Andreas Seyfang

Telomere lengthening early in development

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

Trypanosome glucose transporters.

, by contrast adopts an intracellularenvironment within their mammalian hosts. Inva-sion and passage through different anatomicallocations within insect vectors also distinguishesthe parasites.All trypanosome species use glucose as a crucialsource of energy, and all have specific plasmamembrane transporters to facilitate the uptake ofthis molecule. Four different trypanosome glucosetransporter genes have been cloned, and theirfunction verified by expression in either

Role of an Expanded Inositol Transporter Repertoire in Cryptococcus neoformans Sexual Reproduction and Virulence

ABSTRACT Cryptococcus neoformans and Cryptococcus gattii are globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal that myo-inositol is an important factor for fungal sexual reproduction. That C. neoformans can utilize myo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important for Cryptococcus development and virulence. In accord with this central importance of inositol, an expanded myo-inositol transporter (ITR) gene family has been identified in Cryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members in C. neoformans and at least six members in the sibling species C. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important for myo-inositol stimulation of mating and that functional redundancies among the myo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence. IMPORTANCE Cryptococcus neoformans is an AIDS-associated human fungal pathogen that causes over 1 million cases of meningitis annually and is the leading cause of fungal meningitis in immunosuppressed patients. The initial cryptococcal infection is caused predominantly via inhalation of sexual spores or desiccated yeast cells from the environment. How this fungus completes its sexual cycle and produces infectious spores in nature and why it frequently infects the central nervous system to cause fatal meningitis are critical questions that remain to be understood. In this study, we demonstrate that inositol acquisition is important not only for fungal sexual reproduction but also for fungal virulence. We identified an expanded inositol transporter gene family that contains over 10 members, important for both fungal sexual reproduction and virulence. Our work contributes to our understanding of how fungi respond to the environmental inositol availability and its impact on sexual reproduction and virulence. Cryptococcus neoformans is an AIDS-associated human fungal pathogen that causes over 1 million cases of meningitis annually and is the leading cause of fungal meningitis in immunosuppressed patients. The initial cryptococcal infection is caused predominantly via inhalation of sexual spores or desiccated yeast cells from the environment. How this fungus completes its sexual cycle and produces infectious spores in nature and why it frequently infects the central nervous system to cause fatal meningitis are critical questions that remain to be understood. In this study, we demonstrate that inositol acquisition is important not only for fungal sexual reproduction but also for fungal virulence. We identified an expanded inositol transporter gene family that contains over 10 members, important for both fungal sexual reproduction and virulence. Our work contributes to our understanding of how fungi respond to the environmental inositol availability and its impact on sexual reproduction and virulence.

Aspartate 19 and glutamate 121 are critical for transport function of the myo-inositol/H+ symporter from Leishmania donovani

The protozoan flagellate Leishmania donovani has an active myo-inositol/proton symporter (MIT), which is driven by a proton gradient across the parasite membrane. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopusoocytes and overexpression in Leishmania transfectants to investigate the significance of acidic transmembrane residues for proton relay and inositol transport. MIT has only three charged amino acids within predicted transmembrane domains. Two of these residues, Asp19 (TM1) and Glu121 (TM4), appeared to be critical for transport function of MIT, with a reduction of inositol transport to about 2% of wild-type activity when mutated to the uncharged amides D19N or E121Q and 20% (D19E) or 4% (E121D) of wild-type activity for the conservative mutations that retained the charge. Immunofluorescence microscopy of oocyte cryosections showed that MIT mutants were expressed on the oocyte surface at a similar level as MIT wild type, confirming that these mutations affect transport function and do not prevent trafficking of the transporter to the plasma membrane. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone and dinitrophenol inhibited inositol transport by 50–70% in the wild-type as well as in E121Q, despite its reduced transport activity. The mutant D19N, however, was stimulated about 4-fold by either protonophore and 2-fold by cyanide or increase of pH 7.5 to 8.5 but inhibited at pH 6.5. The conservative mutant D19E, in contrast, showed an inhibition profile similar to MIT wild type. We conclude that Asp19 and Glu121 are critical for myo-inositol transport, while the negatively charged carboxylate at Asp19 may be important for proton coupling of MIT.

