Cell therapy has been extensively investigated in heart disease but less so in the kidney. We considered whether cell therapy also might be useful in diabetic kidney disease. Cognizant of the likely need for autologous cell therapy in humans, we sought to assess the efficacy of donor cells derived from both healthy and diabetic animals. Eight-week-old db/db mice were randomized to receive a single intravenous injection of PBS or 0.5 × 106 early-outgrowth cells (EOCs) from db/m or db/db mice. Effects were assessed 4 weeks after cell infusion. Untreated db/db mice developed mesangial matrix expansion and tubular epithelial cell apoptosis in association with increased reactive oxygen species (ROS) and overexpression of thioredoxin interacting protein (TxnIP). Without affecting blood glucose or blood pressure, EOCs not only attenuated mesangial and peritubular matrix expansion, as well as tubular apoptosis, but also diminished ROS and TxnIP overexpression in the kidney of db/db mice. EOCs derived from both diabetic db/db and nondiabetic db/m mice were equally effective in ameliorating kidney injury and oxidative stress. The similarly beneficial effects of cells from healthy and diabetic donors highlight the potential of autologous cell therapy in the related clinical setting.
Carotid intima-media thickness (IMT) and elasticity have been shown to be independent predictors of cardiovascular disease (CVD). Cardiovascular risk factors (CVRFs) includes hypertension, dyslipidemia, diabetes, overweight and smoking. The objective was to investigate whether the clustering of three or more components of CVRFs has a greater impact on carotid IMT and elasticity than individual components of CVRFs.
One hundred and seventy-three participants without clinical CVD were classified as the multiple CVRFs patients with three or more CVRFs (n = 55) and control group with two or less CVRFs (n = 118). Carotid IMT and elastic modulus were measured by B-mode ultrasound and vessel texture matching method (VTMM), respectively.
The multiple CVRFs conferred a disproportionate increase in carotid IMT (43%, p<0.0001) and elastic modulus (60%, p<0.0001), compared with control group. Multiple regression models, which included age, gender, as well as each individual component of CVRFs as continuous variables, showed that multiple CVRFs was an independent determinant of both IMT (p = 0.042) and elasticity (p = 0.008). In the analysis of variance adjusted with age, subjects with single, double, and multiple CVRFs, increased by 8.1%, 42.2%, and 66% for IMT, 54.6%, 94.3%, and 125.2% for elastic modulus, respectively, compared to subjects without CVRFs.
The clustering of multiple CVRFs has a greater impact on carotid IMT and elasticity than individual components of CVRFs. This suggests that the components of CVRFs interact to synergistically impact carotid IMT and elasticity.
Amounts of empirical evidence, ranging from microbial cooperation to collective hunting, suggests public goods produced often nonlinearly depend on the total amount of contribution. The implication of such nonlinear public goods for the evolution of cooperation is not well understood. There is also little attention paid to the divisibility nature of individual contribution amount, divisible vs. non-divisible ones. The corresponding strategy space in the former is described by a continuous investment while in the latter by a continuous probability to contribute all or nothing. Here, we use adaptive dynamics in finite populations to quantify and compare the roles nonlinearity of public-goods production plays in cooperation between these two contribution mechanisms. Although under both contribution mechanisms the population can converge into a coexistence equilibrium with an intermediate cooperation level, the branching phenomenon only occurs in the divisible contribution mechanism. The results shed insight into understanding observed individual difference in cooperative behavior.
The joint venture of many members is common both in animal world and human society. In these public enterprizes, highly cooperative groups are more likely to while low cooperative groups are still possible but not probable to succeed. Existent literature mostly focuses on the traditional public goods game, in which cooperators create public wealth unconditionally and benefit all group members unbiasedly. We here institute a model addressing this public goods dilemma with incorporating the public resource foraging failure risk. Risk-averse individuals tend to lead a autarkic life, while risk-preferential ones tend to participate in the risky public goods game. For participants, group's success relies on its cooperativeness, with increasing contribution leading to increasing success likelihood. We introduce a function with one tunable parameter to describe the risk removal pattern and study in detail three representative classes. Analytical results show that the widely replicated population dynamics of cyclical dominance of loner, cooperator and defector disappear, while most of the time loners act as savors while eventually they also disappear. Depending on the way that group's success relies on its cooperativeness, either cooperators pervade the entire population or they coexist with defectors. Even in the later case, cooperators still hold salient superiority in number as some defectors also survive by parasitizing. The harder the joint venture succeeds, the higher level of cooperation once cooperators can win the evolutionary race. Our work may enrich the literature concerning the risky public goods games.
