Secondary nucleating sequences affect kinetics and thermodynamics of tau aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)(n)Z, Z(X)(n)Z (n ≥ 2), or (XZ)(n) (n ≥ 2), where X is a hydrophobic residue and Z is a charged or polar residue. N-Acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed seven peptides, in addition to well-known primary nucleating sequences Ac(275)VQIINK (AcPHF6*) and Ac(306)VQIVYK (AcPHF6), formed fibers, tubes, ribbons, or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac(10)VME, AcPHF6*, Ac(375)KLTFR, and Ac(393)VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac(375)KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild-type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics.     

Lack of Alpha-Synuclein Modulates Microglial Phenotype In Vitro

Alpha (α)-synuclein neuronal effects are continually being defined although its role in regulating glial phenotypes remains unclear. An ability to regulate microglial activation was investigated using primary cultures from wild type and α-synuclein deficient mice (Snca ^−/−). Snca ^−/− microglia demonstrated increased secretion of the cytokine tumor necrosis factor-alpha (TNF-α), impaired phagocytic ability, elevated prostaglandin levels, and increased protein levels of key enzymes in lipid-mediated signaling events, cytosolic phospholipase (cPLA₂), cyclooxygenase-2 (Cox-2) and phospholipase D2 (PLD2) when compared to wild type cells. Increased cytokine secretion and cPLA₂ and Cox-2 levels in Snca ^−/− microglia were partially attenuated by inhibiting PLD-dependent signaling with n-butanol treatment.     

Simulation of LV pacemaker lead in marginal vein: potential risk factors for acute dislodgement

Although left ventricular (LV) coronary sinus lead dislodgement remains a problem, the risk factors for dislodgement have not been clearly defined. In order to identify potential risk factors for acute lead dislodgement, we conducted dynamic finite element simulations of pacemaker lead dislodgement in marginal LV vein. We considered factors such as mismatch in lead and vein diameters, velocity of myocardial motion, branch angle between the insertion vein and the coronary sinus, degree of slack, and depth of insertion. The results show that large lead-to-vein diameter mismatch, rapid myocardial motion, and superficial insertion are potential risk factors for lead dislodgement. In addition, the degree of slack presents either a positive or negative effect on dislodgement risk depending on the branch angle. The prevention of acute lead dislodgment can be enforced by inducing as much static friction force as possible at the lead-vein interface, while reducing the external force. If the latter exceeds the former, dislodgement will occur. The present findings underscore the major risk factors for lead dislodgment, which may improve implantation criterion and future lead design.     

Albumin affinity tags increase peptide half-life in vivo

Small organic molecules that bind tightly to serum albumin were applied to the amino terminus of an anticoagulant peptide in an effort to increase its protein binding in vivo. The tagged peptides were evaluated for their ability to be retained on liquid chromatographic columns with serum albumins incorporated into the stationary phase. Those which demonstrated significant affinity were administered intravenously to rabbits and found to have significantly increased plasma half-lives. Novel affinity tags were identified by appending a focused library of compounds to a model tetrapeptide and evaluating the resulting compounds' ability to bind to the serum albumin columns. The most promising were synthesized as the full length peptides and again evaluated in vivo. They were found to have still longer half-lives than the first generation compounds.     

NAR Breakthrough Article: Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.     

Spontaneous hypertension and its sequelae in woolly monkeys (Lagothrix lagotricha)

Arteriolar nephrosclerosis was observed at necropsy in 26 of 38 woolly monkeys (Lagothrix lagotricha). This lesion is the earliest histologic change associated with hypertension in humans. Seventeen of the monkeys had died of congestive heart failure, renal failure or acute cardiovascular accident, complications similar to those seen in human hypertension. All monkeys known to be over 4 years of age were affected. Direct blood pressure measurements in nine otherwise healthy woolly monkeys revealed systolic pressures of 194 +/- 20 mmHg. Our physiologic, clinical and pathologic studies suggest that woolly monkeys develop hypertension spontaneously and could be a useful model for the study of human hypertension.     

Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex

A bovine liver protein which catalyzes the transfer of triglyceride between membranes has previously been isolated from the lumen of the microsomal fraction. When further purified about 100-fold, two polypeptides of molecular mass 58,000 and 88,000 were identified (Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222). We demonstrate here that the two polypeptides (referred to as 58-kDa and 88-kDa, respectively) are associated in a protein-protein complex, and that the triglyceride transfer activity is associated with this complex. Antibodies specific for either polypeptide immunoprecipitated both the 58-kDa and 88-kDa polypeptides as well as the lipid transfer activity. The 58-kDa subunit of the microsomal transfer protein complex was identified as protein disulfide-isomerase (PDI) (EC 5.3.4.1) by 1) a comparison of the amino-terminal sequence of PDI and the 58-kDa subunit of the transfer protein, 2) a comparison of the reverse phase high performance liquid chromatography peptide maps of CNBr digests of PDI and the lipid transfer protein, 3) immunoprecipitation competition experiments in which PDI was found to compete with the lipid transfer protein for immunoprecipitation by the anti-58-kDa polyclonal antibodies, 4) immunological cross-reactivity of the microsomal triglyceride transfer protein complex with polyclonal antibodies raised against PDI, and 5) the appearance of protein disulfide isomerase activity following the dissociation of purified microsomal transfer protein complex with guanidine HCl. In conclusion, the microsomal triglyceride transfer protein has a multi-subunit structure which is unique compared to other intracellular lipid transfer proteins which have been described to be single polypeptides. The unexpected finding that PDI is a component of the microsomal triglyceride transfer protein complex suggests a new previously undescribed role for protein disulfide isomerase.     

Variability of ovarian structures and plasma progesterone profiles in dairy cows with ovarian cysts

Weekly reproductive health examinations were performed on 46 multiparous Holstein cows from 14 to 100 d post partum. Sixteen cows developed 19 nonsimultaneous ovarian cysts, with a mean day of first detection at 34.3 +/- 4.5 d post partum and a mean duration of 31.0 +/- 4.3 d after first detection. Coccygeal blood was collected three times weekly, and plasma progesterone concentrations were determined by radioimmunoassay. Cysts were diagnosed by palpation per rectum or by ultrasonography and classified as follicular or luteal cysts; the cows were not treated. Cows with a mean plasma progesterone concentration of < 1 ng/ml from the first day of detection (Day 1) of a cyst until Day 10 were classified as having a follicular cyst, and those with a mean plasma progesterone concentration of >/= 1 ng/ml from Day 1 to Day 10 were classified as having a luteal cyst. According to this classification, 58% of the cysts were follicular and 42% were luteal. There was an overall 47% agreement between classification by palpation and by ultrasonography on Day 1 with progesterone concentration during Days 1 to 10 after detection of the cyst. Detailed graphs of progesterone concentrations and area of largest follicles or cysts and corpora lutea demonstrate the variability of ovarian structures and progesterone profiles in cystic cows. Detection of a cyst at any one time accompanied by simultaneous measurement of progesterone can lead to different diagnoses of cyst type depending on the method of classification, the presence and age of luteinized tissue in the cyst and undetected corpora lutea.     

A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence

Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities.     

Studies on the effects of selenium on rumen microbial fermentation in vitro

The effects of selenium (Se) on ruminant microbial fermentation were investigated in vitro using rumen microflora collected from a rumen-fistulated dairy cow. First, the effects ofl-selenomethionine (SeMet; at 0.2 or 2 ppm Se) in the presence or absence of wheat bran (WB, 500 mg per incubation flask) were evaluated. Second, the effects of several forms of Se (elemental Se: 50 ppm Se; sodium selenite: 2 ppm Se; SeMet: 2 ppm Se) were compared. Results showed that the amounts of short-chain fatty acids (SCFAs) tended to be increased by SeMet treatment, whereas SeMet in the presence of WB transiently suppressed fermentation. The addition of SeMet tended to increase the production of acetate while reducing the production of butyrate with and without WB supplementation. Among the different Se compounds tested, the amounts of SCFAs were greater with SeMet treatment, which yielded a higher proportion of acetate compared to other treatments. Selenite did not influence the total SCFAs concentrations; however, it increased the relative proportion of butyrate at the expense of acetate. Elemental Se did not significantly affect fermentation. Higher bacterial Se concentrations were observed for selenite than for SeMet. It was concluded that Se supplementation can influence rumen microbial fermentation and that Se compounds differ in this regard.