The alpha-L-iduronidase mutations R89Q and R89W result in an attenuated mucopolysaccharidosis type I clinical presentation

Mucopolysaccharidosis type I (MPS I; McKusick 25280; Hurler syndrome, Hurler-Scheie syndrome and Scheie syndrome) is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase (EC 3.2.1.76). MPS I patients present within a clinical spectrum bounded by the extremes of Hurler and Scheie syndromes. The alpha-L-iduronidase missense mutations R89Q and R89W were investigated and altered an important arginine residue proposed to be a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase. The R89Q alpha-L-iduronidase mutation was shown to result in a reduced level of alpha-L-iduronidase protein (< or =10% of normal control) compared to a normal control level of alpha-L-iduronidase protein that was detected for the R89W alpha-L-iduronidase mutation. When taking into account alpha-L-iduronidase specific activity, the R89W mutation had a greater effect on alpha-L-iduronidase activity than the R89Q mutation. However, overall the R89W mutation produced more residual alpha-L-iduronidase activity than the R89Q mutation. This was consistent with MPS I patients, with an R89W allele, having a less severe clinical presentation compared to MPS I patients with either a double or single allelic R89Q mutation. The effects of the R89Q and R89W mutations on enzyme activity supported the proposed role of R89 as a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase.     

Mutant PrP is delayed in its exit from the endoplasmic reticulum,but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.     

MLL-ENL cooperates with SCF to transform primary avian multipotent cells

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.     

Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone

White adipose tissue is an important endocrine organ involved in the control of whole-body metabolism, insulin sensitivity, and food intake. To better understand these functions, 3T3-L1 cell differentiation was studied by using combined proteomic and genomic strategies. The proteomics approach developed here exploits velocity gradient centrifugation as an alternative to isoelectric focusing for protein separation in the first dimension. A 20- to 30-fold increase in the concentration of numerous mitochondrial proteins was observed during adipogenesis, as determined by mass spectrometry and database correlation analysis. Light and electron microscopy confirmed a large increase in the number of mitochondrion profiles with differentiation. Furthermore, mRNA profiles obtained by using Affymetrix GeneChips revealed statistically significant increases in the expression of many nucleus-encoded mitochondrial genes during adipogenesis. Qualitative changes in mitochondrial composition also occur during adipose differentiation, as exemplified by increases in expression of proteins involved in fatty acid metabolism and of mitochondrial chaperones. Furthermore, the insulin sensitizer rosiglitazone caused striking changes in mitochondrial shape and expression of selective mitochondrial proteins. Thus, although mitochondrial biogenesis has classically been associated with brown adipocyte differentiation and thermogenesis, our results reveal that mitochondrial biogenesis and remodeling are inherent to adipose differentiation per se and are influenced by the actions of insulin sensitizers.     

Pollen tube growth and early ovule development following self- and cross-pollination in<Emphasis Type="Italic"> Eucalyptus nitens</Emphasis>

Controlled self- and cross-pollinations were conducted on flowers of five mature Eucalyptus nitens trees. Levels of self-sterility of the trees ranged from 25.8 to 93.6%. Pollen tube numbers in styles and ovule penetration by pollen tubes was investigated 2 weeks after pollination by fluorescence microscopy. There were no significant differences between treatments in the number of pollen tubes present in styles or in the percentage of ovules penetrated by pollen tubes. Embryology of material harvested 2 and 4 weeks after pollination was investigated by bright-field microscopy. Fertilisation had taken place by 2 weeks after pollination with nearly every ovule showing evidence of fertilisation. Cross-pollination resulted in a greater proportion of healthy, developing ovules, at both 2 and 4 weeks after pollination, compared with self-pollination. The proportion of degenerating ovules increased from 2 to 4 weeks after pollination. The reduced ability of E. nitens to set self-pollinated seed compared with cross-pollinated seed appears to be controlled by a post-zygotic mechanism. Differences in ovule size may potentially assist in the identification of trees incapable of setting self-pollinated seed.     