In recent years, mechanisms favoring fair split in the ultimatum game have attracted growing interests because of its practical implications for international bargains. In this game, two players are randomly assigned two different roles respectively to split an offer: the proposer suggests how to split and the responder decides whether or not to accept it. Only when both agree is the offer successfully split; otherwise both get nothing. It is of importance and interest to break the symmetry in role assignment especially when the game is repeatedly played in a heterogeneous population. Here we consider an adaptive role assignment: whenever the split fails, the two players switch their roles probabilistically. The results show that this simple feedback mechanism proves much more effective at promoting fairness than other alternatives (where, for example, the role assignment is based on the number of neighbors).
Viral vectors have been utilized extensively to introduce genetic material into the central nervous system. In order to investigate gene functions in cardiovascular control regions of rat brain, we applied WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) enhanced-adenoviral (Ad) and adeno-assoicated virus (AAV) type 2 vectors to mediate neuronal gene delivery to the paraventricular nucleus of the hypothalamus, the nucleus tractus solitarius and the rostral ventrolateral medulla, three important cardiovascular control regions known to express renin-angiotensin system (RAS) genes. Ad or AAV2 harboring an enhanced green fluorescent protein (EGFP) reporter gene or the angiotensin type 2 receptor gene were microinjected into these brain regions in adult rats. Our results demonstrated that both AAV2 and Ad vectors elicited long-term neuronal transduction in these regions. Interestingly, we found that the WPRE caused expression of GFP driven by the synapsin1 promoter in pure glial cultures or co-cultures of neurons and glia derived from rat hypothalamus and brainstem. However, in rat paraventricular nucleus WPRE did not cause expression of GFP in glia. This demonstrates the potential use of these vectors in studies of physiological functions of certain genes in the cardiovascular control regions of the brain.
adenoviral vector; adeno-associated viral vector; cardiovascular control regions of brain; neuron; gene transfer.
A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed 15N, 13C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with 15N and 13C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone 15N, 13C and 1H resonance assignments of the r-RGD-Hirudin. The 15N-1H HSQC spectrum of uniformly 15N, 13C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the 15N-lH correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets.
Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P
Mice deficient for VAC14, a scaffolding protein required for PIP2 biosynthesis and linked to human neuropathies, show increased postsynaptic function due to altered AMPA receptor trafficking.
Normal steady-state levels of the signalling lipids PI(3,5)P2 and PI(5)P require the lipid kinase FAB1/PIKfyve and its regulators, VAC14 and FIG4. Mutations in the PIKfyve/VAC14/FIG4 pathway are associated with Charcot-Marie-Tooth syndrome and amyotrophic lateral sclerosis in humans, and profound neurodegeneration in mice. Hence, tight regulation of this pathway is critical for neural function. Here, we examine the localization and physiological role of VAC14 in neurons. We report that endogenous VAC14 localizes to endocytic organelles in fibroblasts and neurons. Unexpectedly, VAC14 exhibits a pronounced synaptic localization in hippocampal neurons, suggesting a role in regulating synaptic function. Indeed, the amplitude of miniature excitatory postsynaptic currents is enhanced in both Vac14−/− and Fig4−/− neurons. Re-introduction of VAC14 in postsynaptic Vac14−/− cells reverses this effect. These changes in synaptic strength in Vac14−/− neurons are associated with enhanced surface levels of the AMPA-type glutamate receptor subunit GluA2, an effect that is due to diminished regulated endocytosis of AMPA receptors. Thus, VAC14, PI(3,5)P2 and/or PI(5)P play a role in controlling postsynaptic function via regulation of endocytic cycling of AMPA receptors.
AMPA receptor; PIKfyve; phosphatidylinositol 3,5-bisphosphate; PtdIns(3,5)P2; synapse
Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP142) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP142 conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP142 self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP142 specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis.
Little is known about mortality among nonagenarians after an earthquake.
