Alpha-1, alpha-2, and beta adrenergic signal transduction in cultured uterine myocytes

Summary The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm² flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10⁻⁸ M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [³H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle. This research was supported by grant HD-22063 from the National Institutes of Health, Bethesda, MD.     

Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.     

Mutations in the heatshock cognate 70 protein (hsc4) modulate Notch signaling

In our effort to dissect the Notch signaling mechanism we have conducted a screen for mutations that reduce Notch signaling activity. We recovered nine complementation groups as modifiers of the hypomorphic Notch allele notchoid. Apart from the known Notch signaling modulators Notch, Delta and mastermind we isolated alleles in vestigial, wingless, scalloped and clipped, genes known to affect wing morphogenesis. In addition, we identified mutations in Bag, the gene encoding clathrin heavy chain and a dominant mutation of the cytosolic 70 kDa heatshock cognate protein encoded by the hsc4 gene, as Notch signaling modifier. We focused our attention on the latter mutation because it displays dramatic genetic interactions with mutations of the Notch receptor as well as several additional Notch signaling pathway elements. We discuss how hsc4, a gene thought to be involved in subcellular trafficking, may affect the number of functional Notch receptors on the cell surface.     

Computer aided screening and evaluation of herbal therapeutics against MRSA infections

Methicillin resistant Staphylococcus aureus (MRSA), a pathogenic bacterium that causes life threatening outbreaks such as community-onset and nosocomial infections has emerged as 'superbug'. The organism developed resistance to all classes of antibiotics including the best known Vancomycin (VRSA). Hence, there is a need to develop new therapeutic agents. This study mainly evaluates the potential use of botanicals against MRSA infections. Computer aided design is an initial platform to screen novel inhibitors and the data finds applications in drug development. The drug-likeness and efficiency of various herbal compounds were screened by ADMET and docking studies. The virulent factor of most of the MRSA associated infections are Penicillin Binding Protein 2A (PBP2A) and Panton-Valentine Leukocidin (PVL). Hence, native structures of these proteins (PDB: 1VQQ and 1T5R) were used as the drug targets. The docking studies revealed that the active component of Aloe vera, β-sitosterol (3S, 8S, 9S, 10R, 13R, 14S, 17R) -17- [(2R, 5R)-5-ethyl-6-methylheptan-2-yl] -10, 13-dimethyl 2, 3, 4, 7, 8, 9, 11, 12, 14, 15, 16, 17- dodecahydro-1H-cyclopenta [a] phenanthren-3-ol) showed best binding energies of -7.40 kcal/mol and -6.34 kcal/mol for PBP2A and PVL toxin, respectively. Similarly, Meliantriol (1S-1-[ (2R, 3R, 5R)-5-hydroxy-3-[(3S, 5R, 9R, 10R, 13S, 14S, 17S)-3-hydroxy 4, 4, 10, 13, 14-pentamethyl-2, 3, 5, 6, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta[a] phenanthren-17-yl] oxolan-2-yl] -2- methylpropane-1, 2 diol), active compound in Azadirachta indica (Neem) showed the binding energies of -6.02 kcal/mol for PBP2A and -8.94 for PVL toxin. Similar studies were conducted with selected herbal compound based on pharmacokinetic properties. All in silico data tested in vitro concluded that herbal extracts of Aloe-vera, Neem, Guava (Psidium guajava), Pomegranate (Punica granatum) and tea (Camellia sinensis) can be used as therapeutics against MRSA infections.     

Junker: An Intergenic Explorer for Bacterial Genomes

In the past few decades, scientists from all over the world have taken a keen interest in novel functional units such as small regulatory RNAs, small open reading frames, pseudogenes, transposons, integrase binding attB/attP sites, repeat elements within the bacterial intergenic regions (IGRs) and in the analysis of those junk regions for ge- nomic complexity. Here we have developed a web server, named Junker, to facilitate the in-depth analysis of IGRs for examining their length distribution, four-quadrant...     

Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators

DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.     

Identification of aspirin and diclofenac binding proteins in the red complex pathogens

Red complex organisms are a group of organisms (Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35405, Tannerella forsythia ATCC 43037) that have been identified for the causation of periodontal diseases. Aspirin and diclofenac have been used as regular analgesics. Therefore, it is of interest to document the identification of aspirin and diclofenac binding proteins in the red complex pathogens using the STITCH v.5 pipeline. The virulence properties of these proteins were analyzed using VICMPred and VirulentPred software. Thus, we document 000 number of proteins having optimal binding features with the known analgesics.     

Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide

Human neutrophil lysosomal cathepsin G (cat G) exerts broad-spectrum antibacterial action in vitro against Gram-negative and -positive bacteria independent of its serine protease activity. We recently determined that an internal peptide of cat G (HPQYNQR), obtained after digestion of cat G with clostripain, possessed broad-spectrum antibacterial action in vitro, displaying an ED50 of 5 x 10(-5) M. In order to evaluate the structure-antibacterial properties of this peptide, synthetic variants with single alanine substitutions at each position were prepared and tested for antibacterial action. We found that alanine substitution for His-1 or Tyr-4, or certain modifications of the His-1 side chain, produced nonbactericidal peptides. A hexapeptide lacking the COOH-terminal Arg-7 but not a pentapeptide lacking both Gln-6 and Arg-7 possessed in vitro bactericidal activity. Interestingly, the cat G bactericidal peptide displays similarity to sequences within other serine proteases, notably the proposed cytotoxic granzymes present in the cytolytic granules of human and mouse cytotoxic T lymphocytes. We now report that an internal peptide of one human granzyme (granzyme B) with the sequence of HPAYNPK also displays bactericidal action in vitro. Our results suggest that an internal antibacterial domain among human serine proteases cat G and granzyme B has been functionally conserved through evolution perhaps for the purpose of host defense against microbial pathogens and targets of cytotoxic T lymphocyte killing.     

An extra cysteine proximal to the transmembrane domain induces differential cross-linking of p185neu and p185neu.

The neu proto-oncogene encodes a receptor tyrosine kinase (p185) that is closely related to the epidermal growth factor receptor. It has been proposed that receptor tyrosine kinases are activated through oligomerization. Because this clustering model predicts that oligomerization of receptors is sufficient to activate them, we determined if p185 can be activated by introducing an extra cysteine proximal to the transmembrane domain. This should induce inter-receptor disulfide bonding and, according to the clustering model, activate the receptor. This amino acid substitution enhanced recovery of both normal and transforming neu proteins as dimers, with normal p185 recovered predominantly as monomers and transforming p185* as dimers. However, the cysteine substitution did not affect the transforming activity of the two proteins.