Thyroid status,but not insulin status,affects expression of avian uncoupling protein mRNA in chicken

The aim of this study was to investigate the hormonal regulation of the avian homolog of mammalian uncoupling protein (avUCP) by studying the impact of thyroid hormones and insulin on avUCP mRNA expression in chickens (Gallus gallus). For 3 wk, chicks received either a standard diet (control group), or a standard diet supplemented with triiodothyronine (T(3); T3 group) or with the thyroid gland inhibitor methimazole (MMI group). A fourth group received injections of the deiodinase inhibitor iopanoic acid (IOP group). During the 4th wk of age, all animals received two daily injections of either human insulin or saline solution. The results indicate a twofold overexpression of avUCP mRNA in gastrocnemius muscle of T3 birds and a clear downregulation (-74%) in MMI chickens compared with control chickens. Insulin injections had no significant effect on avUCP mRNA expression in chickens. This study describes for the first time induction of avUCP mRNA expression by the thermogenic hormone T(3) in chickens and supports a possible involvement of avUCP in avian thermogenesis.     

RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.     

Dipeptidyl peptidase 9 (DPP9) from bovine testes: Identification and characterization as the short form by mass spectrometry

The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.     

Hormonal and ion regulatory response in three freshwater fish species following waterborne copper exposure

We evaluated effects of sublethal copper exposure in 3 different freshwater fish: rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). In a first experiment we exposed these fishes to an equally toxic Cu dose, a Cu level 10 times lower than their 96 h LC₅₀ value: 20, 65, and 150 µg/L Cu. In a second series we exposed them to the same Cu concentration (50 µg/L). Na⁺/K⁺-ATPase activity in gill tissue was disturbed differently in rainbow trout then in common and gibel carp. Rainbow trout showed a thorough disruption of plasma ion levels at the beginning of both exposures, whereas common carp and gibel carp displayed effects only after 3 days. Rainbow trout and common carp thyroid hormones experienced adverse effects in the beginning of the exposure. The involvement of prolactin in handling metal stress was reflected in changes of mRNA prolactin receptor concentrations in gill tissue, with an up regulation of this mRNA in rainbow trout and a down regulation in gibel carp, which was more pronounced in the latter. Overall, rainbow trout appeared more sensitive in the beginning of the exposure, however, when it overcame this first challenge, it handled copper exposure in a better manner then common and gibel carp as they showed more long term impacts of Cu exposure.     

Discovery and Verification of Osteopontin and Beta-2-microglobulin as Promising Markers for Staging Human African Trypanosomiasis

Human African trypanosomiasis, or sleeping sickness, is a parasitic disease endemic in sub-Saharan Africa, transmitted to humans through the bite of a tsetse fly. The first or hemolymphatic stage of the disease is associated with presence of parasites in the bloodstream, lymphatic system, and body tissues. If patients are left untreated, parasites cross the blood-brain barrier and invade the cerebrospinal fluid and the brain parenchyma, giving rise to the second or meningoencephalitic stage. Stage determination is a crucial step in guiding the choice of treatment, as drugs used for S2 are potentially dangerous. Current staging methods, based on counting white blood cells and demonstrating trypanosomes in cerebrospinal fluid, lack specificity and/or sensitivity. In the present study, we used several proteomic strategies to discover new markers with potential for staging human African trypanosomiasis. Cerebrospinal fluid (CSF) samples were collected from patients infected with Trypanosoma brucei gambiense in the Democratic Republic of Congo. The stage was determined following the guidelines of the national control program. The proteome of the samples was analyzed by two-dimensional gel electrophoresis (n = 9), and by sixplex tandem mass tag (TMT) isobaric labeling (n = 6) quantitative mass spectrometry. Overall, 73 proteins were overexpressed in patients presenting the second stage of the disease. Two of these, osteopontin and β-2-microglobulin, were confirmed to be potential markers for staging human African trypanosomiasis (HAT) by Western blot and ELISA. The two proteins significantly discriminated between S1 and S2 patients with high sensitivity (68% and 78%, respectively) for 100% specificity, and a combination of both improved the sensitivity to 91%. The levels of osteopontin and β-2-microglobulin in CSF of S2 patients (μg/ml range), as well as the fold increased concentration in S2 compared with S1 (3.8 and 5.5 respectively) make the two markers good candidates for the development of a test for staging HAT patients.Human African trypanosomiasis (HAT), or sleeping sickness, is caused by an extracellular protozoan parasite of the genus Trypanosoma, which is transmitted through the bite of a tsetse fly (genus Glossina). Two morphologically identical subspecies of the parasite, are responsible for the two geographically and clinically different forms of HAT: a chronic form, widespread in West and Central Africa, caused by T. b. gambiense, and an acute form, endemic in eastern Africa, caused by T. b. rhodesiense (1). In both forms of the disease, parasites are initially localized in the blood stream, lymph, and peripheral tissues; this is the first or hemolymphatic stage (S1). During this stage, patients present generic clinical features that are common to other infectious diseases such as human immunodeficiency virus (HIV), malaria, and tuberculosis (TB), which can coexist with HAT, thus making its early diagnosis difficult (2). If treatment is not carried out, the disease progresses to the second or meningoencephalitic stage (S2) after trypanosomes cross the blood-brain barrier (BBB) and invade the central nervous system (CNS). This phase is characterized by a broad range of neurological signs that are indicative of CNS involvement (1). Diagnosis of HAT is based on parasitological demonstration of parasites in blood or lymph-node aspirate (3). All positive or suspect patients have to undergo a lumbar puncture and cerebrospinal fluid (CSF)1 examination, to determine whether they have second stage disease (4). According to the World Health Organization (WHO) guidelines, the meningoencephalitic stage is defined by the presence of parasites in CSF and/or a white blood cell (WBC) count of more than 5 cells per μl (5). Other parameters, such as intrathecal IgM production could also provide additional information to determine whether the CNS is involved (6, 7).Treatment of HAT patients varies depending on the infecting parasite and the stage of disease (5, 8). S2 drugs in current use, including melarsoprol, eflornithine, and a combination of nifurtimox and eflornithine have several limitations, such as a high rate of toxicity (melarsoprol causes death to 5% of treated patients) (9), complex logistics, and mode of administration (6, 10). Consequently, staging is a vital step in the diagnosis and treatment of HAT. However, the poor specificity or sensitivity of WBC counting and of parasitological techniques for demonstration of parasites in CSF, highlight the need for discovery of better tools for staging the disease.Several attempts have been made during the last decade to identify potential biomarkers able to discriminate between the two stages of sleeping sickness. Most of the efforts focused on cytokines and chemokines, because the patient''s immune system plays a crucial role in the brain pathology (11–14).Proteomic approaches are increasingly being applied in biomedical research and clinical medicine to investigate body fluids as a source of biomarkers (15), including the diagnosis of neurological disorders such as Alzheimer''s disease (16), Parkinson''s disease (17), and multiple sclerosis (18, 19). The protein composition of CSF is strictly regulated and can reflect the physiological or pathological state of the CNS (15). Thus in the present study, we addressed the challenge of staging HAT by analyzing CSF from T. b. gambiense patients using two complementary proteomic strategies: a classical approach based on two-dimensional gel electrophoresis (2-DE), and quantitative mass spectrometry (MS) using isobaric tandem mass tag (TMT) technology (sixplex TMT® MS/MS) (20).     

