Identification of secreted proteins associated with obesity and type 2 diabetes in Psammomys obesus

Objective: Skeletal muscle produces a variety of secreted proteins that have important roles in intercellular communication and affects processes such as glucose homoeostasis. The objective of this study was to develop a novel Signal Sequence Trap (SST) in conjunction with cDNA microarray technology to identify proteins secreted from skeletal muscle of Psammomys obesus that were associated with obesity and type 2 diabetes (T2D).

Design: Secreted proteins that were differentially expressed between lean, normal glucose tolerant (NGT), overweight and impaired glucose tolerant (IGT) and obese, T2D P. obesus were isolated using SST in conjunction with cDNA microarray technology. Subsequent gene expression was measured in tissues from P. obesus by real-time PCR (RT-PCR).

Results: The SST yielded 1600 positive clones, which were screened for differential expression. A total of 91 (B6%) clones were identified by microarray to be differentially expressed between NGT, IGT and T2D P. obesus. These clones were sequenced to identify 51 genes, of which only 27 were previously known to encode secreted proteins. Three candidate genes not previously associated with obesity or type 2 diabetes, sushi domain containing 2, collagen and calcium-binding EGF domains 1 and periostin (Postn), as well as one gene known to be associated, complement component 1, were shown by RT-PCR to be differentially expressed in  skeletal muscle of P. obesus. Further characterization of the secreted protein Postn revealed it to be predominantly expressed in adipose tissue, with higher expression in visceral compared with subcutaneous adipose depots.

Conclusion: SST in conjunction with cDNA microarray technology is a powerful tool to identify differentially expressed secreted proteins involved in complex diseases such as obesity and type 2 diabetes. Furthermore, a number of candidate genes were identified, in particular, Postn, which may have a role in the development of obesity and type 2 diabetes.

ß2-Agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle

Aging is associated with a slowing of skeletal muscle contractile properties, including a decreased rate of relaxation. In rats, the age-related decrease in the maximal rate of relaxation is reversed after 4-wk administration with the β2-adrenoceptor agonist (β2-agonist) fenoterol. Given the critical role of the sarcoplasmic reticulum (SR) in regulating intracellular Ca2+ transients and ultimately the time course of muscle contraction and relaxation, we tested the hypothesis that the mechanisms of action of fenoterol are mediated by alterations in SR proteins. Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) kinetic properties were assessed in muscle homogenates and enriched SR membranes isolated from the red (RG) and white (WG) portions of the gastrocnemius muscle in adult (16 mo) and aged (28 mo) F344 rats that had been administered fenoterol for 4 wk (1.4 mg/kg/day ip, in saline) or vehicle only. Aging was associated with a 29% decrease in the maximal activity (Vmax) of SERCA in the RG but not in the WG muscles. Fenoterol treatment increased the Vmax of SERCA and SERCA1 protein levels in RG and WG. In the RG, fenoterol administration reversed an age-related selective nitration of the SERCA2a isoform. Our findings demonstrate that the mechanisms underlying age-related changes in contractile properties are fiber type dependent, whereas the effects of fenoterol administration are independent of age and fiber type.

Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling

Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling.

Heterotrimeric G proteins facilitate arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling

Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the Gbg dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional Ga or Gb subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in Gb-deficient mutants while Ga-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in Gb-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, Gb-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the Ga-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbg functional subunit but not by Ga. We hypothesize that Gbg acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

Multiple urea transporter proteins in the kidney of holocephalan elephant fish (Callorhinchus milii)

Reabsorption of filtered urea by the kidney is essential for retaining high levels of urea in marine cartilaginous fish. Our previous studies on the shark facilitative urea transporter (UT) suggest that additional UT(s) comprising the urea reabsorption system could exist in the cartilaginous fish kidney. Here, we isolated three cDNAs encoding UTs from the kidney of elephant fish, Callorhinchus milii, and termed them efUT-1, efUT-2 and efUT-3. efUT-1 is orthologous to known elasmobranch UTs, while efUT-2 and efUT-3 are novel UTs in cartilaginous fish. Two variants were found for efUT-1 and efUT-2, in which the NH₂-terminal intracellular domain was distinct between the variants. Differences in potential phosphorylation sites were found in the variant-specific NH2-terminal domains. When expressed in Xenopus oocytes, all five UT transcripts including the efUT-1 and efUT-2 variants induced more than a 10-fold increase in [¹⁴C] urea uptake. Phloretin inhibited dose-dependently the increase of urea uptake, suggesting that the identified UTs are facilitative UTs. Molecular phylogenetic analysis revealed that efUT-1 and efUT-2 had diverged in the cartilaginous fish lineage, while efUT-3 is distinct from efUT-1 and efUT-2. The present finding of multiple UTs in elephant fish provides a key to understanding the molecular mechanisms of urea reabsorption system in the cartilaginous fish kidney.

Plasmodium falciparum induces reorganization of host membrane proteins during intraerythrocytic growth

The virulence of the malaria parasite, Plasmodium falciparum, is due in large part to the way in which it modifies the membrane of its erythrocyte host. In this work we have used confocal microscopy and fluorescence recovery after photo-bleaching to examine the lateral mobility of host membrane proteins in erythrocytes infected with P falciparum at different stages of parasite growth. The erythrocyte membrane proteins band 3 and glycophorin show a marked decrease in mobility during the trophozoite stage of growth. Erythrocytes infected with a parasite strain that does not express the knob-associated histidine-rich protein show similar effects, indicating that this parasite protein does not contribute to the immobilization of the host proteins. Erythrocytes infected with ring-stage parasites exhibit intermediate mobility indicating that the parasite is able to modify its host prior to its active feeding stage.

A conserved region in the EBL proteins Is implicated in microneme targeting of the malaria parasite Plasmodium falciparum

The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBAGFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.

Plasmodium falciparum possesses two GRASP proteins that are differentially targeted to the Golgi complex via a higher- and lower-eukaryote-like mechanism

Plasmodium falciparum, the causative agent of malaria, relies on a complex protein-secretion system for protein targeting into numerous subcellular destinations. Recently, a homologue of the Golgi re-assembly stacking protein (GRASP) was identified and used to characterise the Golgi organisation in this parasite. Here, we report on the presence of a splice variant that leads to the expression of a GRASP isoform. Although the first GRASP protein (GRASP1) relies on a well-conserved myristoylation motif, the variant (GRASP2) displays a different N-terminus, similar to GRASPs found in fungi. Phylogenetic analyses between GRASP proteins of numerous taxa point to an independent evolution of the unusual N-terminus that could reflect unique requirements for Golgi-dependent protein sorting and organelle biogenesis in P. falciparum. Golgi association of GRASP2 depends on the hydrophobic N-terminus that resembles a signal anchor, leading to a unique mode of Golgi targeting and membrane attachment.

Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice

In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E–deficient (apoE^-/-) mice. The apoE^-/- mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE^-/- mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE^+/+) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3⁺ T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.

Biological properties of the Hendra virus envelope proteins

Outbreaks of Hendra virus in Queensland, during 1994-1999, resulted in the deaths of 17 horses and 2 humans from respiratory or neurological disease. Immunological, ultrastructural and expressional studies of the viral envelope proteins using recombinant poxviruses and DNA vectors contributed to improving our understanding of the biology of Hendra virus.

Novel secreted proteins involved in obesity and type 2 diabetes

A signal sequence trap was utilised to identify secreted proteins which were differentially expressed in key metabolic tissues associated with obesity and type 2 diabetes. Decorin and periostin were identified and functionally characterised as novel secreted proteins associated with the pathophysiology of these conditions.