Using secondary data analyses from the 2005 study called the Project of Longevity and Aging in Dujiangyan(n = 870), 1-year mortality rates were compared among a pre-earthquake group and a post-earthquake group of nonagenarians. All participants were from Dujiangyan, 50 km from the epicenter of the May 12, 2008 earthquake, in China. The pre-earthquake group was a subset of the 870 Project of Longevity and Aging in Dujiangyan participants, ages 93–95 years at the beginning of “Time Frame 1” (July 2005 through June 2006; n = 228). The post-earthquake group was a different subset of the 870 Project of Longevity and Aging in Dujiangyan participants, ages 93–95 years and alive at the beginning of Time Frame 2 (July 2008 through June 2009; n = 235). Time Frame 2 excluded a 7-week period following the earthquake in order to account for deaths due to trauma. Pre-earthquake health assessment data from the 2005 Project of Longevity and Aging in Dujiangyan study were used to calculate unadjusted/adjusted hazard ratios (HRs) for mortality.
One-year mortality rates were 8.3% (19/228) and 16.2% (38/235) in the pre-earthquake group and the post-earthquake group, respectively (p =.01). In unadjusted analyses, only “being in the post-earthquake group” was associated with death (HR = 2.04; 95% confidence interval [CI], 1.17–3.53; p = .011). In the multivariable Cox regression model, being in the post-earthquake group continued to be the strongest risk factor associated with mortality (HR = 2.47; 95% CI, 1.39–4.40; p = .002). Other significant risk factors included impaired cognition (HR = 1.97; 95% CI, 1.10–3.53; p = .024), serum albumin (HR = 0.90; 95% CI, 0.82–0.98; p < .015), and serum triglycerides (HR = 1.51; 95% CI, 1.15–1.99; p = .003).
The May 12, 2008 earthquake in Wenchuan, China, was associated with a twofold increase in the 1-year mortality among a group of nonagenarians who lived nearby.
Earthquake; Nonagenarians; Mortality
Tyroserleutide (YSL) is a tripeptide compound that exhibits potent antitumor activity in human tumor xenografts and tumor cell lines. However, the target of YSL on which it exerts its antitumor activity has yet to be identified. Therefore, our study aimed to investigate the subcellular location of YSL in BEL-7402 human hepatocellular carcinoma cells. Using methods of fluorescent tracing and confocal colocalization, we provide evidence that when BEL-7402 cells are treated with YSL, YSL is distributed in the cytoplasm and colocalized with the mitochondria of cancer cells. Furthermore, we observed the effect of YSL on the isolated mitochondria. Using fluorescence spectrophotometry to monitor the Δψ collapse of mitochondria isolated from BEL-7402 cells by reversion of the quenching of tetramethylrhodamine methyl ester (TMRM), we found that the isolated mitochondria reversed the quenching of the fluorescence in the solution containing TMRM and YSL. This indicates that YSL decreases the Δψ of the isolated mitochondria. Another photometry method was used to observe the effect on mitochondrial swelling when YSL acted directly on the isolated mitochondria. We reveal that YSL directly causes mitochondrial swelling in 60 min. In conclusion, this study encloses a preliminary facet of the pharmacological target of YSL, and we speculate that YSL may act directly on the mitochondria to exert its antitumor activity.
tyroserleutide; subcellular location; antitumor; target; mitochondria
Rarefaction of the renal microvasculature correlates with declining kidney function. However, current technologies commonly used for its evaluation are limited by their reliance on endothelial cell antigen expression and assessment in two dimensions. We set out to establish a widely applicable and unbiased optical sectioning method to enable three dimensional imaging and reconstruction of the renal microvessels based on their luminal filling. The kidneys of subtotally nephrectomized (SNx) rats and their sham-operated counterparts were subjected to either routine two-dimensional immunohistochemistry or the novel technique of fluorescent microangiography (FMA). The latter was achieved by perfusion of the kidney with an agarose suspension of fluorescent polystyrene microspheres followed by optical sectioning of 200 µm thick cross-sections using a confocal microscope. The fluorescent microangiography method enabled the three-dimensional reconstruction of virtual microvascular casts and confirmed a reduction in both glomerular and peritubular capillary density in the kidneys of SNx rats, despite an overall increase in glomerular volume. FMA is an uncomplicated technique for evaluating the renal microvasculature that circumvents many of the limitations imposed by conventional analysis of two-dimensional tissue sections.
Recent research has implicated that mutations in the neurexin-1 (NRXN1) gene on chromosome 2p16.3 might play a role in schizophrenia, autism, and nicotine dependence. In order to explore the association of NRXN1 polymorphisms with schizophrenia, we made a case-control association study in Chinese Han population.
