Identification and characterization of the interactive proteins with cytotoxic T-lymphocyte antigen-2α

Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) is a potent inhibitor of cathepsin L-like cysteine proteases. Recombinant CTLA-2α is known to be a potent, competitive inhibitor of cathepsin L-like cysteine proteases. In this study, cathepsin L, cathepsin C, and tubulointerstitial nephritis antigen-related protein 1 (TINAGL1) were identified as novel interactive proteins of CTLA-2α by the yeast two-hybrid screening system. The direct interactions and co-localization of these proteins with CTLA-2α were confirmed using co-immunoprecipitation and immunofluorescence staining, respectively. The disulfide-bonded CTLA-2α/cathepsin L complex was isolated from mouse tissue. CTLA-2α was found to be specific and consistently expressed on the maternal side of the mouse placenta. Double immunofluorescence analysis showed that CTLA-2α was co-localized with cathepsin L, cathepsin C, and TINAGL1 in placenta. A simple cell-based fluorescence assay revealed that CTLA-2α exhibited inhibitory activity toward cathepsin C in live cells, which indicated that CTLA-2α is a novel endogenous inhibitor of cathepsin C.     

N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile

Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood–brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH₂ (DIPP-NH₂) and H-Dmt-TicΨ[CH₂NH]Phe-Phe-NH₂ (DIPP-NH₂[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH₂ and Guan-DIPP-NH₂ in the δ receptor binding site. Lys³-analogues of DIPP-NH₂ and DIPP-NH₂[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction.     

Geographic distribution of microsatellite alleles in geladas (Primates,Cercopithecidae): Evidence for three evolutionary units

The subspecific taxonomy and distribution of geladas (Theropithecus gelada Rüppell, 1835) remains uncertain. Recent molecular studies based on mitochondrial sequence data revealed a geographically structured, three-deme population, suggesting that there are three evolutionary units of geladas. However, mitochondrial distributions do not always recover population relationships, particularly in taxa with a complex history of isolation and gene flow. We therefore analysed the nuclear genetic population structure of the global gelada population based on 20 microsatellite loci in 43 samples from across its geographic range. F_ST values, a STRUCTURE analysis and a principal coordinate analysis (PCoA) confirmed the three-deme population structure corresponding to the mitochondrial population structure. Therefore, our analyses provide additional support for three evolutionary units in geladas, corresponding to (a) a northern (north of Lake Tana, primarily in the Simien Mountains, previously classified as Theropithecus gelada gelada Rüppell, 1835), (b) a central (between Addis Ababa and the highlands east of Lake Tana, previously classified as Theropithecus gelada obscurus Heuglin, 1863) and (c) a southern (south of the Rift Valley, previously tentatively classified as Theropithecus gelada arsi Shotake et al., 2016, Anthropological Science, 124, 157) population. These results pave the way for future conservation decisions and highlight that the gelada population boundaries need more fine-grained genetic sampling and phenotypic analyses, in particular for their taxonomic ranking.     

Death among geladas (Theropithecus gelada): a broader perspective on mummified infants and primate thanatology

Despite intensive study in humans, responses to dying and death have been a neglected area of research in other social mammals, including nonhuman primates. Two recent reports [Anderson JR, Gillies A, Lock LC. 2010. Pan thanatology. Current Biology 20:R349–R351; Biro D, Humle T, Koops K, Souse C, Hayashi M, Matsuzawa T. 2010. Chimpanzee mothers at Bossou, Guinea carry the mummified remains of their dead infants. Current Biology 20:R351–R352] offered exciting new insights into behavior toward dying and dead conspecifics in our closest living relatives—chimpanzees. Here, we provide a comparative perspective on primate thanatology using observations from a more distant human relative—gelada monkeys (Theropithecus gelada)—and discuss how gelada reactions to dead and dying groupmates differ from those recently reported for chimpanzees. Over a 3.75‐year study period, we observed 14 female geladas at Guassa, Ethiopia carrying dead infants from 1 hr to ≥48 days after death. Dead infants were carried by their mothers, other females in their group, and even by females belonging to other groups. Like other primate populations in which extended (>10 days) infant carrying after death has been reported, geladas at Guassa experience an extreme climate for primates, creating conditions which may favor slower rates of decomposition of dead individuals. We also witnessed the events leading up to the deaths of two individuals and the responses by groupmates to these dying individuals. Our results suggest that while chimpanzee mothers are not unique among primates in carrying their dead infants for long periods, seemingly “compassionate” caretaking behavior toward dying groupmates may be unique to chimpanzees among nonhuman primates (though it remains unknown whether such “compassionate” behavior occurs outside captivity). Am. J. Primatol. 73:405–409, 2011. © 2010 Wiley‐Liss, Inc.     

