Characterization of a Giardia lamblia WB C6 clone resistant to the isoflavone formononetin

Giardia lamblia is a common intestinal-dwelling protozoan and causes diarrhoea in humans and animals worldwide. For several years, a small number of drugs such as the 5-nitroimidazole metronidazole (MET) or the thiazolide nitazoxanide (NTZ) have been used for chemotherapy against giardiasis. However, various pre-clinical and clinical investigations revealed that antigiardial chemotherapy may be complicated by emergence of giardial resistance to these drugs. The present study addressed the question if isoflavones with antigiardial activity, such as daidzein (DAI) or formononetin (FOR), may serve as alternative compounds for treatment of giardiasis. For this purpose, the potential of G. lamblia clone WB C6 to form resistance to FOR and related isoflavones was tested in vitro. In the line of these experiments, a clone (C3) resistant to isoflavones, but sensitive to MET and NTZ, was generated. Affinity chromatography on DAI-agarose using cell-free extracts of G. lamblia trophozoites resulted in the isolation of a polypeptide of approximately 40 kDa, which was identified by mass spectrometry as a nucleoside hydrolase (NH) homologue (EAA37551.1). In a nucleoside hydrolase assay, recombinant NH hydrolysed all nucleosides with a preference for purine nucleosides and was inhibited by isoflavones. Using quantitative RT-PCR, the expression of genes that are potentially involved in resistance formation was analysed, namely NH and genes encoding variant surface proteins (VSPs, TSA417). The transcript level of the potential target NH was found to be significantly reduced in C3. Moreover, drastic changes were observed in VSP gene expression. This may indicate that resistance formation in Giardia against isoflavones is linked to, and possibly mediated by, altered gene expression. Taken together, our results suggest FOR or related isoflavones as an alternative antigiardial agent to overcome potential problems of resistance to drugs like MET or NTZ. However, the capacity of Giardia to develop resistance to isoflavones can potentially interfere with this alternative treatment of the disease.

A novel Giardia lamblia nitroreductase, GlNR1, interacts with nitazoxanide and other thiazolides

The nitrothiazole analogue nitazoxanide [NTZ; 2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] represents the parent compound of a class of drugs referred to as thiazolides and exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. NTZ and other thiazolides are active against a wide range of other intracellular and extracellular protozoan parasites in vitro and in vivo, but their mode of action and respective subcellular target(s) have only recently been investigated. In order to identify potential targets of NTZ and other thiazolides in Giardia lamblia trophozoites, we have developed an affinity chromatography system using the deacetylated derivative of NTZ, tizoxanide (TIZ), as a ligand. Affinity chromatography on TIZ-agarose using cell extracts of G. lamblia trophozoites resulted in the isolation of an approximately 35-kDa polypeptide, which was identified by mass spectrometry as a nitroreductase (NR) homologue (EAA43030.1). NR was overexpressed as a six-histidine-tagged recombinant protein in Escherichia coli, purified, and then characterized using an assay for oxygen-insensitive NRs with dinitrotoluene as a substrate. This demonstrated that the NR was functionally active, and the protein was designated GlNR1. In this assay system, NR activity was severely inhibited by NTZ and other thiazolides, demonstrating that the antigiardial activity of these drugs could be, at least partially, mediated through inhibition of GlNR1.

Streptozotocin-induced diabetes mellitus in pigs

We induced, as a precondition for a pancreas transplant, insulin-dependent diabetes mellitus in 67 Yorkshire Landrace pigs by administering streptozotocin. A dosage of 150 mg/kg body weight gave rise to a long-lasting diabetes mellitus that persisted with time (follow-up period: 26 weeks). Consecutive measurements of serum glucose and plasma insulin, before and up to 30 hours after administering streptozotocin, revealed triphasic behavior: initial hyperglycemia (1st to 3rd hour), pronounced hypoglycemia (12th to 18th hour), then hyperglycemia (22nd hour on). IVGTTs done 1 to 7 days after administering streptozotocin revealed a reduction of the K-value (glucose disappearance rate) from 0.3 (day 2) to 0.07 (day 4). Immunohistochemical studies revealed a complete loss of all beta-cells, concomitantly with a relative increase in glucagon- and somatostatin-positive cells. We also observed a complete loss of pp (pancreatic polypeptide)-positive cells. Diabetes induced by streptozotocin at 150 mg/kg body weight is complete and permanent; our mortality rate was 0%. Given the high morbidity rate after pancreatectomy, streptozotocin should be the method of choice for inducing diabetes mellitus in pigs.

Time-related, cross-sectional and prospective follow-up of pancreatic endocrine function after pancreas allograft transplantation in type 1 (insulin-dependent) diabetic patients

It has been established that successful pancreas transplantation in Type 1 (insulin-dependent) diabetic patients results in normal but exaggerated phasic glucose-induced insulin secretion, normal intravenous glucose disappearance rates, improved glucose recovery from insulin-induced hypoglycaemia, improved glucagon secretion during insulin-induced hypoglycaemia, but no alterations in pancreatic polypeptide responses to hypoglycaemia. However, previous reports have not segregated the data in terms of the length of time following successful transplantation and very little prospective data collected over time in individual patients has been published. This article reports that in general there are no significant differences in the level of improvement when comparing responses as early as three months post-operatively up to as long as two years post-operatively when examining the data cross-sectionally in patients who have successfully maintained their allografts. Moreover, this remarkable constancy in pancreatic islet function is also seen in a smaller group of patients who have been examined prospectively at various intervals post-operatively. It is concluded that successful pancreas transplantation results in remarkable improvements in Alpha and Beta cell but not PP cell function that are maintained for at least one to two years.

