Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways

There are several pathways for the incorporation of sugars into glycosphingolipids (GSL). Sugars can be added to ceramide that contains sphinganine (dihydrosphingosine) synthesized de novo (pathway 1), to ceramide synthesized from sphingoid bases produced by hydrolysis of sphingolipids (pathway 2), and into GSL recycling from the endosomal pathway through the Golgi (pathway 3). We reported previously the surprising observation that SW13 cells, a human adrenal carcinoma cell line, synthesize most of their GSL in pathway 2. We now present data on the synthesis of GSL in four additional cell lines. Approximately 90% of sugar incorporation took place in pathway 2, and 10% or less in pathway 1, in human foreskin fibroblasts and NB41A3 neuroblastoma cells. In contrast, approximately 50-90% of sugar incorporation took place in pathway 1 in C2C12 myoblasts. The C2C12 cells divide more rapidly and synthesize 10-14 times as much GSL as the other three cell lines. In C6 glioma cells, approximately 30% of sugar incorporation occurred in pathway 1 and 60% in pathway 2. There was no relation between the utilization of pathways for GSL and sphingomyelin synthesis in foreskin fibroblasts and C2C12 cells. In both cells pathways 1 and 2 each accounted for 50% of incorporation of choline into sphingomyelin. In five of the six cell lines that we have studied, most GSL synthesis takes place in pathway 2. We suggest that when the need for synthesis is relatively low, as in slowly dividing cells, GSL are synthesized predominantly from sphingoid bases salvaged from the hydrolytic pathway. When cells are dividing more rapidly, the need for increased synthesis is met by upregulating the de novo pathway.      

Diabetes-induced changes in the renal cortical proteome assessed with two-dimensional gel electrophoresis and mass spectrometry

To understand the spectrum of proteins affected by diabetes and to characterize molecular functions and biological processes they control, we analyzed the renal cortical proteome of db/db mice using 2-DE combined with MALDI-TOF, MALDI-TOF/TOF, and LC-MS/MS. This approach yielded 278 high confidence identifications whose expression levels were significantly increased or decreased >two-fold by diabetes, of which 170 mapped to gene identifiers representing 147 nonredundant proteins. Gene Ontology classification demonstrated that 80% of these proteins modulated physiological functions, 55% involved metabolism, approximately 25% involved carboxylic and organic acid metabolism, 14% involved biosynthesis or catabolism, and 12% involved fatty acid metabolism. Predominant molecular functions were catalytic (61%), oxidoreductase (20%), and transferase (17%) activities, and nucleotide and ATP binding (11-15%). Twenty eight percent of the proteins identified as significantly altered by diabetes were mitochondrial proteins. The top-ranked network described by Ingenuity Pathway Analysis indicated PPARalpha was the most common node of interaction for the numerous enzymes whose expression levels were influenced by diabetes. These differentially regulated proteins create a foundation for a systems biology exploration of molecular mechanisms underlying the pathophysiology of diabetic nephropathy.     

Genetic structure of Anogeissus dhofarica (Combretaceae) populations endemic to the monsoonal fog oases of the southern Arabian Peninsula

Anogeissus dhofarica (Combretaceae) is an endemic tree of the monsoon affected coastal mountains of the southern Arabian Peninsula, being the character species of the Hybantho durae–Anogeissetum dhofaricae association, a drought deciduous, monsoon forest community found only in the Dhofar region of southern Oman and the eastern Al‐Mahra region of south‐east Yemen. Due to the steep precipitation gradient from the centre to the edges in this monsoon affected area, A. dhofarica is found in two different habitat types: in continuous woodland belts of the Hawf and Dhofar mountains, and in isolated, scattered woodland patches, as found especially in the Fartak Mts (south‐east Yemen). Fifteen populations (212 individuals) from across the whole distribution area of the species were analysed using amplified fragment length polymorphism fingerprinting to: (1) evaluate the consequences of population fragmentation on the genetic diversity harboured in isolated patches versus cohering stands of the species and (2) to reconstruct the phylogeographical pattern of A. dhofarica as a consequence of oscillations in the monsoon activity during the Pleistocene and Holocene. The analysis of among‐population genetic differentiation and within‐population genetic diversity in A. dhofarica populations resulted in a lack of genetic pauperization and genetic differentiation of populations of the distinctly isolated patches of the Fartak Mts compared to the more luxurious forests of the Hawf and Dhofar regions. This is considered to be due to the high buffer capacity against the loss of genetic diversity caused by the long‐lived life‐form of the species combined with the capability to propagate clonally and the relatively recent fragmentation of Anogeissus forests into the described patches rather than due to high values of gene flow among remnant populations caused by bee pollination and anemochorical and hydrochorical diaspore dispersal. The phylogeographical pattern of the species argues for a quite recent fragmentation of a once continuous forest belt of A. dhofarica that is rather connected with climate changes in the Holocene than triggered by aridity–humidity oscillations reported for the Pleistocene. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97 , 40–51.     

