The Fowler Syndrome-Associated Protein FLVCR2 Is an Importer of Heme

Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders.Membrane transporters play essential roles in cellular homeostasis by importing substrates critical for cell growth and differentiation or by exporting substrates that cause toxicity. There are five major categories of membrane transporters consisting of over 550 transporter superfamilies (41). The major facilitator superfamily (MFS) is the largest and most diverse superfamily, consisting of over 10,000 members (31, 41). Transporters in this superfamily consist of 12 to 14 transmembrane (TM)-spanning segments and transport substrates as diverse as sugars, polyols, drugs, neurotransmitters, amino acids, organic/inorganic ions, and peptides (31). Recently, a disruption of MFS transporters that is associated with human diseases has been described, further confirming their role in the maintenance of normal cell homeostasis. The DIRC2 MFS transporter (substrate transported unknown) is disrupted in renal cell carcinoma cosegregating with a t(2;3)(q35;q21) chromosomal translocation (4). Mutations in the thiamine transporter THTR1 have been shown to be responsible for Rogers syndrome (14, 21), a thiamine-responsive megaloblastic anemia. We have recently reported that a disruption in the heme exporter FLVCR1 (MFSD7B) plays a role in Diamond Blackfan anemia (DBA) (40), a fatal infant anemia characterized by a block in erythroid progenitor cell development (3, 12, 13). The abrogation of FLVCR1 function in primary human hematopoietic stem cells (40) or in a human erythroid cell line (37) specifically disrupts erythropoiesis, mimicking the hematological features observed for patients with DBA. We have reported previously that FLVCR1 is disrupted not as a consequence of mutations in the FLVCR1 coding region but due to the aberrant splicing of specific FLVCR1 exons that reduces the expression and cell surface localization of the encoded FLVCR1 protein (40). Interestingly, the THTR1 and FLVCR1 proteins were shown previously to function as receptors for entry by feline leukemia retrovirus (FeLV) subgroup A (FeLV-A) (25) and FeLV-C (36, 46), respectively. These viruses disrupt the cellular function of these proteins in infected cats and can induce diseases that correlate with Rogers syndrome (17) and DBA (1, 28).Recently, mutations in the cell surface protein FLVCR2 (MFSD7C), an MFS transporter member, have been shown to be associated with Fowler syndrome (22, 26), a proliferative vascular disorder of the brain (16). A previous study (6) suggested that FLVCR2 functions as a calcium-chelate transporter based on its expression in murine and human tissues involved in calcium homeostasis. We have shown previously that FLVCR2 is highly related to the heme exporter/retroviral receptor FLVCR1 (7), and we have recently shown it to function as a receptor for the subgroup C FeLV variant FY981 (42). Based on its close sequence relationship to the heme exporter/retroviral receptor FLVCR1 and based on previous reports showing that retroviruses often adapt to use closely related cell surface proteins as receptors for infection (27, 30, 44), we investigated the heme transport function of FLVCR2. Here, we show the physiological function of FLVCR2 as an importer of heme.     

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.     

Variation in resource acquisition and use among host clones creates key epidemiological trade‐offs

Parasites can certainly harm host fitness. Given such virulence, hosts should evolve strategies to resist or tolerate infection. But what governs those strategies and the costs that they incur? This study illustrates how a fecundity‐susceptibility trade‐off among clonally reared genotypes of a zooplankton (Daphnia dentifera) infected by a fungal parasite (Metschnikowia) arises due to variation in resource acquisition and use by hosts. To make these connections, we used lab experiments and theoretical models that link feeding with susceptibility, energetics, and fecundity of hosts. These feeding‐based mechanisms also produced a fecundity‐survivorship trade‐off. Meanwhile, a parasite spore yield–fecundity trade‐off arose from variation in juvenile growth rate among host clones (another index of resource use), a result that was readily anticipated and explained by the models. Thus, several key epidemiological trade‐offs stem from variation in resource acquisition and use among clones. This connection should catalyze the creation of new theory that integrates resource‐ and gene‐based responses of hosts to disease.     

Resuspending ram spermatozoa in seminal plasma after cryopreservation does not improve pregnancy rate in cervically inseminated ewes