Pancreatic endocrine function in cystic fibrosis

To characterize pancreatic endocrine secretion and to examine interrelationships among alterations in alpha, beta, and pancreatic polypeptide cell function in patients with cystic fibrosis (CF), we studied 19 patients with exocrine insufficiency (EXO), including 9 receiving insulin therapy (EXO-IT); 10 patients with no exocrine insufficiency (NEXO); and 10 normal control subjects. First-phase C-peptide response to intravenously administered glucose was significantly impaired in CF patients with exocrine insufficiency (EXO-IT = 0.02 +/- 0.01; EXO = 0.11 +/- 0.02; NEXO = 0.25 +/- 0.05; control subjects = 0.30 +/- 0.04 nmol/L). Lowering fasting glucose levels with exogenous insulin administration in EXO-IT did not improve beta cell responsivity to glucose. The C-peptide response to arginine was less impaired (EXO-IT = 0.12 +/- 0.02; EXO = 0.15 +/- 0.02; NEXO = 0.23 +/- 0.06; control subjects = 0.28 +/- 0.04 nmol/L). Alpha cell function, measured as peak glucagon secretion in response to hypoglycemia, was diminished in EXO but not NEXO (EXO-IT = 21 +/- 10; EXO = 62 +/- 19; NEXO = 123 +/- 29; control subjects = 109 +/- 12 ng/L). Despite diminished glucagon response, EXO patients recovered normally from hypoglycemia. Peak pancreatic polypeptide response to hypoglycemia distinguished CF patients with exocrine insufficiency from those without exocrine insufficiency (EXO-IT = 3 +/- 2; EXO = 3 +/- 1; NEXO = 226 +/- 68; control subjects = 273 +/- 100 pmol/L). Thus CF patients with exocrine disease have less alpha, beta, and pancreatic polypeptide cell function than CF patients without exocrine disease. These data suggest either that exocrine disease causes endocrine dysfunction in CF or that a common pathogenic process simultaneously and independently impairs exocrine and endocrine function.

When small non-coding RNAs meet the ribosome: Tuning the translational machinery

The functions of ribosomes in translation are complex and involve different types of activities critical for decoding the genetic code, linkage of amino acids via amide bonds to form polypeptide chains, as well as the release and proper targeting of the synthesized protein. Non-protein-coding RNAs (ncRNAs) have been recognized to be crucial in establishing regulatory networks.1 However all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. The main goal of this project is to identify potential novel ncRNAs that directly bind and possibly regulate the ribosome during protein biosynthesis. To address this question we applied various stress conditions to the archaeal model organism Haloferax volcanii and deep-sequenced the ribosome-associated small ncRNA interactome. In total we identified 6.250 ncRNA candidates. Significantly, we observed the emersed presence of tRNA-derived fragments (tRFs). These tRFs have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNAs. Here we present evidence that tRFs from H. volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome a 26 residue long fragment originating from the 5’ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production.2 Currently we are investigating the binding site of this tRF on the 30S subunit in more detail.

Functional proteomics and biochemsitry: working together to unravel mitochondrial phenomena of African trypansomes

Eukaryotic cells are compartmentalized into membrane-bound organelles in order to provide sheltered reaction rooms for various specific processes. Organelles are not randomly distributed in a cell or operate isolated from each other. At the contrary — some organelles are closely linked and their functions are tightly orchestrated. The most well-known example of two such organelles acting in concert are the ER and the mitochondrion that work together in order to coordinate cellular lipid biosynthesis, maintain Ca2+-homeostasis, regulate mitochondrial division and control mitochondrial/ER shape as well as to synchronize the movement of these organelles within a cell. To study the mitochondrion and its interface to the ER requires a simplified mitochondrial system. African trypanosomes represent such a system. The unicellular parasite that causes devastating diseases in humans and animals has only one large mitochondrion that does not undergo fission/fusion events except for the context of cell division. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the protozoan. Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal MOM proteome consists of 82 proteins, two thirds of which have never been associated with mitochondria before. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. Interestingly, a putative tethering complex in mammalian cells was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating, if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved. This ER-subdomain associated with mitochondria has been termed mitochondria-associated ER-membranes or MAM. The MAM has recently been implicated to play a key role in Alzheimer’s disease. It is therefore of broad and general interest to establish other eukaryotic model systems in order to investigate the MAM-MOM connection in more detail.

Functional proteomics and biochemsitry: working together to unravel mitochondrial phenomena of African trypansomes

Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of the mitochondrial model organism T. brucei and characterized its proteome. Our results show that the trypanosomal MOM proteome consists of 82 proteins. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. In mammalian cells, a putative tethering complex was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved.

