Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.     

Resolving the micro-heterogeneity and structural integrity of monoclonal antibodies by hybrid mass spectrometric approaches

For therapeutic monoclonal antibodies (mAbs), detailed analysis of the structural integrity and heterogeneity, which results from multiple types of post-translational modifications (PTMs), is relevant to various processes, including product characterization, storage stability and quality control. Despite the recent rapid development of new bioanalytical techniques, it is still challenging to completely characterize the proteoform profile of a mAb. As a nearly indispensable tool in mAb analysis, mass spectrometry (MS) provides unique structural information at multiple levels. Here, we tested a hybrid strategy for the comprehensive characterization of micro-heterogeneity by integrating 2 state-of-the-art MS-based approaches, high-resolution native MS and targeted glycan profiling, to perform complementary analysis at the intact protein level and released glycan level, respectively. We compared the performance of these methods using samples of engineered half-body IgG4s and a panel of mAbs approved for human use. The glycosylation characterization data derived from these approaches were found to be mutually consistent in composition profiling, and complementary in identification and relative-quantitation of low-abundant uncommon glycoforms. In addition, multiple other sources of micro-heterogeneity, such as glycation, lack of glycosylation, and loss of light chains, could be detected by this approach, and the contribution of multiple types of modifications to the overall micro-heterogeneity could be assessed using our superposition algorithm. Our data demonstrate that the hybrid strategy allows reliable and thorough characterization of mAbs, revealing product characteristics that would easily be missed if only a single approach were used.     

Vimentin is important in the neural differentiation of PC12 cells promoted by sialylation

Sialic acid modification is a kind of post-translational modification. To investigate the regulation effect of sialic acid on neural differentiation, we used CycloManN propanyl perac (CycloManN pro), a metabolic precursor of sialic acid, to treat PC12 cells. We noted that CycloManN pro indeed robustly promoted global sialylation detected by MAL II lectin blot in PC12 cells. Simultaneously, we interestingly found that the neurite outgrowth of PC12 cells was significantly promoted by the CycloManN pro treatment. The profile analysis of sialylated proteins showed that a protein band at 55KD was greatly enhanced especially in PC12L cells after CycloManN pro treatment. After enrichment with lectin MAL II, the proteins in this band were analyzed by mass spectrometry. The results showed that 23 proteins were in the band, but the score of vimentin was the highest among them. To investigate further the role of vimentin in the process of neurite differentiation, vimentin construct was transfected into PC12 cells. We interestingly observed that ectopic expression of vimentin significantly enhanced the neurite outgrowth induced by CycloManN pro. However, after three potential glycosylation sites (Ser-7, Thr-33, Ser-34:) of vimentin were mutated to alanine, overexpression of the mutated vimentin completely lost the enhancement activity for the neural differentiation even in the presence of CycloManN pro. Taken together, our study demonstrated that vimentin was important in the induction of neural differentiation by CycloManN pro.     

99mTc-labeled bombesin(7-14)NH2 with favorable properties for SPECT imaging of colon cancer

In this report, we present the synthesis and evaluation of the (99m)Tc-labeled beta-Ala-BN(7-14)NH2 (ABN = beta-Ala-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) as a new radiotracer for tumor imaging in the BALB/c nude mice bearing HT-29 human colon cancer xenografts. The gastrin releasing peptide receptor binding affinity of ABN and HYNIC-ABN (6-hydrazinonicotinamide) was assessed via a competitive displacement of (125)I-[Tyr4]BBN bound to the PC-3 human prostate carcinoma cells. The IC 50 values were calculated to be 24 +/- 2 nM and 38 +/- 1 nM for ABN and HYNIC-ABN, respectively. HYNIC is the bifunctional coupling agent for (99m)Tc-labeling, while tricine and TPPTS (trisodium triphenylphosphine-3,3',3'-trisulfonate) are used as coligands to prepare the ternary ligand complex [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] in very high yield and high specific activity. Because of its high hydrophilicity (log P = -2.39 +/- 0.06), [(99m)Tc(HYNIC-ABN)(tricine)(TPPS)] was excreted mainly through the renal route with little radioactivity accumulation in the liver, lungs, stomach, and gastrointestinal tract. The tumor uptake at 30 min postinjection (p.i.) was 1.59 +/- 0.23%ID/g with a steady tumor washout over the 4 h study period. As a result, it had the best T/ B ratios in the blood (2.37 +/- 0.68), liver (1.69 +/- 0.41), and muscle (11.17 +/- 3.32) at 1 h p.i. Most of the injected radioactivity was found in the urine sample at 1 h p.i., and there was no intact [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] detectable in the urine, kidney, and liver samples. Its metabolic instability may contribute to its rapid clearance from the liver, lungs, and stomach. Despite the steady radioactivity washout, the tumors could be clearly visualized in planar images of the BALB/c nude mice bearing the HT-29 human colon xenografts at 1 and 4 h p.i. The favorable excretion kinetics from the liver, lungs, stomach, and gastrointestinal tract makes [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] a promising SPECT radiotracer for imaging colon cancer.     

