the B-cell lymphoma 2 (BCL-2) family linked to breast cancer. Therefore, it is of interest to document the Molecular docking analysis data of penta galloyl glucose with the bcl-2 family of anti-apoptotic targets (Bcl-2, BCL-XL, Caspase 3, and Caspase 9). Data shows that Pentagalloyl glucose have optimal binding features with Bcl-2, BCL-XL, Caspase 3, and Caspase 9 proteins with binding energy of -8.6,-7,-7.5 and 4.4 kcal/mol respectively for further consideration in this context.     

Indigenous Plants Demonstrating Effective Antioxidant Properties

Biology Bulletin - Increased production of antioxidant herbal supplements utilizing traditional medicinal plants has been attributed to a high margin of economic effectiveness, environmental...     

The synthesis and structure-activity relationships of 3-amino-4-benzylquinolin-2-ones; discovery of novel KCNQ2 channel openers

3-amino-4-benzylquinolin-2-ones have been identified as a novel class of KCNQ2 channel openers. Synthesis and SAR is described along with their electrophysiological evaluation as activators of the cloned mKCNQ2 channel expressed in Xenopus laevis oocytes. The preliminary SAR data suggest the importance of both the trifluoromethylsulfonamido group and electron-withdrawing substituents on the quinolone nucleus for expression of KCNQ2 channel opening properties.     

Pruning of motor neuron branches establishes the DLM innervation pattern in Drosophila

During the Drosophila life-cycle two sets of neuromuscular junctions are generated: the embryonic/larval NMJs develop during the first half, followed by the period of metamorphosis during which the adult counterpart is generated. Development of the adult innervation pattern is preceded by a withdrawal of larval NMJs, which occurs at the onset of metamorphosis, and is followed by adult-specific motor neuron outgrowth to innervate the newly developing adult fibers. Establishment of the adult innervation pattern occurs in the context of a broader restructuring of the nervous system, which results in the development of neural circuits that are necessary to carry out behaviors specific to the adult. In this article, we follow development of the dorsal longitudinal muscle (DLM) innervation pattern through metamorphosis. We find that the initial period of motor neuron elaboration is followed by a phase of extensive pruning resulting in a threefold reduction of neuromuscular contacts. This event establishes the adult pattern of second order branching. Subsequent higher order branching from the second order "contact" points generates the characteristic multiterminal innervation pattern of the DLMs. Boutons begin to appear after the pruning phase, and are much smaller than their larval counterparts. Additionally, we demonstrate that the DLM innervation is altered in the hyperexcitable double mutant, ether a go-go Shaker, and that the phenotype is suppressed by the hypoexcitable mutant, nap(ts1). Our results demonstrate that electrical activity regulates the patterning of DLM innervation during metamorphosis.     

Transcriptional regulation of cyclooxygenase-2 in the Human Microvascular Endothelial Cell Line,HMEC-1: Control by the combinatorial actions of AP2, NF-IL-6 and CRE elements

Interleukin-1beta (IL-1) is a potent inducer of cyclooxygenase-2 (COX-2) and prostaglandin biosynthesis in many types of cells, yet little is known about the molecular mechanisms regulating IL-1 mediated prostanoid biosynthesis in the endothelium of the microvasculature. Therefore, we examined the cis- and trans-acting factors regulating IL-1-induced COX-2 expression in the human microvascular endothelial cell line, HMEC-1. IL-1 enhanced steady state levels of COX-2 protein and mRNA synthesis by approximately 2-fold which preceded a 2-fold increase in PGF(alpha) biosynthesis. Expression of a series of COX-2 promoter-luciferase constructs in IL-1 treated HMEC-1 cells revealed that the 'full length' (-1432/+59 bp) promoter was 10 times more active than the SV-40 promoter/enhancer and that it could be further activated by IL-1. Surprisingly however, all except for the shortest COX-2 promoter construct retained the ability to respond to IL-1 and luciferase activity driven by -191/+59 bp COX-2 promoter was as responsive to IL-1 as the full-length promoter. Moreover, site-directed promoter mutagenesis and electophoretic mobility shift assays (EMSA) indicate that the combinatorial actions of AP2, NF-IL6, and CRE elements are critical for both constitutive and IL-1-inducible COX-2 promoter activity. Understanding the mechanism(s) regulating COX-2 gene expression and prostaglandin biosynthesis in the microvasculature has important implications with regard to inflammation and angiogenesis in vivo.     

Pesticidal effect on soybean-rhizobia symbiosis

Summary Relative compatibility of selected pesticides at two levels of application (recommended rate and 5× or 10 ×) with soybean-rhizobia symbiosis was tested in pot culture experiments using a prepared peat inoculant.PCNB, carboxin and carboxin+captan at recommended level were innocuous to growth, nodulation, N₂-fixation and total N content of shoot. Carboxin and carboxin+captan but not PCNB at 10 times recommended level proved detrimental to nodulation and N₂-fixation. Carbaryl and malathion at recommended level had no adverse effect but at 10 times recommended level severely reduced N₂-fixation but not other parameters. Acephate, diazinon and toxaphene at both levels reduced N₂-fixation and total N content but not growth and nodulation. All five herbicides used at recommended and 5 times recommended level adversely affected nodulation and N₂-fixation. Glyphosate proved least toxic to all parameters. 2,4-DB at recommended level was less harmful to nodulation and N₂-fixation than trifluralin, alachlor and metribuzin.