We examined six tag single nucleotide polymorphisms (SNPs) spanning 116.7 kb of NRXN1 in 768 schizophrenic patients and 738 healthy control subjects. The association of NRXN1 polymorphisms with schizophrenia and the age-at-onset of this disease were explored.
Our results showed that four SNPs of NRXN1 gene were significantly associated with schizophrenia (rs10490168: G > A, p = 0.017; rs2024513: A > G, p = 0.006; rs13382584: T > C, p = 0.009; and rs1558852: G > A, p = 0.031). Furthermore, the association of SNP rs2024513 with schizophrenia remained significance after the Bonferroni correction. Haplotypes consisting of above six SNPs also showed significantly associated with schizophrenia (global chi-square = 14.725, p = 0.022). A protective haplotype AGTGCA remained associated with schizophrenia, even after 10,000 permutation tests (empirical p-value = 0.043). However, we did not find any association with age-at-onset of schizophrenia with NRXN1 polymorphisms.
Our findings suggest that NRXN1 might represent a major susceptibility gene for schizophrenia in Chinese Han population.
Schizophrenia; Neurexin-1 (NRXN1); Haplotype; age-at-onset
Membrane fusion and fission in intracellular trafficking is controlled by both intraluminal Ca2+ release and phosphoinositide signaling. However, the molecular identities of the Ca2+ release channels and the target proteins of phosphoinositides are elusive. Here, by direct patch-clamping of the endolysosomal membrane, we report that PI(3,5)P2, an endolysosome-specific phosphoinositide, binds and activates endolysosome-localized mucolipin TRP (TRPML) channels with specificity and potency. Both PI(3,5)P2-deficient cells and cells that lack TRPML1 exhibited enlarged endolysosomes/vacuoles and trafficking defects in the late endocytic pathway. We find that the enlarged vacuole phenotype observed in PI(3,5)P2-deficient mouse fibroblasts is suppressed by overexpression of TRPML1. Notably, this PI(3,5)P2-dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, hyperosmotic stress induces Ca2+ release from the vacuole. Here, we show that this release requires both PI(3,5)P2 production and a yeast functional TRPML homolog. We propose that TRPMLs regulate membrane trafficking by transducing information about PI(3,5)P2 levels into changes in juxtaorganellar Ca2+, thereby triggering membrane fusion/fission events.
Whole-endolysosome recording; TRP channel; Ca2+ release channel; PIKfyve; Fab1; phosphoinositide; PI(3,5)P2; type IV Mucolipidosis; endosome; lysosome; membrane trafficking; vacuole
Diabetic nephropathy is a complex and poorly understood disease process, and our current treatment options are limited. It remains critical, then, to identify novel therapeutic targets. Recently, a developmental protein and one of the bone morphogenetic protein antagonists, Gremlin, has emerged as a novel modulator of diabetic nephropathy. The high expression and strong co-localization with transforming growth factor- β1 in diabetic kidneys suggests a role for Gremlin in the pathogenesis of diabetic nephropathy. We have constructed a gremlin siRNA plasmid and have examined the effect of Gremlin inhibition on the progression of diabetic nephropathy in a mouse model. CD-1 mice underwent uninephrectomy and STZ treatment prior to receiving weekly injections of the plasmid. Inhibition of Gremlin alleviated proteinuria and renal collagen IV accumulation 12 weeks after the STZ injection and inhibited renal cell proliferation and apoptosis. In vitro experiments, using mouse mesangial cells, revealed that the transfect ion of gremlin siRNA plasmid reversed high glucose induced abnormalities, such as increased cell proliferation and apoptosis and increased collagen IV production. The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid. Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels. We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy.
In this paper we report our efforts to enhance the immunogenicity of Pfs28, a transmission blocking vaccine candidate of Plasmodium falciparum, using a strategy of chemical conjugation. With an improved procedure Pfs28 was covalently coupled to the mutant and nontoxic ExoProtein A of Pseudomonas aeruginosa by the reaction between thiolated antigen and maleimide modified carrier protein. The optimized process resulted in a higher antigen-carrier conjugation ratio, and the conjugation product could be purified using single-step size-exclusion chromatography. A significant increase in immunogenicity measured by ELISA was observed in mice immunized with conjugated Pfs28 as compared to unconjugated Pfs28.
