Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism, but the profile of proteins associated with progesterone synthesis in cyclic and pregnant corpus luteum (CL) is not well-known in cattle. In Experiment 1, plasma progesterone level was monitored in cyclic cows (n = 5) and pregnant cows (n = 6; until d-90). A significant decline in the plasma progesterone level occurred at d-19 of cyclic cows. Progesterone level in abbatoir-derived luteal tissues was also determined at d 1 to 5, 6 to 13 and 14 to 20 of cyclic cows, and d-60 and -90 of pregnant cows (n = 5 each). Progesterone level in d-60 CL was not different from those in d 6 to 13 CL and d-90 CL, although the difference between d 6 to 13 and d-90 was significant. In Experiment 2, protein expression pattern in CL at d-90 (n = 4) was compared with that in CL of cyclic cows at d 6 to 13 (n = 5). Significant changes in the level of protein expression were detected in 32 protein spots by two-dimensional polyacrylamide gel electrophoresis (2-DE), and 23 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six proteins were found only in pregnant CL, while the other 17 proteins were found only in cyclic CL. Among the above 6 proteins, vimentin which is involved in the regulation of post-implantation development was included. Thus, the protein expression pattern in CL was disorientated from cyclic luteal phase to mid pregnancy, and alterations in specific CL protein expression may contribute to the maintenance of pregnancy in Korean native cows.
The correct distinction of carbapenem-resistant Enterobacteriaceae (CRE) and ccarbapenemase producing Enterobacteriaceae (CPE) and the rapid detection of CPE are important for instituting the correct treatment and management of clinical infections. Screening protocols are mainly based on cultures of rectal swab specimens on selective media followed by phenotypic tests to confirm a carbapenem-hydrolyzing activity, the rapid carbapenem inactivation method, lateral flow immunoassay, the matrix-assisted laser desorption ionization-time-of-flight test and molecular methods. The CPE is accurate for detection, and is essential for the clinical treatment and prevention of infections. A variety of phenotypic methods and gene-based methods are available for the rapid detection of carbapenemases, and these are expected to be routinely used in clinical microbiology laboratories. Therefore, to control the spread of carbapenemase, many laboratories around the world will need to use reliable, fast, high efficiency, simple and low cost methods. Optimal effects in patient applications would require rapid testing of CRE to provide reproducible support for antimicrobial management interventions or the treatment by various types of clinicians. For the optimal test method, it is necessary to combine complementary test methods to discriminate between various resistant bacterial species and to discover the genetic diversity of various types of carbapenemase for arriving at the best infection control strategy.
Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as $5.2{\pm}2.0{\mu}M$, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.
In 'solid-phase' PEGylation, the conjugation reaction occurs as the proteins are attached to a solid matrix, and thus it can have distinct advantages over the conventional, solution-phase process. We report a case study: rhIFN-${\alpha}$-2a was first adsorbed to cation exchange resin and then N-terminally PEGylated by aldehyde mPEG of 5, 10, and 20 kD through reductive alkylation. After the PEGylation, salt gradient elution efficiently recovered the mono-PEGylate in a purified form from the unwanted species such as unmodified IFN, unreacted PEG, and others. The mono-PEGylation and its purification were integrated in a single chromatographic step. Depending on the molecular weight of the mPEG aldehyde used, the mono-PEGylation yield ranged 50-64%. We could overcome the major problems of random, or uncontrollable, multi-PEGylation and the post-PEGylation purification difficulties associated with the solution-phase process. N-terminal sequencing and MALDI-TOF MS confirmed that a PEG molecule was conjugated only to the N-terminus. Compared with the unmodified IFN, the mono-PEGylate showed the reduced anti-viral activity as measured by the cell proliferation assay. The bioactivity was reduced more as the higher molecular weight PEG was conjugated. Immunoreactivity, evaluated indirectly by antibody binding activity using a surface plasmon resonance biosensor, also decreased. Nevertheless, trypsin resistance as well as thermal stability was considerably improved.