A Maraviroc-Resistant HIV-1 with Narrow Cross-Resistance to Other CCR5 Antagonists Depends on both N-Terminal and Extracellular Loop Domains of Drug-Bound CCR5

CCR5 antagonists inhibit HIV entry by binding to a coreceptor and inducing changes in the extracellular loops (ECLs) of CCR5. In this study, we analyzed viruses from 11 treatment-experienced patients who experienced virologic failure on treatment regimens containing the CCR5 antagonist maraviroc (MVC). Viruses from one patient developed high-level resistance to MVC during the course of treatment. Although resistance to one CCR5 antagonist is often associated with broad cross-resistance to other agents, these viruses remained sensitive to most other CCR5 antagonists, including vicriviroc and aplaviroc. MVC resistance was dependent upon mutations within the V3 loop of the viral envelope (Env) protein and was modulated by additional mutations in the V4 loop. Deep sequencing of pretreatment plasma viral RNA indicated that resistance appears to have occurred by evolution of drug-bound CCR5 use, despite the presence of viral sequences predictive of CXCR4 use. Envs obtained from this patient before and during MVC treatment were able to infect cells expressing very low CCR5 levels, indicating highly efficient use of a coreceptor. In contrast to previous reports in which CCR5 antagonist-resistant viruses interact predominantly with the N terminus of CCR5, these MVC-resistant Envs were also dependent upon the drug-modified ECLs of CCR5 for entry. Our results suggest a model of CCR5 cross-resistance whereby viruses that predominantly utilize the N terminus are broadly cross-resistant to multiple CCR5 antagonists, whereas viruses that require both the N terminus and antagonist-specific ECL changes demonstrate a narrow cross-resistance profile.Small-molecule CCR5 antagonists are a relatively new class of drugs that block HIV entry into target cells, with the first member of this class, maraviroc (MVC), having been approved for the treatment of HIV-infected patients. These drugs bind to a hydrophobic pocket formed by the transmembrane helices of CCR5, inducing conformational changes in the extracellular loops (ECLs) of the receptor (18, 31, 39, 40, 58, 62, 64). These conformational changes can vary with different drugs, as evidenced by differential chemokine binding and HIV resistance profiles, and block the ability of HIV to use drug-bound CCR5 as a coreceptor for entry (59, 64).As with other antiretroviral agents, HIV can develop resistance to CCR5 antagonists. One pathway by which HIV can become resistant to CCR5 antagonists is via mutations in the viral envelope (Env) protein that enable it to recognize the drug-bound conformation of the coreceptor. Most of our information on this pathway has come from in vitro passaging of HIV-1 in the presence of increasing concentrations of inhibitor (2, 4, 5, 33, 41, 44, 61, 66). In most instances, the viral determinants of resistance are localized to the V3 loop of gp120 (5, 33, 41, 44, 46, 63, 66). This is as expected: the base of the V3 loop interacts with O-sulfated tyrosines in the N terminus of CCR5, while the tip of the V3 loop is thought to contact the ECLs of the receptor (14, 15, 17, 19, 26, 29, 37). Viral resistance to one CCR5 antagonist commonly results in cross-resistance to other drugs in this class, although this is not universally the case (33, 41, 60, 63, 66). Mechanistically, a number of CCR5 antagonist-resistant viruses have been shown to have increased dependence on the N-terminal domain of CCR5 (5, 34, 44, 45, 48), which is largely unaffected by drug binding and may allow viruses to tolerate drug-induced changes in ECL conformation.In contrast to several well-characterized viruses that have evolved resistance to CCR5 antagonists in vitro, few examples of patient-derived CCR5 antagonist-resistant viruses have been reported. One mechanism of resistance that has been described in patients is the outgrowth of CXCR4-tropic HIV isolates that were present at low frequencies prior to the initiation of therapy (22, 23, 35, 36, 42, 65). Due to this finding, patients undergo tropism testing prior to treatment with CCR5 antagonists, with only those harboring exclusively R5-tropic viruses considered candidates for therapy. Patient-derived viruses capable of using drug-bound CCR5 have been reported in studies using vicriviroc and aplaviroc (45, 60, 63). The aplaviroc-resistant viruses were determined to utilize the drug-bound form of the receptor by interacting primarily with the N terminus of CCR5, similar to the viruses derived by serial in vitro passaging (48).In the present study, we report the isolation of MVC-resistant Envs from a treatment-experienced patient who had a viral load rebound while on a regimen containing MVC. Viral Envs isolated from this patient at the time MVC therapy was initiated were fully sensitive to drug. However, resistance evolved over the course of 224 days, culminating in Envs that were completely resistant to inhibition but continued to use CCR5 for entry. The emergence of resistance was dependent upon changes within the V3 loop of the virus, while changes in the V4 loop modulated the magnitude of resistance. The MVC-resistant Envs studied here exhibited several unusual properties. First, while they were cross-resistant to TAK779, they remained sensitive to all other CCR5 antagonists tested, including vicriviroc and aplaviroc. Second, the Envs were particularly adept at utilizing low levels of CCR5 to mediate infection of cells. Third, and in contrast to several recent reports of CCR5 antagonist-resistant viruses, these Envs were dependent upon residues within both the N terminus and ECLs of CCR5 for efficient entry in the presence of drug. When considered in the context of other reports, our data suggest a model in which resistance to multiple CCR5 antagonists can arise if an Env protein becomes highly dependent upon the N-terminal domain of CCR5, the conformation of which appears to be unaffected by drug binding. A more narrow resistance profile results from changes in Env that enable it to use both the N-terminal domain of CCR5 as well as the drug-induced conformation of the CCR5 ECLs.     

