Multiple Glycosaminoglycan-binding Epitopes of Monocyte Chemoattractant Protein-3/CCL7 Enable It to Function as a Non-oligomerizing Chemokine

The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP-3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAG-binding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate that CCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces.     

Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins: EVIDENCE FOR SYNDAPIN I REGULATING SYNAPTIC GLYCINE RECEPTORS*

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.     

The Cleaved N-Terminus of pVI Binds Peripentonal Hexons in Mature Adenovirus

Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen–deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule.     

Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles

Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92 Å, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.     

Factors Influencing Compact–Extended Structure Equilibrium in Oligomers of Aβ1–40 Peptide—An Ion Mobility Mass Spectrometry Study

Oligomers formed by amyloid β (Aβ) peptide are widely believed to be the main neurotoxic agent in Alzheimer's disease. Studies discovered a broad variety of oligomeric forms, which display different levels of toxicity. Some of these forms may further assemble into mature fibrils, while other might be off-pathway from conversion to fibrils and assemble into alternative forms. To better understand a relationship between the structure and toxicity of Aβ oligomers, we require systematic characterization and classification of all possible forms, facilitating rational design of the beneficial modifiers of their activity. In previous ion mobility analysis of Aβ1–40 oligomers, we have detected the coexistence of two alternative structural forms (compact and extended) in a pool of low-order Aβ1–40 oligomers. These forms may represent two pathways of the oligomer evolution, leading either to fibrils or to off-pathway oligomers, which are potential candidates for the neurotoxic species. Here, we have analyzed the impact of incubation time, the presence of selected metal ions and the effect of a series of point mutations on mutual population of alternative forms. We have shown that a salt bridge D23K28 provides stabilization of the compact form whereas G25 is required for the existence of the extended form. We have found that binding of metal ions also stabilizes the compact form. These results improve our understanding of the possible molecular mechanism of the bifurcation of structural evolution of non-monomeric Aβ species into an off-fibril pathway, ultimately leading to the formation of potentially neurotoxic species.     

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.     

Metabolic profiling and biological activities of the aerial parts of Micromeria imbricata Forssk. growing in Saudi Arabia

The hydroalcoholic extract (MIT) of Micromeria imbricata (Forssk.) growing in Saudi Arabia in addition to the chloroform (MIC) and n-butanol (MIB) fractions were investigated for the first time using UPLC-ESI-MS/MS. The analysis revealed the tentative identification of fifty-eight compounds including three organic acids, twenty-five phenolic compounds, three coumarins, two anthocyanins, twenty-one flavonoids, three terpenes, and one miscellaneous. Moreover, the therapeutic potential of M. imbricata (MIT) and its fractions (MIC and MIB) were determined by in vitro evaluation of their cytotoxic, antioxidant, and anti-obesity characteristics. The MIT extract showed the highest phenolic (125.23 ± 0.87 mg gallic acid equivalent/100 gm extract) and flavonoid (112.24 ± 2.45 mg quercetin equivalent/100 gm extract) contents followed by n-butanol and chloroform fractions. The MIT extract revealed a potent cytotoxic activity against HepG-2 (Hepatocellular carcinoma) and MCF-7 (Breast carcinoma) with IC₅₀ 28.5 ± 2.0 and 35.2 ± 1.2 µg/mL, respectively. Additionally, the tested hydroalcoholic extract exhibited a significant DPPH scavenging activity with SC₅₀ 28.4 ± 1.2 µg/mL and a remarkable lipase inhibitory activity with IC₅₀ 54.2 ± 1.2 µg/mL. In conclusion, the current study presents the first insights into the phytochemical constituents and pharmacological properties of M. imbricata extract and its chloroform and n-butanol fractions. The results revealed that M. imbricata hydroalcoholic extract might be a prolific source of bioactive constituents with potent antioxidant, cytotoxic and anti-obesity potential. It might be a natural alternative therapy and nutritional strategy for obesity treatment.     

强壮前沟藻化感物质分析

微藻化感作用是一种极其复杂的生理、生态学现象。选取强壮前沟藻指数生长初期Ⅰ和平台生长初期Ⅱ两个阶段的滤液对中肋骨条藻、海洋原甲藻、锥状斯氏藻及球等鞭金藻生长的影响进行了研究,并萃取了阶段Ⅱ的粗提物,抑藻检测表明其具有"杀藻"效应,通过GC/MS分析该粗提物中具有潜在化感作用的物质种类。研究发现强壮前沟藻两个生长阶段的滤液对中肋骨条藻均产生强烈致死效应(phaseⅠ:F=15.18475,P=0.00298<0.05;phaseⅡ:F=6.24559,P=0.03149<0.05);锥状斯氏藻在强壮前沟藻滤液中生长,实验结束时两个阶段中的细胞密度分别是对照组的79.3%和68.9%;海洋原甲藻在强壮前沟藻生长阶段Ⅱ滤液实验的最后3d,其生长受到显著抑制(F=4.84438,P=0.04925<0.05);而等鞭金藻在强壮前沟藻两个生长阶段滤液中被抑制现象不明显(P>0.05)。强壮前沟藻滤液实验表明,强壮前沟藻能够向微环境中分泌代谢产物来抑制中肋骨条藻和海洋原甲藻的生长,并且这种抑制效应具有种类特殊对应性。上述实验结果还表明,强壮前沟藻生长阶段Ⅱ的滤液具有的生长抑制作用较为明显。采用乙酸乙酯萃取强壮前沟藻生长阶段Ⅱ滤液中的代谢产物,检测发现其代谢粗提物具有溶藻效应,GC/MS分析结果表明粗提物中存在4种可能产生化感抑制作用的物质,其中二丁基羟基甲苯(Butylated Hydroxytoluene BHT)被认为具有抗滤过性病原体和抗微生物活性。