In vitro rescue of FGA deletion by lentiviral transduction of an afibrinogenemic patient's hepatocytes.

BACKGROUND: Congenital afibrinogenemia is a rare inherited autosomal recessive disorder in which a mutation in one of three genes coding for the fibrinogen polypeptide chains Aα, Bβ and γ results in the absence of a functional coagulation protein. A patient with congenital afibrinogenemia, resulting from an FGA homozygous gene deletion, underwent an orthotopic liver transplant that resulted in complete restoration of normal hemostasis. The patient's explanted liver provided a unique opportunity to further investigate a potential novel treatment modality. OBJECTIVE: To explore a targeted gene therapy approach for patients with congenital afibrinogenemia. METHODS AND RESULTS: At the time of transplant, the patient's FGA-deficient hepatocytes were isolated and transduced with lentiviral vectors encoding the human fibrinogen Aα-chain. FGA-transduced hepatocytes produced fully functional fibrinogen in vitro. CONCLUSIONS: Orthotopic liver transplantation is a possible rescue treatment for failure of on-demand fibrinogen replacement therapy. In addition, we provide evidence that hepatocytes homozygous for a large FGA deletion can be genetically modified to restore Aα-chain protein expression and secrete a functional fibrinogen hexamer.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro.

Pestivirus N(pro) is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N(pro) blocks the host׳s interferon response by inducing degradation of interferon regulatory factor-3. N(pro׳)s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N(pro)-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N(pro) proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N(pro׳)s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N(pro) does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus.

Purification and characterization of a specific antigen from Echinococcus multilocularis.

A polypeptide (Em2a) purified by affinity chromatography from the Echinococcus multilocularis metacestode showed a high degree of purity as assayed by SDS-PAGE and analytical isoelectrical focusing. A minor contamination with host albumin was revealed. Estimation of relative mol. mass gave a value of 54,000. The isoelectric point was found to be 4.8. Antigenic activity of the polypeptide was demonstrated by immunoprecipitation and western blotting. In these assays the protein was recognized only by homologous sera from patients infected with larval E. multilocularis. This antigen (Em2a) did not react in the ELISA with sera from patients infected with heterologous helminths; these sera were highly cross-reacting with antigen from E. granulosus hydatid fluid. Seventy-three (94%) from 78 investigated patients (alveolar echinococcosis) showed a seropositive reaction with the polypeptide Em2a.

The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein

The polypeptide composition of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone messenger RNA (mRNA) 3' end formation has recently been elucidated. In contrast to spliceosomal snRNPs, which contain a ring-shaped assembly of seven so-called Sm proteins, in the U7 snRNP the Sm proteins D1 and D2 are replaced by U7-specific Sm-like proteins, Lsm10 and Lsm11. This polypeptide composition and the unusual structure of Lsm11, which plays a role in histone RNA processing, represent new themes in the biology of Sm/Lsm proteins. Moreover this structure has important consequences for snRNP assembly that is mediated by two complexes containing the PRMT5 methyltransferase and the SMN (survival of motor neurons) protein, respectively. Finally, the ability to alter this polypeptide composition by a small mutation in U7 snRNA forms the basis for using modified U7 snRNA derivatives to alter specific pre-mRNA splicing events, thereby opening up a new way for antisense gene therapy.

Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein.

U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.

Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2

The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report.

Identification of Sleep-Modulated Pathways Involved in Neuroprotection from Stroke.

STUDY OBJECTIVES Sleep deprivation (SDp) performed before stroke induces an ischemic tolerance state as observed in other forms of preconditioning. As the mechanisms underlying this effect are not well understood, we used DNA oligonucleotide microarray analysis to identify the genes and the gene-pathways underlying SDp preconditioning effects. DESIGN Gene expression was analyzed 3 days after stroke in 4 experimental groups: (i) SDp performed before focal cerebral ischemia (IS) induction; (ii) SDp performed before sham surgery; (iii) IS without SDp; and (iv) sham surgery without SDp. SDp was performed by gentle handling during the last 6 h of the light period, and ischemia was induced immediately after. SETTINGS Basic sleep research laboratory. MEASUREMENTS AND RESULTS Stroke induced a massive alteration in gene expression both in sleep deprived and non-sleep deprived animals. However, compared to animals that underwent ischemia alone, SDp induced a general reduction in transcriptional changes with a reduction in the upregulation of genes involved in cell cycle regulation and immune response. Moreover, an upregulation of a new neuroendocrine pathway which included melanin concentrating hormone, glycoprotein hormones-α-polypeptide and hypocretin was observed exclusively in rats sleep deprived before stroke. CONCLUSION Our data indicate that sleep deprivation before stroke reprogrammed the signaling response to injury. The inhibition of cell cycle regulation and inflammation are neuroprotective mechanisms reported also for other forms of preconditioning treatment, whereas the implication of the neuroendocrine function is novel and has never been described before. These results therefore provide new insights into neuroprotective mechanisms involved in ischemic tolerance mechanisms.