The objectives of this study were to compare the protein expression patterns and degrees and identify the protein function of disomic addition lines (DAs) in Leymus racemosus, in order to improve the quality of wheat. Upon SDS-PAGE, L. racemosus showed two major protein bands whereas Chinese Spring (CS) had four major protein bands of high molecular weight. The DA(s) generally showed a similar protein expression pattern to that of CS, because 42 chromosomes were from CS and two chromosomes were from L. racemosus. However, only the L.r[J] line showed two protein bands of between 15 and 20 kDa, like L. racemosus. Image analysis based on 2-DE revealed that L.r[F] had the most upregulated protein spots, whereas L.r[N] had the least upregulated protein spots. For L.r[I], the frequency of the downregulated protein spots was higher than that of the upregulated ones. Using MALDI-TOF MS, the protein function was identified for each protein spot on the 2-DE polyacrylamide gel. The protein spots were classified into 11 groups according to protein function. Among the 11 groups, most protein spots of the DA(s) were identified as proteins related to metabolism. Additionally, unique protein spots of the DA(s) were related to abiotic stressors such as cold and heat. Those proteins are useful for improving wheat quality with resistance against abiotic stressors.
Glycans are attached to proteins as in glycoproteins and proteoglycans. They are found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotic cells but may also be found in prokaryotes. The interaction of cell surface glycans with complementary glycan binding proteins located on neighboring cells, other cell types, pathogens like virus, or bacteria is crucial in biologically and biomedically important processes like pathogen recognition, cell migration, cell-cell adhesion, development, and infection. Their implication in pathological condition, suggests an important role for glycans as disease markers. In addition, a great amount of research has been shown that appropriate glycosylation of a recombinant therapeutic protein is critical for product solubility, stability, pharmacokinetics and pharmacodynamics, bioactivity, and safety. Besides, cancer-associated glycosylation changes often involve sialic acid in glycan branch which play important roles in cell-cell interaction, recognition and immunological response. This review aims at giving a comprehensive overview of the glycan's biological function and describing the relevance among the glycosylation, disease diagnosis and treatment methods. Furthermore, the high-throughput analytic methods available to measure the profile changing patterns of glycan in the blood serum as well as possible underlying biochemical mechanisms.
BACKGROUND/OBJECTIVES: This study aimed to establish a mother and child cohort in the Chinese population, and investigate human breastmilk (HBM) composition and its relationship with maternal body mass index (BMI) and infant growth during the first 3 mon of life. SUBJECTS/METHODS: A total of 101 Chinese mother and infant pairs were included in this prospective cohort. Alterations in the milk macronutrients of Chinese mothers at 1 mon (T1), 2 mon (T2), and 3 mon (T3) lactation were analyzed. HBM fatty acid (FA) profiles were measured by gas chromatography (GC), and HBM proteomic profiling was achieved by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). RESULTS: During the first 3 mon of lactation (P < 0.05), significant decreases were determined in the levels of total energy, fat, protein, and osteopontin (OPN), as well as ratios of long-chain saturated FA (including C16:0, C22:0 and C24:0), monounsaturated FA (including C16:1), and n-6 poly unsaturated FA (PUFA) (including C20:3n-6 and C20:4n-6, and n-6/n-3). Conversely, butyrate, C6:0 and n-3 PUFA C18:3n-3 (α-linolenic acid, ALA) were significantly increased during the first 3 mon (P < 0.05). HBM proteomic analyses distinguished compositional protein differences over time (P = 0.001). Personalized motherinfant analyses demonstrated that HBM from high BMI mothers presented increased total energy, fat, protein and OPN, and increased content of n-6 PUFA (including C18:3n-6, C20:3n-6 and n-6/n-3 ratio) as compared with low BMI mothers (P < 0.05). Furthermore, BMI of the mothers positively correlated with the head circumference (HC) of infants as well as the specific n-6 PUFA C20:3n-6 over the 3 time points examined. Infant HC was negatively associated with C18:0. CONCLUSION: This study provides additional evidence to the Chinese HBM database, and further knowledge of FA function. It also helps to establish future maternal strategies that support the healthy growth and development of Chinese infants.