CD4+CD28null T cells in autoimmune disease: pathogenic features and decreased susceptibility to immunoregulation

To determine the role of expanded CD4(+)CD28(null) T cells in multiple sclerosis and rheumatoid arthritis pathology, these cells were phenotypically characterized and their Ag reactivity was studied. FACS analysis confirmed that CD4(+)CD28(null) T cells are terminally differentiated effector memory cells. In addition, they express phenotypic markers that indicate their capacity to infiltrate into tissues and cause tissue damage. Whereas no reactivity to the candidate autoantigens myelin basic protein and collagen type II was observed within the CD4(+)CD28(null) T cell subset, CMV reactivity was prominent in four of four HC, four of four rheumatoid arthritis patients, and three of four multiple sclerosis patients. The level of the CMV-induced proliferative response was found to be related to the clonal diversity of the response. Interestingly, our results illustrate that CD4(+)CD28(null) T cells are not susceptible to the suppressive actions of CD4(+)CD25(+) regulatory T cells. In conclusion, this study provides several indications for a role of CD4(+)CD28(null) T cells in autoimmune pathology. CD4(+)CD28(null) T cells display pathogenic features, fill up immunological space, and are less susceptible to regulatory mechanisms. However, based on their low reactivity to the autoantigens tested in this study, CD4(+)CD28(null) T cells most likely do not play a direct autoaggressive role in autoimmune disease.     

Folate fortification of rice by metabolic engineering

Rice, the world's major staple crop, is a poor source of essential micronutrients, including folates (vitamin B9). We report folate biofortification of rice seeds achieved by overexpressing two Arabidopsis thaliana genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway from a single locus. We obtained a maximal enhancement as high as 100 times above wild type, with 100 g of polished raw grains containing up to four times the adult daily folate requirement.     

Recombinant expression systems: the obstacle to helminth vaccines?

The need for alternative ways to control helminth parasites has in recent years led to a boost in vaccination experiments with recombinant antigens. Despite the use of different expression systems, only a few recombinants induced high levels of protection against helminths. This is often attributed to the limitations of the current expression systems. Therefore, the need for new systems that can modify and glycosylate the expressed antigens has been advocated. However, analysis of over 100 published vaccine trials with recombinant helminth antigens indicates that it is often not known whether the native parasite antigen itself can induce protection or, if it does, which epitopes are important. This information is vital for a well-thought-out strategy for recombinant production. So, in addition to testing more expression systems, it should be considered that prior evaluation and characterization of the native antigens might help the development of recombinant vaccines against helminths in the long term.