The role of seminal plasma (SP) components on the maintenance of motility, viability and fertilising ability of frozen-thawed spermatozoa is of considerable interest. However, differences observed in constituents of SP among males could explain differences in fertility obtained in vivo. Two experiments were designed to examine the effects of seminal plasma on fertility from cervically inseminated frozen-thawed semen. The objective of Experiment 1 was to investigate if source or type of SP influences pregnancy rate. Seminal plasma was collected from rams previously classified as having either High (HSP; n=3) or Low (LSP; n=3) fertility in vivo. Artificial SP (fructose/sodium solution with 10% BSA; ASP) was made. Frozen semen from the same 6 rams was thawed and inseminated (Control) or resuspended either in HSP, LSP or ASP (20% in semen) prior to insemination of ewes (n=284, over 2 farms). The overall pregnancy rate was 28.1%. Treatments (Control, ASP, HSP and LSP) were not significantly different (P>0.3). There was no difference between HSP and LSP (P>0.5), and no effect of using ASP compared to ram SP (P>0.7), on pregnancy rate. As there was no effect of SP on pregnancy rate a repeat experiment (Experiment 2) was designed to test the effect of washing and selecting motile sperm prior to resuspending in phosphate-buffered saline (PBS) containing SP on pregnancy rate. Frozen-thawed semen from each of 2 rams was centrifuged through a density gradient, pellets were centrifuged through a wash medium and the sperm concentration/ram was counted. Sperm cells were resuspended in: (1) control PBS, (2) PBS containing 30% HSP or (3) PBS containing 30% LSP to give 100 x 10(6) motile sperm in 0.25 mL. Control straws were thawed and inseminated directly. Ewes (n=223 over 2 farms) were inseminated 57 h post-sponge withdrawal and those not returning to oestrus were slaughtered 29-50 days post-insemination for pregnancy determination. In Experiment 2, the pregnancy rate for Control, PBS, HSP and LSP were 15.4%, 2.3%, 0% and 0%, respectively, for Farm 1 (P>0.05) and 17.8%, 11.0%, 3.9% and 12.4%, respectively, for Farm 2. Under the conditions of the current study, addition of SP from different donors of either High or Low fertility status to frozen-thawed ram semen post-thawing did not improve pregnancy rate in ewes. ASP had no effect on pregnancy rate in ewes when added to frozen-thawed semen. Washing and selection of motile sperm prior to resuspension in PBS with or without SP (30%) before insemination had a negative effect on pregnancy rate in cervically inseminated ewes. Hence, the addition of seminal plasma or some of its constituents to semen does not appear to improve pregnancy rate in cervically inseminated ewes.     

Postnatal exposure to octylphenol decreases semen quality in the adult ram

The aim of this experiment was to determine if maternal exposure to octylphenol pre- and/or postnatally influenced FSH concentrations and semen quantity and quality in postpubertal rams. Rams were born to ewes that received twice-weekly s.c. injections of octylphenol equivalent to 1000microg/kg/day for one of the following periods: (1) day 70 of gestation (D70) to weaning (at 20 weeks postnatally; n=4); (2) D70 to birth (n=6); (3) birth to weaning (n=7), controls received corn oil from D70 to weaning (n=5). Rams were blood-sampled weekly and semen characteristics were evaluated at 1 year of age. Maternal exposure to octylphenol, pre- and/or postnatally did not affect FSH concentrations, semen volume, concentration, percentage live, motility or IVM/IVF characteristics. However, exposure to octylphenol from birth to weaning increased the number of morphologically abnormal sperm cells in the ejaculates of these rams.     

Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease

Congenital transmission of Trypanosoma cruzi may occur in some or all the gestations from a T. cruzi-infected mother. Variable rates of congenital transmission have been reported in different geographical areas where different parasitic strains predominate, suggesting that parasitic genotypes might play a role in the risk of congenital transmission. Moreover, in cases of transmission it is unknown if the whole maternal T. cruzi population or certain clones are preferentially transmitted by the transplacental route. In this study, bloodstream T. cruzi lineages were identified in blood samples from congenitally infected children, transmitting and non-transmitting mothers and unrelated Chagas disease patients, using improved PCR strategies targeted to nuclear genomic markers. T. cruzi IId was the prevalent genotype among 36/38 PCR-positive congenitally infected infants, 5/5 mothers who transmitted congenital Chagas disease, 12/13 mothers who delivered non-infected children and 28/34 unrelated Chagas disease patients, all coming from endemic localities of Argentina and Bolivia. These figures indicate no association between a particular genotype and vertical transmission. Furthermore, minicircle signatures from the maternal and infants' bloodstream trypanosomes were profiled by restriction fragment length polymorphism of the 330-bp PCR-amplified variable regions in seven cases of mothers and congenitally infected infants. Minicircle signatures were nearly identical between each mother and her infant/s and unique to each mother-infant/s case, a feature that was also observed in twin deliveries. Moreover, allelic size polymorphism analysis of microsatellite loci from populations transmitted to twins showed that all clones from the maternal polyclonal population were equally infective to both siblings.     

The use of angler diary surveys to evaluate long-term changes in muskellunge populations on Lake of the Woods, Ontario

Reported improvements in the muskellunge angling fishery on Lake of the Woods, Ontario over the last two decades have paralleled an increase in the practice of catch and release angling, and an increase in minimum size regulations for this species. The overall status of muskellunge populations in this large, complex lake has proven difficult to monitor using standard assessment methods. A volunteer muskellunge angler diary program, established in 1988, has provided a cost effective method of gathering a large amount of information with which to track this fishery and associated populations. Results from angling diaries indicated increased angling effort, catch and success rates for muskellunge on Lake of the Woods since the early 1990s. Although the month of July accounted for the majority of angling effort and catch, angling success rates and sizes of fish reported in diaries improved monthly into the late fall. Angling success rates were consistently higher in angling diaries than from creel surveys, but both survey types showed similar long-term trends in the fishery. Angler diary data, incorporating both the numbers of fish caught and/or seen by anglers, were used to calculate catch equality indices which proved to be sensitive to changes in population abundance. Increased minimum length regulations for muskellunge during 1987–2001 have been largely responsible for a decline in harvest rates from an estimated 36% in 1986 to 0% since 1999. Although higher size limits have yet to produce more quality-sized fish in angler catches, diary survey data, supported by recent improvements in catch rates from assessment gill nets, would indicate that muskellunge recruitment has increased. This article concludes with a brief review of how muskellunge angler diary data has been used in the past, including recommendations to minimize biases associated with this survey method.