The heterodimerization of platelet-derived chemokines

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.     

Cyanobacterial bioactive metabolites—A review of their chemistry and biology

Cyanobacterial blooms occur when algal densities exceed baseline population concentrations. Cyanobacteria can produce a large number of secondary metabolites. Odorous metabolites affect the smell and flavor of aquatic animals, whereas bioactive metabolites cause a range of lethal and sub-lethal effects in plants, invertebrates, and vertebrates, including humans. Herein, the bioactivity, chemistry, origin, and biosynthesis of these cyanobacterial secondary metabolites were reviewed. With recent revision of cyanobacterial taxonomy by Anagnostidis and Komárek as part of the Süβwasserflora von Mitteleuropa volumes 19(1–3), names of many cyanobacteria that produce bioactive compounds have changed, thereby confusing readers. The original and new nomenclature are included in this review to clarify the origins of cyanobacterial bioactive compounds.Due to structural similarity, the 157 known bioactive classes produced by cyanobacteria have been condensed to 55 classes. This review will provide a basis for more formal procedures to adopt a logical naming system. This review is needed for efficient management of water resources to understand, identify, and manage cyanobacterial harmful algal bloom impacts.     

Heat shock proteins: A dual carrier-adjuvant for an anti-drug vaccine against heroin

Heroin is a highly abused opioid that has reached epidemic status within the United States. Yet, existing therapies to treat addiction are inadequate and frequently result into rates of high recidivism. Vaccination against heroin offers a promising alternative therapeutic option but requires further development to enhance the vaccine’s performance. Hsp70 is a conserved protein with known immunomodulatory properties and is considered an excellent immunodominant antigen. Within an antidrug vaccine context, we envisioned Hsp70 as a potential dual carrier-adjuvant, wherein immunogenicity would be increased by co-localization of adjuvant and antigenic drug hapten. Recombinant Mycobacterium tuberculosis Hsp70 was appended with heroin haptens and the resulting immunoconjugate granted anti-heroin antibody production and blunted heroin-induced antinociception. Moreover, Hsp70 as a carrier protein surpassed our benchmark Her-KLH cocktail through antibody-mediated blockade of 6-acetylmorphine, the main mediator of heroin’s psychoactivity. The work presents a new avenue for exploration in the use of hapten-Hsp70 conjugates to elicit anti-drug immune responses.     

Phenolic and anthocyanin fractions from wild blueberries (V. angustifolium) differentially modulate endothelial cell migration partially through RHOA and RAC1

The present study investigates the effect of anthocyanin (ACN), phenolic acid (PA) fractions, and their combination (ACNs:PAs) from wild blueberry powder (Vaccinum angustifolium) on the speed of endothelial cell migration, gene expression, and protein levels of RAC1 and RHOA associated with acute exposure to different concentrations of ACNs and PAs. Time-lapse videos were analyzed and endothelial cell speed was calculated. Treatment with ACNs at 60 μg/mL inhibited endothelial cell migration rate ( P ≤ 0.05) while treatment with PAs at 0.002 μg/mL ( P ≤ 0.0001), 60 μg/mL ( P ≤ 0.0001), and 120 μg/mL ( P ≤ 0.01) significantly increased endothelial cell migration rate compared with control. Moreover, exposure of HUVECs to ACNs:PAs at 8:8 μg/mL ( P ≤ 0.05) and 60:60 μg/mL increased ( P ≤ 0.001) endothelial cell migration. Gene expression of RAC1 and RHOA significantly increased 2 hours after exposure with all treatments. No effect of the above fractions was observed on the protein levels of RAC1 and RHOA. Findings suggest that endothelial cell migration is differentially modulated based on the type of blueberry extract (ACN or PA fraction) and is concentration-dependent. Future studies should determine the mechanism of the differential action of the above fractions on endothelial cell migration.     

A two-dimensional proteome map of maize endosperm

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.     