Framingham Ten-Year General Cardiovascular Disease Risk: Agreement between BMI-Based and Cholesterol-Based Estimates in a South Asian Convenience Sample

The goal of this analysis was to determine the agreement between body mass index-based and cholesterol-based ten-year Framingham general cardiovascular disease risk scores among a convenience sample of 773 South Asian Canadian adults attending community-based screening clinics. Scores were calculated using age, systolic blood pressure, antihypertensive use, current smoking, diabetes, and total cholesterol and high density lipoprotein (for cholesterol-based risk) or height and weight (for body mass index-based risk). Mean risk score differences (body mass index-based risk minus cholesterol-based risk) were estimated using paired t-tests. Bland-Altman plots were used to assess agreement between scores. Finally, agreement across risk categories (low [<10%], moderate [10% to <20%], high [> = 20%]) was examined using the kappa statistic. Average agreement between the two risk scores was quite good overall (mean differences of 0.6% for men and 0.5% for women), but increased to about 3% among participants 60–74 years of age. However, Bland-Altman plots revealed that the differences between the two scores and the variability of the differences increased with increasing average 10-year risk. In terms of clinical importance, the limits of agreement were reasonable for women < 60 years (95% confidence interval: -3.2% to 3.1%), but of concern for women 60-74 years (95% confidence interval: -6.0% to 12.3%), men < 60 years (95% confidence interval: -7.1% to 7.3%) and men 6-074 years (95% confidence interval: -13.8% to 18.8%). Agreement across categories was moderate for most sex and age groups examined (kappa values: 0.51 for women < 60 years, 0.50 for women 60-74 years, 0.65 for men < 60 years), except for men 60-74 years, where agreement was only fair (kappa = 0.26). In light of these disagreements, evaluation of a participant’s change in cardiovascular disease risk over time will necessitate use of the same risk score (i.e., either body mass index-based or cholesterol-based) at all screening sessions.     

Blood-Based Protein Biomarker Panel for the Detection of Colorectal Cancer

Background

The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test.

Principal Findings

In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2).

Conclusions

Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.     

Viral Dose and Immunosuppression Modulate the Progression of Acute BVDV-1 Infection in Calves: Evidence of Long Term Persistence after Intra-Nasal Infection

Bovine viral diarrhoea virus (BVDV) infection of cattle causes a diverse range of clinical outcomes from being asymptomatic, or a transient mild disease, to producing severe cases of acute disease leading to death. Four groups of calves were challenged with a type 1 BVDV strain, originating from a severe outbreak of BVDV in England, to study the effect of viral dose and immunosuppression on the viral replication and transmission of BVDV. Three groups received increasing amounts of virus: Group A received 10^2.55TCID₅₀/ml, group B 10^5.25TCID₅₀/ml and group C 10^6.7TCID ₅₀/ml. A fourth group (D) was inoculated with a medium dose (10^5.25TCID₅₀/ml) and concomitantly treated with dexamethasone (DMS) to assess the effects of chemically induced immunosuppression. Naïve calves were added as sentinel animals to assess virus transmission. The outcome of infection was dose dependent with animals given a higher dose developing severe disease and more pronounced viral replication. Despite virus being shed by the low-dose infection group, BVD was not transmitted to sentinel calves. Administration of dexamethasone (DMS) resulted in more severe clinical signs, prolonged viraemia and virus shedding. Using PCR techniques, viral RNA was detected in blood, several weeks after the limit of infectious virus recovery. Finally, a recently developed strand-specific RT-PCR detected negative strand viral RNA, indicative of actively replicating virus, in blood samples from convalescent animals, as late as 85 days post inoculation. This detection of long term replicating virus may indicate the way in which the virus persists and/or is reintroduced within herds.     

Assessment of Blood Collection from the Lateral Saphenous Vein for Microfilaria Counts in Mongolian Gerbils (Meriones unguiculatus) Infected with Brugia pahangi