Conjugation; Pfs28; rEPA; Malaria; Transmission blocking vaccine
Previously, we identified a Plasmodium yoelii YM 140-kDa merozoite protein, designated PyP140, which formed a complex with apical membrane antigen 1 (AMA1). Furthermore, we produced a nonprotective monoclonal antibody (MAb), 48F8, that immunoprecipitated metabolically labeled PyP140 and localized the protein to the merozoite's apical end and, less frequently, to the merozoite surface, as observed by immunofluorescence assay (IFA). Here, using MAb 48F8, we have identified the pyp140 gene by screening a P. yoelii λ-Zap cDNA expression library. The pyp140 cDNA covers approximately 90% of the putative open reading frame (ORF) of PY02159 from the P. yoelii NL genome sequencing project. Analysis of the complete gene identified the presence of two introns. The ORF encodes a 102,407-Da protein with an amino-terminal signal sequence, a series of three unique types of repeats, and a cysteine-rich region. The binding site of MAb 48F8 was also identified. A BLAST search with the deduced amino acid sequence shows significant similarity with the Toxoplasma gondii RON4 protein and the Plasmodium falciparum RON4 protein, and the sequence is highly conserved in other Plasmodium species. We produced the cysteine-rich domain of PyP140/RON4 by using the Pichia pastoris expression system and characterized the recombinant protein biochemically and biophysically. BALB/c mice immunized with the protein formulated in oil-in-water adjuvants produced antibodies that recognize parasitized erythrocytes by IFA and native PyP140/RON4 by immunoblotting but failed to protect against a lethal P. yoelii YM infection. Our results show that PyP140/RON4 is located within the rhoptries or micronemes. It may associate in part with AMA1, but the conserved cysteine-rich domain does not appear to elicit inhibitory antibodies, a finding that is supported by the marked sequence conservation in this protein within Plasmodium spp., suggesting that it is not under immune pressure.
The signalling lipid PI(3,5)P2 is generated on endosomes and regulates retrograde traffic to the trans-Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P2 levels. Mutations that lower PI(3,5)P2 cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P2 was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P2 regulatory complex by direct contact with the known regulators of PI(3,5)P2: Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P2 regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P2. Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse.
pale tremor; phosphatidylinositol 3,5-bisphosphate; PIKfyve; Saccharomyces cerevisiae
Phosphatidylinositol 3-phosphate (PI(3)P) plays an important role in insulin-stimulated glucose uptake. Insulin promotes the production of PI(3)P at the plasma membrane by a process dependent on TC10 activation. Here, we report that insulin-stimulated PI(3)P production requires the activation of Rab5, a small GTPase that plays a critical role in phosphoinositide synthesis and turnover. This activation occurs at the plasma membrane and is downstream of TC10. TC10 stimulates Rab5 activity via the recruitment of GAPEX-5, a VPS9 domain–containing guanyl nucleotide exchange factor that forms a complex with TC10. Although overexpression of plasma membrane-localized GAPEX-5 or constitutively active Rab5 promotes PI(3)P formation, knockdown of GAPEX-5 or overexpression of a dominant negative Rab5 mutant blocks the effects of insulin or TC10 on this process. Concomitant with its effect on PI(3)P levels, the knockdown of GAPEX-5 blocks insulin-stimulated Glut4 translocation and glucose uptake. Together, these studies suggest that the TC10/GAPEX-5/Rab5 axis mediates insulin-stimulated production of PI(3)P, which regulates trafficking of Glut4 vesicles.
Conjugation of polysaccharides to carrier proteins has been a successful approach for producing safe and effective vaccines. In an attempt to increase the immunogenicity of two malarial vaccine candidate proteins of Plasmodium falciparum, apical membrane antigen 1 (AMA1) for blood stage vaccines and surface protein 25 (Pfs25) for mosquito stage vaccines, each was chemically conjugated to the mutant, nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA). AMA1 is a large (66 kD) relatively good immunogen in mice; Pfs25 is a poorly immunogenic protein when presented on alum to mice. Mice were immunized on days 0 and 28 with AMA1 or Pfs25 rEPA conjugates or unconjugated AMA1 or Pfs25, all formulated on Alhydrogel. Remarkably, sera from mice 14 days after the second immunization with Pfs25-rEPA conjugates displayed over a 1000 fold higher antibody titers as compared to unconjugated Pfs25. In contrast, AMA1 conjugated under the same conditions induced only a 3 fold increase in antibody titers. When tested for functional activity, antibodies elicited by the AMA1-rEPA inhibited invasion of erythrocytes by blood-stage parasites and antibodies elicited by the Pfs25-rEPA conjugates blocked the development of the sexual stage parasites in the mosquito midgut. These results demonstrate that conjugation to rEPA induces a marked improvement in the antibody titer in mice for the poor immunogen (Pfs25) and for the larger protein (AMA1). These conjugates now need to be tested in humans to determine if mice are predictive of the response in humans.