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4+ T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient''s pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4⁺ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N′ terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4⁺ T cells in the presence of APL, with relative sparing of the central memory CD4⁺ T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T_CM subset of CD4⁺ T cells and result in improved T cell homeostasis and immune function.Entry of human immunodeficiency virus (HIV) into target cells is a complex, multistep process that is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4, which trigger conformational changes in gp120 that form and orient the coreceptor binding site (9, 24). Upon binding to coreceptor, which is either CCR5 or CXCR4 for primary HIV isolates, Env undergoes further conformational changes resulting in insertion of the gp41 fusion peptide into the host cell membrane and gp41-mediated membrane fusion (8, 15, 26). Targeting stages of the HIV entry process with antiretroviral drugs is a productive method of inhibiting HIV replication, as demonstrated by the potent antiviral effects of small-molecule CCR5 antagonists and fusion inhibitors (23, 35, 49). As with other antiretroviral drugs, HIV can develop resistance to entry inhibitors, and a detailed understanding of viral and host determinants of resistance will be critical to the optimal clinical use of these agents.The coreceptor binding site that is induced by CD4 engagement consists of noncontiguous regions in the bridging sheet and V3 loop of gp120 (4, 18, 42, 43, 50). Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sulfated tyrosine residues in the N′ terminus of CCR5, and (ii) the crown of the V3 loop is thought to engage the extracellular loops (ECLs), particularly ECL2, of CCR5 (10-12, 14, 18, 28). Small-molecule CCR5 antagonists bind to a hydrophobic pocket in the transmembrane helices of CCR5 and exert their effects on HIV by altering the position of the ECLs, making them allosteric inhibitors of HIV infection (13, 31, 32, 46, 52). The conformational changes in CCR5 that are induced by CCR5 antagonists vary to some degree with different drugs, as evidenced by differential binding of antibodies and chemokines to various drug-bound forms of CCR5 (47, 54).CCR5 antagonists are unusual among antiretroviral agents in that they bind to a host protein rather than a viral target, and therefore the virus cannot directly mutate the drug binding site to evade pharmacologic pressure. Nevertheless, HIV can escape susceptibility to CCR5 antagonists. One mechanism by which this occurs is the use of the alternative HIV coreceptor, CXCR4. In vivo, this has most often been manifest as the outgrowth of R5/X4-tropic HIV isolates that were present in the patient''s circulating viral swarm prior to therapy (17, 27, 55). A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1, 2, 22, 33, 36, 51, 56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21, 48). The Envs from this patient were cross resistant to the CCR5 antagonists AD101, TAK779, SCH-C, and maraviroc. Surprisingly, this antiretroviral-naïve patient harbored Envs resistant to aplaviroc prior to the initiation of therapy. In the present study, we have examined viral and host factors that contribute to aplaviroc resistance and examined the consequences of resistance for viral tropism. Aplaviroc resistance determinants were located within the V3 loop of gp120, although additional residues diffusely spread throughout the gp120 and gp41 proteins modulated the magnitude of drug resistance. The resistant virus displayed altered interactions between gp120 and CCR5 such that the virus became critically dependent upon the N′ terminus of drug-bound CCR5. This differential recognition of CCR5 in the presence of aplaviroc was also associated with increased dependence on a higher CCR5 receptor density for efficient virus infection and a tropism shift toward effector memory cells on primary CD4⁺ T cells.     

