Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon

Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.     

Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties.     

Two distinct heme distal site states define Cerebratulus lacteus mini-hemoglobin oxygen affinity

The nerve tissue hemoglobin of Cerebratulus lacteus (CerHb) is the smallest naturally occurring known hemoglobin. Stabilization of the diatomic bound species (e.g., O(2)) is achieved through a network of hydrogen bonds based on three key residues TyrB10, GlnE7, and ThrE11. The first two residues are typically associated in hemoglobins with enhanced O(2) affinity, related to hydrogen bond stabilization of the heme-bound O(2) resulting in a decrease of the ligand dissociation rates. In contrast to the above observations, the affinity of CerHb for O(2) is only moderate, and the rate of O(2) dissociation is unexpectedly high. To gain insight on the diverse molecular mechanisms controlling ligand affinities, we have analyzed w.t. CerHb and its ThrE11-->Val mutant by means of joint molecular dynamics and quantum mechanics simulation techniques, complementing recent site-directed mutagenesis experiments. Our results suggest that the observed O(2) dissociation rates can only be explained through a dynamic equilibrium between high and low affinity states of the w.t. CerHb heme distal site.     

Nitrogen Cycling and Mass Balance for a Forested Catchment in the Italian Alps. Assessment of Nitrogen Status

During 1999–2001 the chemical composition and fluxes were measured in rainfall, throughfall, soil solution and stream water in a remote forested site in the Italian Alps. The analysis of temporal patterns revealed the differential behaviour of nitrogen and sulphur and suggested that different mechanisms controlled their flux. No important changes in sulphate concentration and fluxes emerged as the solution passed through the various components of the forest ecosystem, and temporal variations of SO₄ in the soil solution and stream were likely driven by the physical process of dilution. The availability of nitrate and ammonia, by contrast, was drastically reduced as throughfall water entered the soil and passed through the mineral layers, irrespective of season. The calculated hydrochemical budget based on throughfall and soil solution N fluxes revealed that ~80% N retention in the forest soil, corresponding to 12 kg ha⁻¹ yr⁻¹, despite a relatively high N deposition loading (15 kg ha⁻¹ yr⁻¹). Most of the leached nitrogen (90%) was in the organic form. Indicators of the N status of this ecosystem, such as C/N ratio in solid and solution phase of the soil and N foliage content as well as land use history were examined. Despite the strong N retention in the forested part of the catchment, the stream water N–NO₃ levels were consistently above 10 μg l⁻¹ suggesting that the Val Masino catchment as a whole was less efficient in processing atmospheric N inputs. This contrasting N behaviour illustrates the role of landscape features, such as the soil cover and vegetation type, that is characteristic of an alpine catchment.     

Positive reinforcement training affects hematologic and serum chemistry values in captive chimpanzees (Pan troglodytes)

Positive reinforcement training (PRT) techniques have received considerable attention for their stress reduction potential in the behavioral management of captive nonhuman primates. However, few published empirical studies have provided physiological data to support this position. To address this issue, PRT techniques were used to train chimpanzees (Pan troglodytes) to voluntarily present a leg for an intramuscular (IM) injection of anesthetic. Hematology and serum chemistry profiles were collected from healthy chimpanzees (n=128) of both sexes and various ages during their routine annual physical examinations over a 7-year period. Specific variables potentially indicative of acute stress (i.e., total white blood cell (WBC) counts, absolute segmented neutrophils (SEG), glucose (GLU) levels, and hematocrit (HCT) levels) were analyzed to determine whether the method used to administer the anesthetic (voluntary present for injection vs. involuntary injection) affected the physiological parameters. Subjects that voluntarily presented for an anesthetic injection had significantly lower mean total WBC counts, SEG, and GLU levels than subjects that were involuntarily anesthetized by more traditional means. Within-subjects analyses revealed the same pattern of results. This is one of the first data sets to objectively demonstrate that PRT for voluntary presentation of IM injections of anesthetic can significantly affect some of the physiological measures correlated with stress responses to chemical restraint in captive chimpanzees.     

Cyanide binding and heme cavity conformational transitions in Drosophila melanogaster hexacoordinate hemoglobin

The reason for the presence of hemoglobin-like molecules in insects, such as Drosophila melanogaster, that live in fully aerobic environments has yet to be determined. Heme endogenous hexacoordination (where HisE7 and HisF8 axial ligands to the heme Fe atom are both provided by the protein) is a recently discovered mechanism proposed to modulate O(2) affinity in hemoglobins from different species. Previous results have shown that D. melanogaster hemoglobin 1 (product of the glob1 gene) displays heme endogenous hexacoordination in both the ferrous and ferric states. Here we present kinetic data characterizing the exogenous cyanide ligand binding process, and the three-dimensional structure (at 1.4 A resolution) of the ensuing cyano-met D. melanogaster hemoglobin. Comparison with the crystal structure of the endogenously hexacoordinated D. melanogaster hemoglobin shows that the transition to the cyano-met form is supported by conformational readjustment in the CD-D-E region of the protein, which removes HisE7 from the heme. The structural and functional features of D. melanogaster hemoglobin are examined in light of previous results achieved for human and mouse neuroglobins and for human cytoglobin, which display heme endogenous hexacoordination. The study shows that, despite the rather constant value for cyanide association rate constants for the ferric hemoproteins, different distal site conformational readjustments and/or heme sliding mechanisms are displayed by the known hexacoordinate hemoglobins as a result of exogenous ligand binding.