2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing

The barley proteins have been the subject of interests of many research groups dealing with barley grains, malt and beer. The proteins which remain intact after harsh malting conditions influence the quality and flavor of beer. The characteristic feature of the proteins present in malt and beer is their extensive modification with carbohydrates, mainly glucose that comes from the starch degradation during technological processes. The degree of the protein glycation has an effect on the quality of malt and beer and on the properties of the beer foam. A combination of two-dimensional high performance liquid chromatography (2D-HPLC) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS) was used for the analysis of the protein extracts that were reduced, alkylated, and degraded enzymatically without prior protein separation. This so-called "shot-gun" approach enabled us to determine glycation sites in one third of the proteins identified in the study and to propose potential glycation markers for fast and efficient monitoring during malting.     

反相C18固相萃取小柱用于蛋白质组分析中少量样品预处理

针对少量且复杂蛋白质组样品,开发一种耗时短、操作简便的分离方法。以人的脑海马组织蛋白样品为研究对象,采用反相C18固相萃取小柱,采用不同乙腈浓度对酶切后样品进行梯度洗脱,与反相高效液相色谱法对比分离效果。通过比较不同乙腈梯度洗脱方案所鉴定到的谱图数、非冗余多肽数、蛋白数和各洗脱组分间重复率分析,确定一种样品量少、简单易行、分离效果好的实验方案。虽然反相C18固相萃取小柱法鉴定蛋白总数占高效液相色谱法的85.5%,但其操作简单,耗时少。通过4种不同乙腈浓度方案比较,确定乙腈洗脱浓度优化为5%、15%、20%和90%时,分离30μg人的海马多肽混合物可以得到较优的分离效果。其蛋白鉴定数为反相液相色谱法的67.0%,且重复性良好。该结果证实反相C18固相萃取小柱分离效果比反相高效液相色谱法稍差,但此方法可以分离少量复杂蛋白质组样品。该方法分离样品充分,操作易行,耗时短,是进行蛋白质组学分析中少量样品的一个简便预处理方法。     

Isolation and characterization of fluorescence-enhancing RNA tags

Methods for the visualization of RNAs are urgently needed for studying a wide variety of cellular processes. Here we report on-bead screening of RNA libraries and its application to the isolation of specific fluorescence-enhancing RNA sequences. A one-bead-one-compound combinatorial RNA library with over one million different sequences was synthesized using the split-and-mix method. Solid-phase synthesis of 30 mer RNAs was performed on 15 ??m and 60 ??m diameter polystyrene beads bearing a non-cleavable linker. The RNA-derivatized beads were incubated with the well-established FlAsH pre-fluorophore and then screened for fluorescence enhancement, either by manually picking the brightest beads under a fluorescence microscope or by sorting with a FACS instrument. A protocol was established for sequence determination from single beads. While numerous RNA sequences showed increased fluorescence when immobilized, only few of them influenced the fluorescence properties of the FlAsH dye when detached from the beads. One of these sequences was found to induce a bathochromic shift in the excitation (from 492 to 510 nm) and emission (from 512 to 523 nm) maxima. This shift was accompanied by a 3.6-fold fluorescence enhancement of FlAsH fluorescence intensity. Mutation studies on the sequence revealed a rather robust structural motif.     

Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry

Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins.     

Structure based drug design and in vitro metabolism study: Discovery of N-(4-methylthiophenyl)-N,2-dimethyl-cyclopenta[d]pyrimidine as a potent microtubule targeting agent

We report a series of tubulin targeting agents, some of which demonstrate potent antiproliferative activities. These analogs were designed to optimize the antiproliferative activity of 1 by varying the heteroatom substituent at the 4′-position, the basicity of the 4-position amino moiety, and conformational restriction. The potential metabolites of the active compounds were also synthesized. Some compounds demonstrated single digit nanomolar IC₅₀ values for antiproliferative effects in MDA-MB-435 melanoma cells. Particularly, the S-methyl analog 3 was more potent than 1 in MDA-MB-435 cells (IC₅₀?=?4.6?nM). Incubation of 3 with human liver microsomes showed that the primary metabolite of the S-methyl moiety of 3 was the methyl sulfinyl group, as in analog 5. This metabolite was equipotent with the lead compound 1 in MDA-MB-435 cells (IC₅₀?=?7.9?nM). Molecular modeling and electrostatic surface area were determined to explain the activities of the analogs. Most of the potent compounds overcome multiple mechanisms of drug resistance and compound 3 emerged as the lead compound for further SAR and preclinical development.