Biodegradation of vinyl chloride, <Emphasis Type="Italic">cis</Emphasis>-dichloroethene and 1,2-dichloroethane in the alkene/alkane-oxidising <Emphasis Type="Italic">Mycobacterium</Emphasis> strain NBB4

Mycobacterium chubuense strain NBB4 can grow on both alkanes and alkenes as carbon sources, and was hypothesised to be an effective bioremediation agent for chlorinated aliphatic pollutants. In this study, the ability of NBB4 to biodegrade vinyl chloride (VC), cis-dichloroethene (cDCE) and 1,2-dichloroethane (DCA) was investigated under pure-culture conditions and in microcosms. Ethene-grown NBB4 cells were capable of biodegrading VC and cDCE, while ethane-grown cells could biodegrade cDCE and DCA. The stoichiometry of inorganic chloride release (1 mol/mol in each case) indicated that VC was completely dechlorinated, while cDCE and DCA were only partially dechlorinated, yielding chloroacetate in the case of DCA, and unknown metabolites in the case of cDCE. The apparent maximum specific activities (k) of whole cells against ethene, cDCE, ethane and DCA were 93 ± 4.6, 89 ± 18, 39 ± 5.5, and 4.8 ± 0.9 nmol/min/mg protein, respectively, while the substrate affinities (K_S) of whole cells with the same substrates were 2.0 ± 0.15, 46 ± 11, 11 ± 0.33 and 4.0 ± 3.2 μM, respectively. In microcosms containing contaminated aquifer sediments and groundwater, NBB4 cells removed 85-95% of the pollutants (cDCE or DCA at 2 mM) within 24 h, and the cells remained viable for >1 month. Due to its favourable kinetic parameters, and robust survival and biodegradation activities, strain NBB4 is a promising candidate for bioremediation of chlorinated aliphatic pollutants.     

Analysis of urine cytology tests in 120 paired cases

OBJECTIVE: To evaluate the smear quality and diagnostic accuracy of ThinPrep processing in comparison to conventional Cytospin technique for urinary cytology. STUDY DESIGN: ThinPrep and Cytospin techniques were retrospectively evaluated by 2 observers in a double-blinded, randomized fashion. Each quality parameter was scored using a semi-quantitative score of 1-3. Diagnostic accuracy indices were calculated with biopsy histology as the gold standard. RESULTS: Quality of cellular distribution and cell preservation were better with Cytospin preparations, whereas ThinPrep smears were superior in terms of stain distribution and cleaner slide background. However, the only significant differences observed were in cellular distribution and a clean background (p < 0.05). Sensitivity and positive and negative predictive values were higher with Cytospin than the ThinPrep technique (90.0%, 94.7% and 71.4% vs. 80.0%, 94.1% and 55.6%, respectively). Conversely, the specificity of both techniques was comparable. CONCLUSION: The Cytospin smears were of better quality than those prepared by the ThinPrep technique. Although both techniques resulted in similar diagnostic accuracies in negative cases, the ThinPrep preparations were not found to be superior to Cytospin smears in diagnosing positive urinary cytology.     

CODAS Syndrome Is Associated with Mutations of LONP1, Encoding Mitochondrial AAA+ Lon Protease

CODAS syndrome is a multi-system developmental disorder characterized by cerebral, ocular, dental, auricular, and skeletal anomalies. Using whole-exome and Sanger sequencing, we identified four LONP1 mutations inherited as homozygous or compound-heterozygous combinations among ten individuals with CODAS syndrome. The individuals come from three different ancestral backgrounds (Amish-Swiss from United States, n = 8; Mennonite-German from Canada, n = 1; mixed European from Canada, n = 1). LONP1 encodes Lon protease, a homohexameric enzyme that mediates protein quality control, respiratory-complex assembly, gene expression, and stress responses in mitochondria. All four pathogenic amino acid substitutions cluster within the AAA⁺ domain at residues near the ATP-binding pocket. In biochemical assays, pathogenic Lon proteins show substrate-specific defects in ATP-dependent proteolysis. When expressed recombinantly in cells, all altered Lon proteins localize to mitochondria. The Old Order Amish Lon variant (LONP1 c.2161C>G[p.Arg721Gly]) homo-oligomerizes poorly in vitro. Lymphoblastoid cell lines generated from affected children have (1) swollen mitochondria with electron-dense inclusions and abnormal inner-membrane morphology; (2) aggregated MT-CO2, the mtDNA-encoded subunit II of cytochrome c oxidase; and (3) reduced spare respiratory capacity, leading to impaired mitochondrial proteostasis and function. CODAS syndrome is a distinct, autosomal-recessive, developmental disorder associated with dysfunction of the mitochondrial Lon protease.     

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.     

The Microbiological and Clinical Characteristics of Invasive Salmonella in Gallbladders from Cholecystectomy Patients in Kathmandu,Nepal

Gallbladder carriage of invasive Salmonella is considered fundamental in sustaining typhoid fever transmission. Bile and tissue was obtained from 1,377 individuals undergoing cholecystectomy in Kathmandu to investigate the prevalence, characteristics and relevance of invasive Salmonella in the gallbladder in an endemic area. Twenty percent of bile samples contained a Gram-negative organism, with Salmonella Typhi and Salmonella Paratyphi A isolated from 24 and 22 individuals, respectively. Gallbladders that contained Salmonella were more likely to show evidence of acute inflammation with extensive neutrophil infiltrate than those without Salmonella, corresponding with higher neutrophil and lower lymphocyte counts in the blood of Salmonella positive individuals. Antimicrobial resistance in the invasive Salmonella isolates was limited, indicating that gallbladder colonization is unlikely to be driven by antimicrobial resistance. The overall role of invasive Salmonella carriage in the gallbladder is not understood; here we show that 3.5% of individuals undergoing cholecystectomy in this setting have a high concentration of antimicrobial sensitive, invasive Salmonella in their bile. We predict that such individuals will become increasingly important if current transmission mechanisms are disturbed; prospectively identifying these individuals is, therefore, paramount for rapid local and regional elimination.