Deletion of Ubiquitin Fold Modifier Protein Ufm1 Processing Peptidase Ufsp in L. donovani Abolishes Ufm1 Processing and Alters Pathogenesis

Previously, we showed Leishmania donovani Ufm1 has a Gly residue conserved at the C-terminal region with a unique 17 amino acid residue extension that must be processed prior to conjugation to target proteins. In this report, we describe for the first time the isolation and characterization of the Leishmania Ufm1-specific protease Ufsp. Biochemical analysis of L. donovani Ufsp showed that this protein possesses the Ufm1 processing activity using sensitive FRET based activity probes. The Ufm1 cleavage activity was absent in a mutant Ufsp in which the active site cysteine is altered to a serine. To examine the effects of abolition of Ufm1 processing activity, we generated a L. donovani null mutant of Ufsp (LdUfsp^−/−). Ufm1 processing activity was abolished in LdUfsp^−/− mutant, and the processing defect was reversed by re-expression of wild type but not the cys>ser mutant in the LdUfsp^−/− parasites. Further LdUfsp^−/− mutants showed reduced survival as amastigotes in infected human macrophages but not as promastigotes. This growth defect in the amastigotes was reversed by re-expression of wild type but not the cys>ser mutant in the Ufsp^−/− indicating the essential nature of this protease for Leishmania pathogenesis. Further, mouse infection experiments showed deletion of Ufsp results in reduced virulence of the parasites. Additionally, Ufsp activity was inhibited by an anti-leishmanial drug Amphotericin B. These studies provide an opportunity to test LdUfsp^−/− parasites as drug and vaccine targets.     

Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) chemical control: insecticide efficacy associated with the three consecutive spray strategy

Abstract  After the initial 1993 discovery of Frankliniella occidentalis , western flower thrips, in Australia, temporary chemical control permits were issued to facilitate their insecticidal control. However, these permits were based on established endemic pests of Australian horticulture rather than demonstrated efficacy against F. occidentalis , and laboratory bioassays of Australian field-collected F. occidentalis suggested that some permit application rates were too low to be effective. In 2002 and 2003 field-based trials were carried out in strawberry and lettuce to collect field-generated efficacy data to verify the laboratory conclusions. It was found that by increasing field application rates above the existing permit rate, efficacy was increased, but not for all insecticides and not for all thrips life stages. Increasing the rate of abamectin and endosulfan in strawberry increased control of adults and larvae and increasing the rate of endosulfan in lettuce increased efficacy against larvae only. Spinosad was effective at both rates tested in strawberry, suggesting that the current permit rate could be reduced. The ability to use reduced rates may be useful if used in combination with Integrated Pest Management programs.     

Comparison of three kinetic models of HIV-1 infection: implications for optimization of treatment

Clinical markers in the peripheral blood guide the treatment of human immunodeficiency virus type 1 (HIV-1). Likewise, many of the theoretical models developed to simulate infection only incorporate variables in the blood. To test the suitability of blood-only models, three distinct models of HIV infection kinetics are compared: "full model" including latently and actively infected cells and virus in the peripheral blood and lymphoid tissue (LT); "reduced model", including peripheral blood and LT without latent cells; and "blood model" including only actively infected cells and virus in the peripheral blood. Using the same parameter values for all three, qualitative differences are demonstrated between the blood model and its more inclusive counterparts. Additionally, optimization studies show that the reduced and blood models generate progressively lower optimal treatment levels relative to the full model when constant-level treatment is considered. These findings indicate that including the lymphoid tissue and latently infected cells into kinetic models may lead to differing conclusions with regard to optimal treatment and could be useful in guiding therapy even when plasma viral levels are below detectable limits.     

Ubiquitous expression of a gene encoding for uncoupling protein isolated from the thermogenic inflorescence of the dead horse arum Helicodiceros muscivorus

Uncoupling proteins (UCPs) are a family of mitochondrial inner membrane proteins that have been implicated in heat production in mammalian cells. The inflorescences of several members of the arum lily family (Araceae) have also been shown to produce heat during flowering, but the involvement of UCP-mediated heat production in plants is not known. In this work a gene has been isolated termed HmUCPa that encodes for a putative uncoupling protein from Helicodiceros muscivorus, a highly thermogenic arum lily. RT-PCR analysis revealed that the expression of HmUCPa was ubiquitously found, both in thermogenic male florets and appendix, and the non-thermogenic female florets, spathe and club-shaped organs of the spadix. These results suggest that HmUCPa is not primarily involved in organ-specific heat production in H. muscivorus.     

The Drosophila cinnamon gene is functionally homologous to Arabidopsis cnx1 and has a similar expression pattern to the mammalian gephyrin gene.

Molybdoenzymes are involved in a variety of essential pathways including nitrate assimilation, sulfur and/or purine metabolism and abscisic acid biosynthesis. Most organisms produce several such enzymes requiring a molybdopterin cofactor for catalytic function. Mutations that result in a lack of the molybdopterin cofactor display a pleiotropic loss of molybdoenzyme activities, and this phenotype has been used to identify genes involved in cofactor biosynthesis or utilization. Although several cofactor genes have been analyzed in prokaryotes, much less is known concerning eukaryotic molybdenum cofactor (MoCF) genes. This work is focused on the Drosophila MoCF gene cinnamon (cin) which encodes a multidomain protein, CIN, that shows significant similarity to three proteins encoded by separate prokaryotic MoCF genes. These domains are also present in the product of cnx1, an Arabidopsis MoCF gene, and in GEPHYRIN, a rat protein thought to organize the glycine receptor, GlyR, within the postsynaptic membrane. Since this apparent consolidation of separate prokaryotic genes into a single eukaryotic gene is a feature of other conserved metabolic pathways, we wished to determine whether the protein's function is also conserved. This report shows that the plant gene cnx1 can rescue both enzymatic and physiological defects of Drosophila carrying cin mutations, indicating that the two genes serve similar or identical functions. In addition, we have investigated the relationship between CINNAMON and GEPHYRIN, using immunohistochemical methods to localize the CIN protein in Drosophila embryos. Most of the CIN protein, like GEPHYRIN in the rat CNS, is localized to the cell borders and shows a tissue-specific pattern of expression. In a parallel study, antibody to GEPHYRIN revealed the same tissue-specific expression pattern in fly embryos. Both antibodies show altered staining patterns in cin mutants. Taken together, these results suggest that GEPHYRIN may also carry out a MoCF-related function. Received: 14 September 1998 / Accepted: 8 March 1999     

Plasma proteome profiling of healthy individuals across the life span in a Sicilian cohort with long‐lived individuals

The study of healthy human aging is important for shedding light on the molecular mechanisms behind aging to promote well‐being and to possibly predict and/or avoid the development of age‐related disorders such as atherosclerosis and diabetes. Herein, we have employed an untargeted mass spectrometry‐based approach to study age‐related protein changes in a healthy Sicilian plasma cohort including long‐lived individuals. This approach confirmed some of the previously known proteins correlated with age including fibulin‐1, dystroglycan, and gamma‐glutamyl hydrolase. Furthermore, our findings include novel proteins that correlate with age and/or with location and uric acid, which could represent a unique signature for healthy aging.