GDF11 prevents the formation of thoracic aortic dissection in mice: Promotion of contractile transition of aortic SMCs

Thoracic aortic dissection (TAD) is an aortic disease associated with dysregulated extracellular matrix composition and de-differentiation of vascular smooth muscle cells (SMCs). Growth Differentiation Factor 11 (GDF11) is a member of transforming growth factor β (TGF-β) superfamily associated with cardiovascular diseases. The present study attempted to investigate the expression of GDF11 in TAD and its effects on aortic SMC phenotype transition. GDF11 level was found lower in the ascending thoracic aortas of TAD patients than healthy aortas. The mouse model of TAD was established by β-aminopropionitrile monofumarate (BAPN) combined with angiotensin II (Ang II). The expression of GDF11 was also decreased in thoracic aortic tissues accompanied with increased inflammation, arteriectasis and elastin degradation in TAD mice. Administration of GDF11 mitigated these aortic lesions and improved the survival rate of mice. Exogenous GDF11 and adeno-associated virus type 2 (AAV-2)-mediated GDF11 overexpression increased the expression of contractile proteins including ACTA2, SM22α and myosin heavy chain 11 (MYH11) and decreased synthetic markers including osteopontin and fibronectin 1 (FN1), indicating that GDF11 might inhibit SMC phenotype transition and maintain its contractile state. Moreover, GDF11 inhibited the production of matrix metalloproteinase (MMP)-2, 3, 9 in aortic SMCs. The canonical TGF-β (Smad2/3) signalling was enhanced by GDF11, while its inhibition suppressed the inhibitory effects of GDF11 on SMC de-differentiation and MMP production in vitro. Therefore, we demonstrate that GDF11 may contribute to TAD alleviation via inhibiting inflammation and MMP activity, and promoting the transition of aortic SMCs towards a contractile phenotype, which provides a therapeutic target for TAD.     

The Expression Alteration of BC1 RNA and its Interaction with Eukaryotic Translation Initiation Factor eIF4A Post-Status Epilepticus

Abnormal dendritic sprouting and synaptic remodelling are important pathological features of temporal lobe epilepsy. BC1 RNA is a translation repressor involved in the regulation of the dendritic protein synthesis and mRNA transport, which is essential for dendritic development and plasticity. The expression alteration of BC1 RNA in the pilocarpine induced epilepsy model remains unknown. It is unclear if the interactions between BC1 RNA and eukaryotic initiation factor 4A (eIF4A) exists in this model. The purpose of this study was to investigate the expression changes of BC1 RNA and its interactions with eIF4A post-status epilepticus (SE). Chloride lithium and pilocarpine were used to induce the SE rat model. Either a whole brain or hippocampus tissues were collected at different time points after SE. The expression patterns of BC1 was detected by qPCR and in situ hybridization. The levels of eIF4AI/II protein expression were analyzed via western blotting and immunohistochemistry. The BC1 RNA-eIF4AI/II interaction was determined by electrophoretic mobility shift assay (EMSA). We found that the BC1 RNA levels decreased in hippocampus 3d, 1w and 2w post-SE before the levels recovered. The eIF4AI/II began to rise 3d post-SE and reached the maximum level 1w post-SE. After 1w post-SE the levels decreased in the hippocampal CA1, CA3 and DG subregions. EMSA analysis showed that BC1 RNA specifically interacted with the eIF4AI/II. The BC1 RNA-eIF4AI/II complex reduced to the lowest level 1w post-SE. Our results suggested that BC1 has a negative regulatory correlation with eIF4AI/II, where BC1 RNA could be involved in epileptogenesis by regulating dendritic protein synthesis.     

Mammalian <Emphasis Type="Italic">CYP2E1</Emphasis> gene triggered changes of relative ions fluxes,CaM content and genes expression profiles in <Emphasis Type="Italic">Petunia hybrida</Emphasis> cells to enhance resistance to formaldehyde

The influence of light quality and cytokinin content in media on growth, development, photosynthetic pigments and secondary metabolite content of Myrtus communis L. was evaluated in an in vitro culture. Various treatments with light emitting diodes (LEDs): 100% blue (B), a mix of 70% red and 30% blue (RB) and 100% red were applied and compared with a traditional fluorescent lamp as control. Axillary shoots were incubated on Murashige and Skoog medium with 30 g dm^?3 sucrose, 0.5% BioAgar, 0.5 μM 1-naphthaleneacetic acid and different concentrations of 6-benzyladenine (BA): 1, 2.5 and 5 µM. Cultures were maintained for 6 weeks in 23/21?±?1 °C (day/night), 80% relative humidity and 16/8 h photoperiod; photosynthetic photon flux density (PPFD) was 35 µmol m^?2 s^?1 in all treatments. Light spectra and BA content in media affected biometrical and phytochemical M. communis properties. Red LEDs and 5 µM BA resulted in the highest multiplication rate. The highest shoots were obtained under red LEDs, but with the lowest concentration of cytokinin in media. Fresh weight was greatest on LEDs containing blue light in the spectrum (B and RB); moreover, 5 µM BA increased dry weight. Photosynthetic pigment levels were lower under LED light compared to control lamps. Phenolic acids and flavonoids were identified in M. communis leaf extracts. Myricetin was the major constituent with highest concentration under red LEDs and highest BA level.