Identification of agonists and antagonists of the human melanocortin-4 receptor from piperazinebenzylamines

SAR studies of a series of piperazinebenzylamines resulted in identification of potent agonists and antagonists of the human melanocortin-4 receptor. Thus, the 1,2,3,4-tetrahydroisoquinolin-1-ylacetyl compound 12e and the quinolin-3-ylcarbonyl analogue 12l possessed K(i) values of 6.3 and 4.5 nM, respectively. Interestingly, 12e was a full agonist with an EC(50) value of 31 nM, and 12l was a weak partial agonist (IA=17%) and functioned as an antagonist (IC(50)=300 nM).     

Alterations in redox homeostasis and prostaglandins impair endothelial-dependent vasodilation in euglycemic autoimmune nonobese diabetic mice

We report herein the novel observation that alterations in oxidant/antioxidant balance are evident and cause vascular dysfunction in aortae of prediabetic nonobese-diabetic mice (NOD). We found that nitrotyrosine, a biochemical marker of oxidant stress, was higher in the NOD aortae when compared to age-matched non-autoimmune BALB/c controls or the diabetes-resistant NOD congenic strain, NOD.Lc7. The oxidant stress was localized to the intimal and medial layers, and endothelium-dependent relaxation to acetylcholine was decreased in isolated aortic rings from NOD mice. Inhibition of nitric oxide synthesis caused an endothelium-dependent contraction, and treatment with either a selective thromboxane A2/prostaglandin H2 receptor antagonist or a non-isozyme-specific cyclooxygenase inhibitor reversed this effect. Aortic rings from NOD.Lc7 did not display the paradoxical vasoconstriction. Furthermore, the vascular dysfunction was caused by oxidative stress, as treatment with a superoxide dismutase mimetic in vivo or with native antioxidant enzymes ex vivo inhibited the tissue oxidant stress and restored endothelium-dependent relaxation. Endothelial function was also restored by the inhibitors of NAD(P)H oxidase, diphenylene iodonium or apocynin. Our studies indicate that an oxidant stress that occurs prior to the onset of diabetes in this mouse model contributes to endothelial dysfunction independently of overt diabetes.     

In vitro and in vivo modulation of vascular barrier integrity by sphingosine 1-phosphate: mechanistic insights

Sphingosine 1-phosphate (S1P), a biologically active lipid growth factor, induces robust endothelial cell activation resulting in cellular locomotion, vascular maturation and angiogenesis. Recent work by our laboratory has demonstrated S1P to enhance the cellular barrier function of the vascular endothelium. S1P-induced modulation of vascular permeability is effected through profound cytoskeletal reorganization initiated by cell surface receptor-mediated G protein activation and downstream signaling via the Rho family of small GTPases. The details of the downstream signaling mechanism remain an active area of in vitro investigation. Translational investigation suggests a profound impact of S1P administration in the modulation of edema formation in disease state manifest as acute inflammatory lung injury in which increased vascular permeability is a hallmark feature. These data support an exciting potential therapeutic role for S1P in vascular barrier enhancement necessary for the treatment of critically ill patients.     

Recent Hits Acquired by BLAST (ReHAB): A tool to identify new hits in sequence similarity searches

Background  

Sequence similarity searching is a powerful tool to help develop hypotheses in the quest to assign functional, structural and evolutionary information to DNA and protein sequences. As sequence databases continue to grow exponentially, it becomes increasingly important to repeat searches at frequent intervals, and similarity searches retrieve larger and larger sets of results. New and potentially significant results may be buried in a long list of previously obtained sequence hits from past searches.     

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Background  

Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.     

Rapid cryopreservation of five mammalian and one mosquito cell line at −80°C while attached to flasks in a serum free cryopreservative

Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at −80°C while attached to the substratum of 25cm² tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines.     

The post-translational modifications of the nuclear encoded subunits of complex I from bovine heart mitochondria

Bovine complex I is an assembly of 46 different proteins. Seven of them are encoded in mitochondrial DNA, and the rest are nuclear gene products that are imported into the organelle. Fourteen of the nuclear encoded subunits have modified N termini. Many of these post-translational modifications have been deduced previously from intact protein masses. These assignments have been verified by mass spectrometric analysis of peptides. Thirteen of them are N-alpha-acetylated, and a 14th, subunit B18, is N-alpha-myristoylated. Subunit B18 forms part of the membrane arm of the complex, and the myristoyl group may attach subunit B18 to the membrane. One subunit, B12, has a particularly complex pattern of post-translational modification that has not been analyzed before. It is a mixture of the N-alpha-acetylated form and the form with a free N terminus. In addition, it has one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations. The predominant form is methylated on residues 4 and 6. There is no evidence for the methylation of histidine 2. Subunit B12 is also part of the membrane arm of complex I, and it probably spans the membrane once, but as its orientation is not known, the methylation sites could be in either the matrix or the intermembrane space. These experiments represent another significant step toward establishing the precise chemical composition of mammalian complex I.     

1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome

Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.     

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.