Multi-residue analysis of eight anticoagulant rodenticides in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry

The determination of C-terminal peptide sequence is critical since the C-terminal peptide contains biologically relevant information and often undergoes post-translational processing. Another important application is in estimating purity of the biopharmaceuticals, especially for determining the presence of ragged processed ends and for N-terminally blocked polypeptides and proteins. In this paper, different isotope coding strategies in combination with reversed phase chromatography (RPC) coupled with electrospray ionization-mass spectrometry (ESI-MS) were evaluated to detect the C-terminal peptide from proteolytic digests. These were (i) O18 (ii) acylation and (iii) esterification based isotope coding strategies. Using reversed phase chromatography, the C-terminal peptide was resolved from other internal peptides. The isotope coding approaches specifically rendered a characteristic MS signature to the C-terminal peptide, thereby facilitating its detection. The unique MS signature, along with accurate mass data for the C-terminal peptide was found to be sufficient for its detection and identification. The advantages and limitations of the three approaches will be discussed.     

Activation of PKR causes amyloid ß-peptide accumulation via de-repression of BACE1 expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer''s disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.     

Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2

The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots. Here, we show that a recombinant derivative of R. leguminosarum bv. trifolii ANU843 that constitutively overproduces the CelC2 enzyme has increased competitiveness in occupying aberrant nodule-like root structures on clover that are inefficient in nitrogen fixation. This aberrant symbiotic phenotype involves an extensive uncontrolled degradation of the host cell walls restricted to the expected infection sites at tips of deformed root hairs and significantly enlarged infection droplets at termini of wider IT within the nodule infection zone. Furthermore, signs of elevated plant host defense as indicated by reactive oxygen species production in root tissues were more evident during infection by the recombinant strain than its wild-type parent. Our data further support the role of the rhizobial CelC2 cell wall-degrading enzyme in primary infection, and show evidence of its importance in secondary symbiotic infection and tight regulation of its production to establish an effective nitrogen-fixing root nodule symbiosis.     

Central NPY-Y5 receptors activation plays a major role in fasting-induced pituitary-thyroid axis suppression in adult rat

Neuropeptide Y (NPY) inhibits TRH neurons in fed state, and hypothalamic NPY higher expression during fasting has been proposed to be involved in fasting-induced suppression of the hypothalamus-pituitary-thyroid (HPT) axis. We investigated the role of central Y5 receptors in the control of thyrotropin (TSH) and thyroid hormone (TH) secretion. Fed and fasting rats received twice daily central injections (3rd ventricle) of Y5 receptor antagonist (CGP71683; 15nmol/rat) for 72h. Fasted rats also received a single central injection of CGP71683 (15nmol/rat) at the end of 72h of fasting. In fed rats, Y5 receptor blockade reduced total food intake by 32% and body mass by almost 10% (p<0.01), corroborating the role of this receptor in food intake control. 72h-fasted rats exhibited a 4-fold increase in serum TSH (p<0.001), 1h after a single injection of Y5 antagonist. Also with multiple injections during 72h of fasting, Y5 blockade resulted in activation of thyroid axis, as demonstrated by a 3-times rise in serum T4 (p<0.001), accompanied by unchanged TSH and T3. In fed rats, the chronic central administration of CGP71683 resulted in reduced total serum T4 without changes in free T4 and TSH. Serum leptin and PYY were not altered by the NPY central blockade in both fed and fasted rats, suggesting no role of these hormones in the alterations observed. Therefore, the inhibition of central Y5 neurotransmission resulted in activation of thyroid axis during fasting suggesting that NPY-Y5 receptors contribute to fasting-induced TSH and TH suppression.     

Introduction of bacteriophage Mu into Pseudomonas solanacearum and Rhizobium meliloti using the R factor RP4.

Phage Mu-1 and a thermoinducible derivative, Mu-1 cts 62 were inserted into the broad host range R factor RP4. These hybrid plasmids were transferred by conjugation to a phytopathogenic bacterium Pseudomonas solanacearum GMI 1000 and a legume-root nodule bacterium Rhizobium meliloti 2011. The Mu genome is transcribed and tranlated in these new hosts: P. solanacearum (RP4:Mu cts) cultures have a spontaneous production of about 5 X 10(5) plaque-forming units ml-1 which is similar to the frequency of spontaneous Mu production in E. coli; the Mu production of R. meliloti is lower (about 10(2) plaque-forming units ml-1).     

A peptidyl alpha-amidation activity in chromaffin granules of bovine adrenal medulla

Peptidyl alpha-amidation activity in bovine adrenal medulla has been localized in chromaffin granules by density gradient centrifugation. The activity was found to be both soluble and membrane-associated. Both enzymatic activities were stimulated by the addition of Cu2+ and ascorbate. The pH maximum for alpha-amidation in the chromaffin granules in pH 8.0-8.5. By gel filtration, the soluble enzyme activity appeared as a protein of approx. 40 kDa. It is suggested that this enzyme is involved in the carboxyl-terminal amidation of metorphamide, amidorphin and neuropeptide Y.     

Beta 1 and beta 2 adrenergic agonists in exocrine pancreatic secretion in the rabbit

The effect of Dobutamine (a beta 1-adrenergic agonist) and Terbutaline (a beta 2-adrenergic agonist) on exocrine pancreatic secretion was studied in anaesthetized rabbits, simultaneously controlling pancreatic blood flow and blood pressure. The secretion of fluid and ions (bicarbonate, sodium and potassium) was unaffected by the infusion of Dobutamine (8 micrograms.kg-1.min-1) or Terbutaline (10 micrograms.kg-1.min-1). Neither were pancreatic blood flow or mean blood pressure altered. Dobutamine or Terbutaline depress the function of the acinar cells, amylase secretion being more affected by the action of Terbutaline. The results show that beta 1 and beta 2-adrenergic stimulation has no effect on the ductular cells but does decrease the secretion by the acinar cells.     

Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.Microtubules (MTs)¹ in vertebrate somatic cells are involved in intracellular transport and distribution of membranous organelles. Fundamental to this role are their tightly controlled, polarized organization, and unusual dynamic properties (Hirokawa, 1994) and their interaction with a complex set of MT-based motor proteins (Hirokawa, 1996; Sheetz, 1996; Goodson et al., 1997). During mitosis, they contribute to the motility of centrosomes, the construction of spindle poles (Karsenti et al., 1996; Merdes and Cleveland, 1997), and the dynamic movements of kinetochores (Rieder and Salmon, 1994) and chromosome arms (Barton and Goldstein, 1996; Vernos and Karsenti, 1996). The motor protein cytoplasmic dynein, drives the transport toward MT minus-ends of a variety of subcellular organelles (Schnapp and Reese, 1989; Schroer et al., 1989; Holzbaur and Vallee, 1994). Dynactin is a molecular complex originally identified as being essential for dynein-mediated movement of salt-washed vesicles in vitro (reviewed in Schroer, 1996; Schroer and Sheetz, 1991). Genetic studies in fungi, yeast, and flies have shown that the two complexes function together to drive nuclear migration, spindle and nuclear positioning and to permit proper neuronal development (Eshel et al., 1993; Clark and Meyer, 1994; Muhua et al., 1994; Plamann et al., 1994; McGrail et al., 1995; Karsenti et al., 1996). Biochemical studies suggest a direct interaction between certain subunits of dynein and dynactin (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995). In vivo, the two molecules may bind one another transiently, since they have not been isolated as a stable complex.There is good evidence indicating that the dynein/dynactin complex, together with other motors (Eg5, and a minus-end oriented kinesin-related protein) and a structural protein (NuMa), drive the focusing of free microtubule ends into mitotic spindle poles (Merdes and Cleveland, 1997; Waters and Salmon, 1997)_. A trimolecular complex composed of NuMa and dynein/dynactin may be crucial in this process in both acentriolar (Merdes et al., 1996), and centriolar spindles (Gaglio et al., 1997). A number of findings also indicate that the combined actions of dynein and dynactin at the kinetochore contribute to chromosome alignment in vertebrate somatic cells. First, the initial interaction between polar spindle MTs and kinetochores seems to involve a tangential capture event (Merdes and De Mey, 1990; Rieder and Alexander, 1990) which is followed by a poleward gliding along the surface lattice of the MT (Hayden et al., 1990). Both in vivo and in vitro (Hyman and Mitchison, 1991) this gliding movement appears similar to the dynein-mediated retrograde transport of vesicular organelles along MTs. Consistent with this is the finding that both dynein (Pfarr et al., 1990; Steuer et al., 1990) and its activator, dynactin (Echeverri et al., 1996), are present at prometaphase kinetochores. Overexpression of dynamitin, a 50-kD subunit of the dynactin complex, results in the partial disruption of the dynactin complex and in the loss, from kinetochores, of dynein, as well as dynactin. Therefore, it has been proposed that dynactin mediates the association of dynein with kinetochores. Abnormal spindles with poorly focused poles are observed and the cells become arrested in pseudoprometaphase (Echeverri et al., 1996). Despite these findings, rigorous proof for a role of the dynein motor complex in kinetochore motility is still lacking, and its role may differ between lower and higher eucaryotes, and between mitosis and meiosis.CLIP-170 (Rickard and Kreis, 1996) is needed for in vitro binding of endocytic transport vesicles to MTs (Pierre et al., 1992). It is a nonmotor MT-binding protein that accumulates preferentially in the vicinity of MT plus ends and on early endosomes and endocytic transport vesicles in nondividing cells (Rickard and Kreis, 1990; Pierre et al., 1992). Like many MT-binding proteins, CLIP-170 is a homodimer whose NH₂-terminal head domains and COOH-terminal tail domains flank a central α-helical coiled-coil domain. The binding of CLIP-170 to MTs involves a 57–amino acid sequence present twice in the head domain (Pierre et al., 1992) and is regulated by phosphorylation (Rickard and Kreis, 1991). The COOH-terminal domain has been proposed to participate in targeting to endocytic membranes (Pierre et al., 1994). The fact that the latter move predominantly toward microtubule minus ends in a process most likely mediated by cytoplasmic dynein and dynactin (Aniento and Gruenberg, 1995), suggests that CLIP-170 may act in concert with this motor complex, and may be subject to regulated interactions with one or more dynactin or dynein subunits at the vesicle membrane.Here we report that during mitosis, CLIP-170 codistributes with dynein and dynactin at kinetochores, but not spindle poles. Evidence is presented that the COOH-terminal domain of CLIP-170 is responsible for its kinetochore targeting, and that this may be mediated by the complex of dynein and dynactin. The effects on mitotic progression of overexpression of wild type and several deletion mutants of CLIP-170 provide evidence for the involvement of CLIP-170 in kinetochore function early in mitosis. We also present in vivo evidence that neither CLIP-170 nor the complex of dynein and dynactin are required for formation of kinetochore fibers.