The NIH guidelines for survival bleeding of mice and rats note that using the retroorbital plexus has a greater potential for complications than do other methods of blood collection and that this procedure should be performed on anesthetized animals. Lateral saphenous vein puncture has a low potential for complications and can be performed without anesthesia. Mongolian gerbils (Meriones unguiculatus) are the preferred rodent model for filarial parasite research. To monitor microfilaria counts in the blood, blood sampling from the orbital plexus has been the standard. Our goal was to refine the blood collection technique. To determine whether blood collection from the lateral saphenous vein was a feasible alternative to retroorbital sampling, we compared microfilaria counts in blood samples collected by both methods from 21 gerbils infected with the filarial parasitic worm Brugia pahangi. Lateral saphenous vein counts were equivalent to retroorbital counts at relatively high counts (greater than 50 microfilariae per 20 µL) but were significantly lower than retroorbital counts when microfilarial concentrations were lower. Our results indicate that although retroorbital collection may be preferable when low concentrations of microfilariae need to be enumerated, the lateral saphenous vein is a suitable alternative site for blood sampling to determine microfilaremia and is a feasible refinement that can benefit the wellbeing of gerbils.Abbreviations: FR3, Filariasis Research Reagent Resource CenterLymphatic filariasis a major threat to human health worldwide. More than one billion people in more than 90 countries around the globe are at risk from lymphatic filariasis, and between 120 and 150 million people are infected.^9,11,25 Infection with the filarioid parasitic worms Brugia malayi and Wuchereria bancrofti can result in severe sequelae, including elephantiasis and hydrocoele formation.^3,11,15,25 In addition to the clinical manifestations of filariasis are the potential associated psychologic, social, and cultural effects in persons exhibiting visible signs of infection.^9,23,34The life cycle of filarioid nematodes requires an arthropod intermediate host and a definitive vertebrate host. Within the definitive host, dioecious adult filarial nematodes reproduce sexually. Inseminated adult female worms then release live, sheathed microfilariae into the lymph that circulate in the peripheral blood.²¹ In the case of B. malayi and W. bancrofti, the intermediate host is the mosquito.²¹ When an uninfected mosquito ingests a blood meal from an infected human, ingested microfilariae unsheathe to penetrate the midgut of the mosquito to reach the thoracic muscles and molt twice, to become the infectious third-stage larvae. The third-stage larvae then migrate to the mosquito''s proboscis and can infect another human when the mosquito takes a blood meal.^10,11 The third-stage larvae enter the new host''s lymphatic system which is their final location, where they undergo 2 molts into adults.Because of the complexity of filarioid life cycles, research involving these parasites can be logistically challenging. Although mice can be infected with W. bancrofti, they do not maintain the infection.³⁵ Furthermore, there is no suitable nonhuman host that can maintain a patent infection, with the exception of the silvered leaf monkey (Trachypithecus cristatus).⁹ Because the closely related parasites B. malayi and B. pahangi have more extensive host ranges than does W. bancrofti, they are easier to maintain in a research setting. Domestic cats (Felis catus) can be experimentally infected with B. malayi and develop a patent infection, and both domestic cats and dogs (Canis familiaris) can be experimentally infected with B. pahangi^13,29,37 and are suitable for obtaining microfilaremic blood for experimental feeding of mosquitoes. The Mongolian gerbil can be infected with B. pahangi. Because replacing a phylogenetically higher species with a lower species is preferable³⁶ and because performing experiments involving dogs and cats can be logistically difficult and cost-prohibitive, many researchers prefer a rodent model, specifically gerbils.The Filariasis Research Reagent Resource Center (FR3) is an NIH center whose mandate is to support filariasis research worldwide. The FR3 provides parasitic and molecular resources, as well as training in animal procedures, to researchers from many nations. The FR3 maintains both B. malayi and B. pahangi, and researchers occasionally require gerbils with patent infections. Because the required level of microfilaria counts varies among investigators, an accurate microfilaria count must be obtained prior to the shipment of gerbils. For example, some experiments require that live mosquitoes feed directly on infected gerbils, and when the microfilaria level is too low, the mosquitoes do not become infected. Conversely when the level is too high, the migration of microfilariae and the later larval stages can kill the mosquitoes. Historically, the FR3 has used retroorbital sampling under general anesthesia to obtain the blood for microfilaria counts.²⁸ Although this method has been fairly successful, the FR3 has encountered occasional complications secondary to the procedure, including exophthalmia and, rarely, death under anesthesia. The NIH guidelines for survival bleeding of mice and rats notes that compared with other blood collection methods, retroorbital sampling has a greater potential for complications. The guidelines recommend a 10- to 14-d period between retroorbital blood collections and state that the procedure is “…best conducted under general anesthesia.”³¹ By comparison, collecting blood from the lateral saphenous vein is considered to have a low potential for complications or tissue damage, can be performed without general anesthesia,^12,18,31 and can be performed repeatedly, even daily.³¹In the current study, we proposed to refine the blood collection method being used by FR3 by developing sampling from the lateral saphenous vein as the new standard blood-collection method for monitoring microfilaremia. Our goal was to assess blood collection from the lateral saphenous vein as a feasible refinement technique to potentially replace retroorbital sampling by determining whether the microfilaria counts in blood collected from the lateral saphenous vein without anesthesia were sufficiently similar to those from retroorbital blood sampling with anesthesia to provide adequate information about the microfilaremia level.