The modular structure of haemagglutinin/adhesin regions in gingipains of Porphyromonas gingivalis

High-molecular-weight arginine- and lysine-specific (Kgp) gingipains are essential virulence factors expressed by the oral pathogen Porphyromonas gingivalis. Haemagglutinin/adhesin (HA) regions of these proteases have been implicated in targeting catalytic domains to biological substrates and in other adhesive functions. We now report the crystal structure of the K3 adhesin domain/module of Kgp, which folds into the distinct β-jelly roll sandwich topology previously observed for K2. A conserved structural feature of K3, previously observed in the Kgp K2 module, is the half-way point anchoring of the surface exposed loops via an arginine residue found in otherwise highly variable sequences. Small-angle X-ray scattering data for the recombinant construct K1K2K3 confirmed a structure comprising a tandem repeat of three homologous modules, K1, K2 and K3 while also indicating an unusual 'y'-shape arrangement of the modules connected by variable linker sequences. Only the K2 and K3 modules and a K1K2 construct were observed to be potently haemolytic. K2, K3 and the K1K2 construct showed preferential recognition of haem-albumin over albumin whereas only low affinity binding was detected for K1 and the K1K2K3 construct. The data indicate replication of some biological functions over the three adhesin domains of Kgp while other functions are restricted.     

2-36[K4,RYYSA(19-23)]PP a novel Y5-receptor preferring ligand with strong stimulatory effect on food intake

Members of the neuropeptide Y (NPY) family regulate many physiological processes via interaction with at least four functional, pharmacologically distinct Y-receptors. However, selective antagonists developed for several subtypes have not been useful in defining particular Y-receptor functions in vivo. To identify critical residues within members of the NPY family required for Y-receptor subtype-selectivity we have determined the contribution of each residue within NPY to receptor binding by replacing them with L-alanine. In a second study, chimeric peptides where single or stretches of residues were interchanged between members of the NPY family were generated and tested in radioligand binding studies. Overall, substituted alanine analogues exhibited similar orders of affinities at each Y-receptor subtype with no obvious subtype-selectivity. Residues of particular interest are Leu30 which exhibited selectivity for the Y4-receptor, whereas Asp16 does not appear to play any role in ligand binding. Several chimeric peptides, e.g., [K4]pancreatic polypeptide ([K4]PP) and [RYYSA(19-23)]PP clearly showed higher affinity at the Y4 and Y5 subtypes compared to the Y1 and Y2 subtypes. In addition, the transfer of a proline residue from position 14 to 13 in peptide YY decreases its affinity at the Y1-, Y4- and Y5-receptors but is unchanged at the Y2 subtype. Combining these results, and with the help of molecular modelling, second generation chimeras were designed. The most significant improvement was achieved in chimera 2-36[K4,RYYSA(19-23)]PP where the affinity for the Y5 subtype increased by ninefold over that from NPY. Several of these compounds were also tested for their ability to stimulate food intake in a rat model. Interestingly, again 2-36[K4,RYYSA(19-23)]PP showed the most dramatic effect with a major increase on food intake over a range of doses compared to NPY suggesting a possible synergistic effect of several Y-receptors on feeding behaviour.     

Quantifying DNA-protein interactions by double-stranded DNA arrays.

We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA-protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2'-deoxyribonucleoside 5'-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with DpnI, which cleaves when its recognition site is methylated. This digestion demonstrated that the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.     

Two-bond C-C spin-coupling constants in carbohydrates: effect of structure on coupling magnitude and sign

An empirical projection method is described to predict the magnitudes and signs of two-bond ¹³C-¹³C spin-coupling constants (²J_CC) in aldopyranosyl rings. The method has been applied primarily to the interpretation of ²J_CCC values, although the behavior of ²J_COC has also been examined in light of the new approach, producing results which may prove useful in the conformational analysis of O-glycosidic linkages in oligosaccharides. High-level ab initio calculations of ²J_CC values in model compounds were found to be in agreement with the predictions.     

Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.     

Targeted delivery of NRASQ61R and Cre-recombinase to post-natal melanocytes induces melanoma in Ink4a/Arflox/lox mice

We have developed a somatic cell gene delivery mouse model of melanoma that allows for the rapid validation of genetic alterations identified in this disease. A major advantage of this system is the ability to model the multi-step process of carcinogenesis in immune-competent mice without the generation and cross breeding of multiple strains. We have used this model to evaluate the role of RAS isoforms in melanoma initiation in the context of conditional Ink4a/Arf loss. Mice expressing the tumor virus A (TVA) receptor specifically in melanocytes under control of the dopachrome tautomerase (DCT) promoter were crossed to Ink4a/Arf^lox/lox mice and newborn DCT-TVA/Ink4a/Arf^lox/lox mice were injected with retroviruses containing activated KRAS, NRAS and/or Cre-recombinase. No mice injected with viruses containing KRAS and Cre or NRAS alone developed tumors; however, more than one-third of DCT-TVA/Ink4a/Arf^lox/lox mice injected with NRAS and Cre viruses developed melanoma and two-thirds developed melanoma when NRAS and Cre expression was linked.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

Melanoma central nervous system metastases: An update to approaches,challenges, and opportunities

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.     

Effect of hypercapnia on intracellular pH regulation in a rainbow trout hepatoma cell line,RTH 149

Fish exposed to elevated water CO₂ experience a rapid increase in blood CO₂ levels (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. While the mechanisms associated with extracellular pH regulation have been well explored, much less is known about intracellular pH (pH_i) regulation. There is great interest in developing non-animal models for research. One such model is the rainbow trout hepatoma cell line (RTH 149), which has been used to study a wide range of topics; however, no studies have investigated its potential use in pH_i regulation. Employing the pH-sensitive fluoroprobe BCECF, the present study examined pH_i regulation in RTH 149 under normocapnia and during extracellular acidification induced by either elevated CO₂ or 1 M HCl. During exposure to hypercapnia, RTH 149 cells were acidified without recovery as long as the elevated CO₂ was maintained. In addition, rates of pH_i recovery from NH₄Cl-induced acidosis were significantly lower in cells exposed to hypercapnia or HCl compared to that in normocapnic cells, indicating that elevated CO₂ indirectly impeded pH_i recovery through a reduction in pH_e and/or pH_i. Moreover, pH_i regulation in RTH 149 was EIPA-sensitive, suggesting that an NHE may be involved. Overall, RTH 149 may have the potential for identifying transporters likely to play a role in pH_i regulation in fish. However, it should not be used as a complete replacement for in vivo studies, especially to quantify acid–base regulatory ability at whole animal level, since RTH 149 appeared to have enhanced pH_i recovery rates relative to primary hepatocytes.