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.     

Association of alpha- and beta-subunits during the biosynthesis of beta-hexosaminidase in cultured human fibroblasts

Subunit association of beta-hexosaminidase was studied in intact fibroblasts using antisera that discriminate between free and associated alpha-chains. These were anti-beta-hexosaminidase A (anti-alpha beta), which precipitated all alpha-chains, free or associated; anti-beta-hexosaminidase B (anti-beta beta), which precipitated those alpha-chains that were associated with beta; and anti-alpha-chains, which recognized only monomeric alpha-chains. After biosynthetic labeling, beta-hexosaminidase or its free alpha-subunit were immuno-precipitated from extracts of cells and medium with the aid of protein A-bearing Staphylococcus aureus, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Pulse-chase labeling showed that the alpha-chains existed predominantly in the monomeric precursor form during the first 5 h, and then began to accumulate in the mature (lysosomal) associated alpha beta form. Precursor alpha beta complexes were secreted, along with some precursor alpha monomers; the latter were catalytically inert. Both alpha- and beta-chains were phosphorylated (a Golgi modification) prior to association. Thus alpha-beta association probably occurred in the Golgi area before transfer to lysosomes and before secretion. Cycloheximide inhibited the association and subsequent maturation of preformed alpha-chains, perhaps by causing a depletion of a pool of beta-chain precursor upstream from the site of subunit association. In fibroblasts from a patient with Sandhoff disease, that produced no beta-chains, the alpha-chains self-associated but their maturation was markedly decreased. We suggest that association with beta-chains is necessary not only for acquisition of catalytic activity but also for transport of alpha-chains to lysosomes.     

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis

Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphorylated by the actions of two S1P-specific phosphatases, sphingosine-1-phosphate phosphatases 1 and 2. To identify the physiological functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1^−/− mice appeared normal at birth, but during the 1st week of life they exhibited stunted growth and suffered desquamation, with most dying before weaning. Both Sgpp1^−/− pups and surviving adults exhibited multiple epidermal abnormalities. Interestingly, the epidermal permeability barrier developed normally during embryogenesis in Sgpp1^−/− mice. Keratinocytes isolated from the skin of Sgpp1^−/− pups had increased intracellular S1P levels and displayed a gene expression profile that indicated overexpression of genes associated with keratinocyte differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis.     

UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib

HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC₅₀) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC₅₀s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC₅₀>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.Constant proteotoxic stress is a frequent occurrence in cancer cells and is derived from an adverse external microenvironment (hypoxia, acidosis) and internally from conformationally aberrant oncoproteins, high reactive oxygen species (ROS) levels, genomic instability, and stoichiometric imbalances in multi-protein machines. This stress condition raises the need for massive heat-shock chaperone support, especially from the heat-shock protein 90 (HSP90) system, to prevent protein aggregation and illicit interactions and promote tumor cell survival. Cancer-associated factors, such as mutant p53,^{1, 2} ErbB2,³ AKT,⁴ and macrophage migration inhibitory factor (MIF),^{5, 6} among others, represent HSP90 clients and require HSP90 for their stabilization in tumors. Hence, the multi-component HSP90 chaperone is highly upregulated and activated specifically in cancer cells as an adaptive response to malignancy.⁷HSP90 inhibitors have emerged as a highly promising class of anti-cancer compounds because of their ability to interfere with broadly active molecular networks, rather than a narrowly defined signaling pathway^{8, 9} and they enhance proteotoxic stress.¹⁰ Geldanamycin-based compounds represented the mainstay of HSP90 inhibition for the last 20 years.⁸ Clinically, however, these compounds proved to be of limited value due to their inherent liver and ocular toxicity coupled with only modest potency in vivo.¹¹ Major advances came with recently developed second-generation synthetic inhibitors such as the resorcinol containing compounds ganetespib (STA-9090)¹² and NVP-AUY922¹³ that are considerably more potent and less toxic. These compounds are currently being tested in phase II/III trials for their efficacy against various cancer types. Anaplastic lymphoma kinase (ALK)-driven NSCLC cancers showed particular clinical responsiveness to ganetespib.^{14, 15}Colorectal carcinoma (CRC) represents one of the most frequent malignancies worldwide, with a correspondingly high death toll. Moreover, the identification of predictive markers for patient stratification has proven to be difficult.¹⁶ HSP90 inhibition might be an attractive strategy for therapeutic improvement. However, this requires studies on how HSP90 inhibitors act on tumor cells in this cancer type.One approach to identify genes that determine cancer drug response was provided by highly parallel analysis of many cancer cell lines, specifically comparing their response with a large variety of small compounds with their gene expression patterns.^{17, 18} This strategy led to the identification of genes with expression patterns correlating with drug sensitivity. Notably, for many anticancer drugs this approach did not yield strong candidates, but in the case of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) a strong correlation was seen between drug sensitivity and the expression of functional NAD(P)H dehydrogenase quinone 1 (NQO1), an enzyme that can metabolize and activate 17-AAG.^{19, 20} Hence, correlating drug sensitivity and gene expression patterns in cell lines can identify mechanisms that determine drug response.Drugs are subjected to metabolic turnover, and a major route of excretion from the body consists in conjugation with a hydrophilic sugar moiety within the liver parenchyma, followed by secretion into the bile. A major group of enzymes that carry out such conjugations are the UDP glucuronosyltransferases (UGTs).^{21, 22, 23} These enzymes are the products of gene clusters that cover various substrate specificities. UGT substrates include bilirubin, amines, and phenol structures.²⁴ The existence of such mechanisms for drug conjugation in the liver raises the question if and under what circumstances they can be found directly in tumor cells, and presumably cause drug resistance when highly expressed.Here, we show that human CRC-derived cell lines fall into ganetespib-sensitive and -resistant groups. While the majority of CRC lines were sensitive, two lines were highly resistant. Importantly, resistant cancer cells show a high expression of the UGT1A gene, and high levels of UGT1A were shown to be critical for ganetespib turnover, drug inactivation, and cell resistance. Thus, UDP glucuronosyl conjugation detoxifies ganetespib not only in the liver but also in a subset of CRC cells, representing a potential predictive biomarker for ganetespib response in CRC and possibly other tumor types.     