In this study, we described the production of an antibody to living modified organisms (LMOs) containing the gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens strain CP4 EPSPS provides resistance to the herbicide glyphosate (N- (phosphonomethyl) glycine). These LMOs were approved and have recently been used in the feed, food production, and processing industries in South Korea. Highly efficient monoclonal antibody (mAb) production is crucial for developing assays that enable the proper detection and quantification of the CP4 EPSPS protein in LMOs. This study describes the purification and characterization of recombinant CP4 EPSPS protein in E. coli BL21 (DE3) based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The production of mAbs was undertaken based on the standard operating procedure of Abclon, Inc.(South Korea), and the purity of the mAbs was assessed using SDS-PAGE. The following five mAb clones were produced: 2F2, 4B9, 6C11, 10A9, and 10G9. To verify the efficiency and specificity of the five developed mAbs, we performed Western blotting analysis using the LM (living modified) cotton crude extracts. All mAbs could detect the CP4 EPSPS protein in the LM cotton traits MON1445 and MON88913 with high specificity, but not in any other LM cottons or non-LM cottons. These data indicate that these five mAbs to CP4 EPSPS could be successfully used for the further development of antibody-based detection methods to target CP4 EPSPS protein in LMOs.
Background: Gastric carcinogenesis is a complicated process that involves environmental and genetic factors like interleukin-4 (IL-4) and IL-8. Single nucleotide polymorphisms in their genes are associated with changed levels of gene expression. Here, we investigated the association between IL4-590 C>T and IL8-251T>A and gastric cancer (GC) risk in Sichuan of Southwestern China. Materials and Methods: We surveyed the research subjects using a self-designed questionnaire with questions on demographic factors and putative risk factors. Approximately 2-5ml of whole blood was collected after field survey to analyze IL4-590 C>T and IL8-251T>A genotypes using MALDI-TOF MS. Results: Our study recruited 308 pairs of GC patients and controls, including 224 (72.7%) men and 84 (27.3%) women in each group. There were 99 cardia and 176 noncardia GC patients in the case group. The case and control groups had an average age of $57.7{\pm}10.6$ ($mean{\pm}SD$) and $57.6{\pm}11.1$ years. GC patients reported a significantly greater proportion of family history of cancer (29.9% vs 10.7%, p<0.01) and drinking (54.6% vs 43.2%, p<0.01) than did controls. Variant genotypes of IL-4-590 C>T and IL-8-251 T>A were not associated with overall GC risk (adjusted OR, 0.89; 95%CI, 0.61-1.28 for CT or CC vs TT; adjusted OR, 1.14; 95%CI, 0.86-1.79 for TA or AA vs TT). Stratification analysis of two SNPs for risk by subsites only found that variant IL-8-251 TA or AA genotype was associated with increased noncardia GC risk (adjusted OR, 2.58; 95%CI, 1.19-5.57). We did not observe interactions between the IL-8-251 T>A genotype and smoking (adjusted OR, 0.38; 95%CI, 0.08-1.79) or drinking (adjusted OR, 0.36; 95%CI, 0.08-1.65) for risk of noncardia GC. Conclusions: Our data indicate no association between the two SNPs of IL-4-590 and IL-8-251 with overall GC risk, while the IL-8-251 TA or AA genotype conferred risk of cardia GC. Our findings contribute to the evidence body for risk of SNPs associated with the development of gastric cancer in this region.
Immuno-modulatory activities of peptides from Asterias amurensis were investigated using a nano-encapsulation process. The molecular weights of the peptides in the range of 5-7 kDa were separated using Sephadex G-75 gel filtration. Eighty-five percent of the nano-particles were in the 300 nm range using dynamic light scattering. The cytotoxicity of the A. amurensis nano-particles against CCD-986sk human dermal fibroblast cells was 11.64% after adding 1.0 mg/mL of the samples, which was lower than that from the control (13.28% collagen). The secretion of $NO^-$ from macrophages was estimated as $40\;{\mu}M$ after adding 1.0 mg/mL of gelatin nano-particles, which was higher than the others. Prostaglandin $E_2$ production from UV-induced human skin cells decreased greatly to 860 pg/mL after adding 1.0 mg/mL of the samples. Confocal microscopy revealed that nano-particles effectively penetrated the cells within 1 hour. From these results, we consider that nano-encapsulation of the peptides from A. amurensis can improve their biological functions.
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