The boronic acid functional group plays very important roles in sugar recognition, catalysis, organic synthesis, and supramolecular assembly. Therefore, understanding the unique properties of this functional group is very important. 8-Quinolineboronic acid (8-QBA) is found to be capable of self-assembling in solid state through a unique intermolecular B-N bond mechanism reinforced by intermolecular boronic anhydride formation, π-π stacking, and hydrogen bond formation. NMR NOE and diffusion studies indicate that intermolecular B-N interaction also exists in solution with 8-QBA. In contrast, a positional isomer of 8-QBA, 5-quinolineboronic acid (5-QBA) showed very different behaviors in crystal packing and in solution and therefore different supramolecular network. Understanding the structural features of this unique 8-QBA assembly could be very helpful for the future design of new sugar sensors, molecular catalysts, and supramolecular assemblies.
Quino lineboronic acid; Self-assembly; Crystal structure; NMR
Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells1. Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a phospholipid present in small quantities that regulates membrane trafficking in the endosome–lysosome axis in yeast2. Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the ‘pale tremor’ mouse. Positional cloning identified insertion of ETn2β (early transposon 2β)3 into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC (suppressor of actin) domain PtdIns(3,5)P2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns(3,5)P2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosome–lysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated patients with hereditary motor and sensory neuropathy. This novel form of autosomal recessive Charcot–Marie–Tooth disorder is designated CMT4J.
Although staphylokianse (SAK) is among the most promising blood dissolving agents, it is far from ideal. It is interesting to hypothesize that the clot lysis efficacy of SAK can be enhanced with direct active platelet binding ability, and at the same time the rethrombosis complication after successful recanalization can be minimized with an antiplatelet aggregation activity. The present study was performed to characterize the functional properties of RGD-SAK, a novel mutant of staphylokinase (SAK).
By using site-directed mutagenesis, an Arg-Gly-Asp (RGD) motif was engineered in the staphylokinase (SAK). This mutant of SAK designated RGD-SAK was expressed, purified and characterized. Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK. Measurement of platelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK. In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis and the concentration required to obtain 50% clot lysis (C50) were reduced significantly across different concentrations of RGD-SAK comparing with SAK. Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation.
RGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted thrombolytic and antiplatelet aggregation activities in compared with SAK.
Collectrin, a homologue of angiotensin converting enzyme 2 (ACE2), is a type I transmembrane protein, and we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells. Recently, two independent studies of targeted disruption of collectrin in mice resulted in severe and general defects in renal amino acid uptake. Collectrin has been reported to be under the transcriptional regulation by HNF-1α, which is exclusively expressed in proximal tubules and localized at the luminal side of brush border membranes. The deficiency of collectrin was associated with reduction of multiple amino acid transporters on luminal membranes. In the current study, we describe that collectrin is a target of HNF-1β and heavily expressed in the primary cilium of renal collecting duct cells. Collectrin is also localized in the vesicles near the peri-basal body region and binds to γ-actin-myosin II-A, SNARE, and polycystin-2-polaris complexes, and all of these are involved in intracellular and ciliary movement of vesicles and membrane proteins. Treatment of mIMCD3 cells with collectrin siRNA resulted in defective cilium formation, increased cell proliferation and apoptosis, and disappearance of polycystin-2 in the primary cilium. Suppression of collectrin mRNA in metanephric culture resulted in the formation of multiple longitudinal cysts in ureteric bud branches. Taken together, the cystic change and formation of defective cilium with the interference in the collectrin functions would suggest that it is necessary for recycling of the primary cilia-specific membrane proteins, the maintenance of the primary cilia and cell polarity of collecting duct cells. The transcriptional hierarchy between HNF-1β and PKD (polycystic kidney disease) genes expressed in the primary cilia of collecting duct cells has been suggested, and collectrin is one of such HNF-1β regulated genes.
Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.