Systematics within Gyps vultures: a clade at risk

Background  

Populations of the Oriental White-backed Vulture (Gyps bengalensis) have declined by over 95% within the past decade. This decline is largely due to incidental consumption of the non-steroidal anti-inflammatory veterinary pharmaceutical diclofenac, commonly used to treat domestic livestock. The conservation status of other Gyps vultures in southern Asia is also of immediate concern, given the lack of knowledge regarding status of their populations and the continuing existence of taxonomic uncertainties. In this study, we assess phylogenetic relationships for all recognized species and the majority of subspecies within the genus Gyps. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits to Gyps bengalensis. Therefore, an accurate assessment of the phylogenetic relationships among Gyps vultures should aid in their conservation by clarifying taxonomic uncertainties, and enabling inference of their respective relatedness to susceptible G. bengalensis.     

Proteolytic inactivation of alpha 1-proteinase inhibitor in vivo: detection, characterization and quantitation of the main fragment excreted in the urine of leukemia patients.

During the search for a therapy response parameter in patients with acute myeloid leukemia, we observed the appearance of a 41 kDa glycoprotein band in the urines of these patients under therapy. To investigate the nature of this molecule and to develop a specific detection system, the protein was isolated and antibodies were raised. Urines and sera of patients and healthy subjects were screened for crossreacting proteins by immunoblotting. Only the leukemia patients showed the urinary 41 kDa protein plus a 53 kDa band. In all sera, including those from healthy donors, a 53 kDa protein was intensely stained. Isolation of the plasma protein and sequence analysis of the urinary protein revealed that alpha 1-proteinase inhibitor is the crossreacting plasma protein and that the 41 kDa molecule is proteolytically modified alpha 1-PI, which has lost its antitryptic activity. Cleavage occurred in the N-terminal part as well as in the reactive site loop of the inhibitor. The 41 kDa truncated inhibitor was also found in the leukemic blast cells. A densitometric method is described for the quantitation of the molecule in the nanomolar range.     

Five novel locations of Neocentromeres in human: 18q22.1, Xq27.1∼27.2, Acro p13, Acro p12, and heterochromatin of unknown origin

Since the first report in 1993, an ectopic centromere, i.e. neocentromere formation, has been reported in more than 100 small supernumerary marker chromosomes (sSMC), in 7 instances of centromere repositioning, and in about a dozen cases with more complex chromosomal rearrangements. Here we report 2 new cases with centromere repositioning and 3 neocentric sSMC consisting exclusively of heterochromatic material. Yet, no centromere formation was reported for the regions 18q22.1 and Xq27.1～27.2 as it was observed in the 2 cases with centromere repositioning here; in both cases, cytogenetically an inversion was suggested. Two of the 3 neocentric sSMC were derived from a short arm of an acrocentric chromosome. The remainder neocentric sSMC case was previously reported and was stainable only by material derived from itself.     

Development and evaluation of 4-(pyrrolidin-3-yl)benzonitrile derivatives as inhibitors of lysine specific demethylase 1

As part of our ongoing efforts to develop reversible inhibitors of LSD1, we identified a series of 4-(pyrrolidin-3-yl)benzonitrile derivatives that act as successful scaffold-hops of the literature inhibitor GSK-690. The most active compound, 21g, demonstrated a K_d value of 22 nM and a biochemical IC₅₀ of 57 nM. In addition, this compound displayed improved selectivity over the hERG ion channel compared to GSK-690, and no activity against the related enzymes MAO-A and B. In human THP-1 acute myeloid leukaemia cells, 21g was found to increase the expression of the surrogate cellular biomarker CD86. This work further demonstrates the versatility of scaffold-hopping as a method to develop structurally diverse, potent inhibitors of LSD1.     

Acetylcholinesterase secretion by parasitic nematodes. II. Trichostrongylus spp

Unusually high levels of acetylcholinesterase (AChE) were found in the nematode parasites Trichostrongylus axei, T. colubriformis and T, retortaeformis. In T. colubriformis the enzyme was located in the oesophageal and excretory glands of the parasitic stages. The highest level/unit wt was found in the fourth-stage larvae, which per worm had a comparable level to that in adult worms because the excretory gland was fully developed in the fourth-stage larvae. In acrylamide gel electrophoresis, T. axei and T. colubriformis AChE and esterases were similar but differed from those present in T. retortaeformis. Globulins prepared from the sera of sheep and guinea-pigs infected with T. colubriformis complexed with T. colubriformis and T. axei AChE, but not with esterases nor with AChE from T. retortaeformis, Nippostrongylus brasiliensis, Oesophagostomum radiatum or O. venulosum. Complexing of AChE to globulins did not inhibit the enzymic function of this enzyme.     

Diadenosine 5′,5‴-P1,P3-triphosphate in eukaryotic cells: Identification and quantitation

A highly sensitive enzymatic assay for diadenosine 5′,5?-P¹,P³-triphosphate (Ap₃A) has been established on the basis of the coupled luminescence assay for diadenosine 5′,5?-P¹,P³-tetraphosphate (A. Ogilvie (1981)Anal. Biochem.115, 302–307). Snake venom phosphodiesterase splits Ap₃A into AMP plus ADP which can be measured in a luminescence reaction containing pyruvate kinase, phosphoenolpyruvate and luciferin-luciferase. The procedure is linear with Ap₃A levels ranging from 0.1 to 2 pmol. The assay has been used to measure Ap₃A in various eukaryotic cells after ion-exchange chromatography and high-performance liquid chromatography of acidic extracts of the cells. The level of diadenosine triphosphate was higher in all instances than the level of diadenosine tetraphosphate. When growing in the abdominal cavity of mice, Ehrlich ascites tumor cells contained high amounts of Ap₃A ($\text{0.1}\text{nmol}\text{10}{}^6\text{cells}$), allowing direct optical determination in the HPLC chromatography. The quantitative measurement of Ap₃A with the luminescence assay gave identical results. Ap₃A extracted from Ehrlich cells was also chromatographed with authentic nucleotide in two thin-layer systems providing additional proof for the existence of Ap₃A in biological material.