Kidney sulfatides in mouse models of inherited glycosphingolipid disorders: determination by nano-electrospray ionization tandem mass spectrometry

Sulfatides show structural, and possibly physiological similarities to gangliosides. Kidney dysfunction might be correlated with changes in sulfatides, the major acidic glycosphingolipids in this organ. To elucidate their in vivo metabolic pathway these compounds were analyzed in mice afflicted with inherited glycosphingolipid disorders. The mice under study lacked the genes encoding either beta-hexosaminidase alpha-subunit (Hexa-/-), the beta-hexosaminidase beta-subunit (Hexb-/-), both beta-hexosaminidase alpha and beta-subunits (Hexa-/- and Hexb-/-), GD3 synthase (GD3S-/-), GD3 synthase and GalNAc transferase (GD3S-/- and GalNAcT-/-), GM2 activator protein (Gm2a-/-), or arylsulfatase A (ASA-/-). Quantification of the sulfatides, I(3)SO(3)(-)-GalCer (SM4s), II(3)SO(3)(-)-LacCer (SM3), II(3)SO(3)(-)-Gg(3)Cer (SM2a), and IV(3,) II(3)-(SO(3)(-))(2)-Gg(4)Cer (SB1a), was performed by nano-electrospray tandem mass spectrometry. We conclude for the in vivo situation in mouse kidneys that: 1) a single enzyme (GalNAc transferase) is responsible for the synthesis of SM2a and GM2 from SM3 and GM3, respectively. 2) In analogy to GD1a, SB1a is degraded via SM2a. 3) SM2a is hydrolyzed to SM3 by beta-hexosaminidase S (Hex S) and Hex A, but not Hex B. Both enzymes are supported by GM2-activator protein. 4) Arylsulfatase A is required to degrade SB1a. It is probably the sole sphingolipid-sulfatase cleaving the galactosyl-3-sulfate bond. In addition, a human Tay-Sachs patient's liver was investigated, which showed accumulation of SM2a along with GM2 storage. The different ceramide compositions of both compounds indicated they were probably derived from different cell types. These data demonstrate that in vivo the sulfatides of the ganglio-series follow the same metabolic pathways as the gangliosides with the replacement of sulfotransferases and sulfatases by sialyltransferases and sialidases. Furthermore, a novel neutral GSL, IV(6)GlcNAcbeta-Gb(4)Cer, was found to accumulate only in Hexa-/- and Hexb-/- mouse kidneys. From this we conclude that Hex S also efficiently cleaves terminal beta1-6-linked HexNAc residues from neutral GSLs in vivo.     

Sphingosine-1-phosphate receptors and the development of the vascular system

Extracellular sphingolipid signaling has been implicated as an essential event in vascular development. Sphingosine-1-phosphate (S1P), through interactions with G protein-coupled receptors, regulates functions of endothelial and smooth muscle cells (SMCs)-the major cell types of the vasculature. The knockout of the gene encoding the S1P1 receptor (formally known as Edg-1) in mice blocks vascular maturation, the process where SMCs and pericytes envelop nascent endothelial tubes. The question that remains is how stimulation of S1P receptors controls this critical event in the developmental sequence leading to the formation of functional blood vessels.     

Proteome analysis of the rat cornea during angiogenesis

Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. However, the study of this complex process is often hampered by the lack of a suitable cell-based model and the tools to study the biochemical events that lead to new blood vessel growth. The most widely accepted model for angiogenesis is the in vivo rat corneal model. In this model, the cornea, which is normally an avascular tissue, is stimulated to undergo angiogenesis in response to silver nitrate cauterization or to the implantation of an exogenous angiogenic agent. The physical changes associated with the new vessel growth can be readily monitored visually, but the regulated biochemical events that result in the growth and remodeling of the new vessels are much more challenging to decipher. In this report, a proteomics approach is evaluated for its utility in deciphering the biochemical events during a time course of angiogenic stimulation. At various time points post-silver nitrate cautery, corneas were harvested, solubilized, and analyzed by two-dimensional gel electrophoresis. Protein expression profiles at the various stages of angiogenesis were compared to those of control corneas. One hundred and eleven differentially-expressed proteins were identified by either matrix-assisted laser desorption/ionization-time of flight mass spectrometry or liquid chromatography-coupled electrospray ionization tandem mass spectrometry. Many of the proteins up-regulated during the angiogenesis process were identified as blood-related proteins, thus validating the development of functional blood vessels in the normally avascular tissue of the cornea. Furthermore, detection of differentially-regulated proteins in cauterized versus control tissue clearly validated the utility of a proteomics approach to study this model of angiogenesis. However, in order to get at the key regulatory proteins in the angiogenesis process, it is clear that additional scale-up and enrichment approaches will be required.