Xiphograptus and the evolution of virgella‐bearing graptoloids

Abstract: The virgellar spine is one of the most consistent features of the graptolite sicula. It is present in a large number of graptoloid groups, but evolved separately and independently in these as it is seen from the presence of the spine in either ventral (Axonophora) or dorsal (Phyllograptus, Xiphograptus) position. The evolution of the virgellar spine in the Pan‐Bireclinata in the Upper Dapingian to Lower Darriwilian time interval is known to follow four main steps, from a simple rutellum, through a lamelliform rutellum and a lanceolate virgella to the true virgellar spine. For the xiphograptids and in Phyllograptus, the origin and early development is less well documented, but appears to follow a similar path. However, the individual stages are condensed, and a true virgellar spine emerges already in the Floian time interval. A virgellar spine was found in Didymograptellus bifidus, necessitating a revision of the diagnosis of the genus Didymograptellus. A number of species of the virgellate genera Xiphograptus, Yutagraptus and Didymograptellus are described from isolated material for the first time. The species are useful for the biostratigraphic correlation of endemic mid‐continent North American faunas with the Pacific Type faunal realm. Xiphograptus artus sp. nov., Didymograptellus primus sp. nov. and Didymograptellus cowheadensis sp. nov. from the Cow Head Group of western Newfoundland are described as new.     

Characterization of novel microsatellite loci for red clover (Trifolium pratense L.) from enriched genomic libraries

Twenty‐seven polymorphic microsatellite markers were isolated from red clover (Trifolium pratense). Allelic variability and cross‐species amplification were assessed on 24 red clover and eight white clover (Trifolium repens) genotypes. The number of alleles detected in red clover ranged from two to 25. Observed and expected heterozygosities were high with average values of 0.71 and 0.88, respectively. Five of the 27 loci were also successfully amplified from white clover, where two to 13 alleles were detected. These highly polymorphic microsatellite loci provide powerful tools for population genetic studies as well as for marker‐assisted selection in this important forage legume species.     

In Vitro Studies on the Biosynthesis of the Surface Glycoprotein of Trypanosoma congolense1,2

Tritiated leucine, glucosamine, mannose, and galactose were incorporated into the variant specific surface glycoprotein (VSG) of Trypanosoma congolense in vitro. The uptake of the precursors is shown by SDS-polyacrylamide electrophoresis and fluorography, by assay of the radioactivity in immunoprecipitates obtained with specific antisera, and by the isolation of the labeled antigens by affinity chromatography on concanavalin A-sepharose and isoelectric focusing. The in vitro labeled VSG exhibits the same degree of microheterogeneity as that observed in the VSG isolated from trypanosomes grown in animals. Analysis of the incorporated sugars after hydrolysis of the glycoprotein showed that glucosamine and mannose were utilized in biosynthesis of the carbohydrate moiety directly whereas galactose was converted possibly to other intermediates before being incorporated into the antigen. Tunicamycin completely prevented the incorporation of the radiolabeled sugars into the surface glycoprotein. The unglycosylated VSG with a molecular weight of 47 kDa had completely lost its size heterogeneity.     

Reversal of the silver inhibition of microorganisms by agar.

Increasing use of silver in the treatment of water has necessitated an examination of microbiological methods for the measurement of silver inactivation of microorganisms. Three common agar media were tested for their ability to neutralize the bacteriostatic effects of silver. Results suggested that growth media differed in their neutralizing capacity; that is, the non-inhibitory media tryptone glucose agar and Trypticase soy agar showed more neutralizing capacity than eosin methylene blue agar. Furthermore, the neutralizing effect appeared to be a function of the soluble component of the media and not of the agar itself.