Substrate depletion upregulates uptake of myo-inositol, glucose and adenosine in Leishmania

Leishmania flagellates undergo a digenetic life cycle in the gut of the sandfly insect vector and in macrophage phagolysosomes of the mammalian host. This involves vast changes of the environment to which the parasite has to adapt, including temperature, pH and concentration of nutrients between different types of meals of the insect vector or within the enclosed intracellular environment of the phagolysosome. The regulation of transporters for important organic substrates in Leishmania donovani, Leishmania mexicana and Leishmania enriettii has been investigated. A pronounced upregulation of inositol (25-fold), adenosine (11-fold) or glucose (5-fold) uptake activities was found when cells were depleted of the respective substrates during culture. Inositol-depleted cells showed a half-maximal uptake rate at nanomolar inositol concentration. Depletion of inositol only affected inositol uptake but did not affect uptake of glucose analog or proline in control experiments, indicating the specificity of the mechanism(s) underlying transport regulation. Adenosine-depleted cells showed an approximately 10-fold increase in both adenosine and uridine uptake, both mediated by the L. donovani nucleoside transporter 1 (LdNT1), but no change in guanosine uptake, which is mediated by the L. donovani nucleoside transporter 2 (LdNT2). These results suggest that extracellular adenosine concentration specifically regulates LdNT1 transport activity and does not affect LdNT2. The data imply that upregulation of transport activities by substrate depletion is a general phenomenon in protozoan flagellates, which is in remarkable contrast to bacteria where upregulation typically follows an increase of extracellular organic substrate. Hence, the parasites can maximize the uptake of important nutrients from the host even under limiting conditions, whereas bacteria often have dormant stages (spores) to overcome unfavorable environmental conditions or are heterotrophic for organic substrates.

Relationship Between Toxoplasma gondii and Mood Disturbance in Women Veterans

BACKGROUND Toxoplasma gondii is an intracellular protozoan parasite with zoonotic potential that causes acute and chronic diseases, which has been associated with schizophrenia, depression, bipolar disorder, and suicidal behavior. Military personnel may be at increased risk for exposure to the parasite when deployed to countries with high prevalence rates. METHODS Women Veterans were recruited to participate in the study at an event to recognize women Veterans and later through e-mails. Blood samples were collected from 70 women Veterans (mean age: 47 years) and analyzed for T. gondii IgG titer. Participants completed a demographic instrument, Center for Epidemiologic Studies Depression scale, Profile of Mood States (POMS), and Post-Traumatic Stress Disorder Checklist-Military. RESULTS The infectivity rate was lower than the rate in the United States (11.4% [8 out of 70 were seropositive], but 6 of the 8 [75%] had been deployed outside the United States. Pearson correlations and t tests showed significant relationships between T. gondii seropositivity and Center for Epidemiologic Studies Depression score), POMS-depression, POMS-confusion, and POMS-anger subscale scores, and total mood disturbance score. CONCLUSIONS This study is the first to describe biobehavioral relationships between chronic T. gondii infection, depression, and dysphoric moods in a military veteran population.

Trypanosoma brucei sspp.: Cleavage of variant specific and common glycoproteins during exposure of live cells to trypsin

Intact bloodstream forms of Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense and procyclic forms of T.b. brucei and T.b. gambiense were incubated in trypsin, solubilized for gel electrophoresis, and analyzed for removal of surface molecules. Silver-stained gels and transfer blots probed with horseradish peroxidase-conjugated or radiolabeled lectins revealed that only three glycoproteins, Gp120p, Gp91p, and Gp23p, were removed from the surface of procyclic forms by trypsin. The variant specific glycoproteins, Gp23b, Gp120b, and in some clones Gp91b were surface molecules cleaved from bloodstream forms. Greater than 90% of the variant specific glycoprotein (VSG) was removed from the surface of all clones studied within 1 hr following the addition of trypsin. The removal of VSG was coincident with appearance of 37 to 50 kDa glycopeptide fragments of VSG with different clones yielding different sized fragments. Detailed kinetic analysis of proteins from whole cell extracts and supernatants of the DuTat 1.1 clone of T.b. rhodesiense using concanavalin A (Con A) and polyclonal antibodies revealed that three major VSG fragments were released during trypsinization. The electrophoretic mobility of the three VSG fragments of DuTat 1.1 was not altered when samples were boiled in sodium dodecyl sulfate to inhibit the endogenous phospholipase C. Antiserum to the cross-reactive determinant bound to intact VSG, but did not bind VSG fragments. Thus, the major Con A binding fragments of DuTat 1.1 VSG and perhaps those of the other clones we studied were probably derived from the N-terminal domain of the molecule. The data suggest that VSG is cleaved by trypsin in situ at the hinge region, but remains attached to the cell surface via weak interaction with neighboring molecules.

Assessment of an Adjusted vs. Fixed Pass Line for Student Performance in a Medical School Curriculum

Starting in Fall 2011, USF Morsani College of Medicine has implemented a new pass-fail grading system using an adjusted pass line for the assignment of grades in Year 1 basic science courses, which incorporates the class mean and distribution of exam grades (‘mean minus two standard deviations’ pass line). This replaces the prior fixed pass line of 70% correct answers. In recent reports, improved psychological well-being (reduced stress and anxiety) and decreased competitiveness, combined with an environment that fosters cooperative learning were discussed as major factors and principal attractions in moving toward a simple pass-fail grading system. 1,2 Here, we report on resultant changes in class performance during the academic year 2011-2012.