The Bacteroid Periplasm in Soybean Nodules Is an Interkingdom Symbiotic Space.

The functional role of the periplasm of nitrogen-fixing bacteroids has not been determined. Proteins were isolated from the periplasm and cytoplasm of Bradyrhizobium diazoefficiens bacteroids and were analyzed using liquid chromatography tandem mass spectrometry proteomics. Identification of bacteroid periplasmic proteins was aided by periplasm prediction programs. Approximately 40% of all the proteins identified as periplasmic in the B. diazoefficiens genome were found expressed in the bacteroid form of the bacteria, indicating the periplasm is a metabolically active symbiotic space. The bacteroid periplasm possesses many fatty acid metabolic enzymes, which was in contrast to the bacteroid cytoplasm. Amino acid analysis of the periplasm revealed an abundance of phosphoserine, phosphoethanolamine, and glycine, which are metabolites of phospholipid metabolism. These results suggest the periplasm is a unique space and not a continuum with the peribacteroid space. A number of plant proteins were found in the periplasm fraction, which suggested contamination. However, antibodies to two of the identified plant proteins, histone H2A and lipoxygenase, yielded immunogold labeling that demonstrated the plant proteins were specifically targeted to the bacteroids. This suggests that the periplasm is an interkingdom symbiotic space containing proteins from both the bacteroid and the plant.

Development of target delivery system based on actinomycin class drugs and recombinant alpha-fetoprotein.

A recombinant alpha-fetoprotein (rAFP) was obtained in the yeast P. pastoris system, and its functional activity was confirmed. A method for producing polymer particles loaded with dactinomycin was developed, and a conjugate of these nanoparticles with rAFP was synthesized. The efficiency of the obtained conjugate on the HeLa, SKOV3, and MG-63 tumor cells and the absence of toxicity on the normal cells was shown. Experiments in vivo demonstrated a significant increase in the antitumor efficacy of the conjugate at a lower general toxicity as compared to the commercially available dactinomycin.

[Accumulation of doxorubicin conjugates with dendritic polymer and vector protein in normal and tumor cells in vitro].

Accumulation of doxorubicin (Dox), its conjugates with the second generation dendritic polymer (G2-Dox) and vector pro- tein (recombinant third domain of alpha-fetoprotein - 3D-G2- Dox) in normal and tumor cells was studied in vitro within the framework of the development of selective transport system of anticancer drugs to the target cells. The objects of the study were cells of peripheral blood mononuclear fraction of healthy donors and cells of breast adenocarcinoma lines MCF-7 and MCF-7/MDR1, differing in chemosensitivity. G2-Dox and 3D-G2-Dox accumulated in tumor cells of the both lines better than free Dox (p<0,05). However removal of these drugs out of cells MCF-7 and MCF-7/MDR1 was significantly different: in the latter case all free Dox was excluded from the cells for 24 hours while Dox, accumulated in composition with dendrimers, still remained in the cells. It was important that 3D-G2-Dox (unlike the G2-Dox) accumulated in normal cells worse than free Dox (p<0.01). Thus, the results indicate that the use of 3D-G2-Dox is the most promising because it accumulates in tumor cells better and in normal cells worse than free Dox. Furthermore it can be assumed that the use of 3D-G2-Dox would be especially useful in cases of multi-drug resistance associated with the high expression of P-glycoprotein.

Proteomic mapping for legume nodule organogenesis.

While genetic screens have identified mutants of the model legume Lotus japonicus that can nodulate in the absence of rhizobia, the lack of a proteome map is a major hindrance to understanding the functional protein networks associated with this nodulation process. In this issue of Proteomics, Dam et al. (Proteomics 2014, 14, 230-240) developed 2D gel-based reference maps of nodules and roots of Lotus and a spontaneous nodule formation mutant (snf1). Comparative proteomic analysis of roots and two developmental stages of nodules provide useful insights into tissue-specific mechanisms underlying nodule organogenesis. Additionally, a comparison of interspecies nodule proteomes displays that overlapping and individual mechanisms are associated with legume nodulation.

Telomerase as a tumor marker in diagnosis of prostatic intraepithelial neoplasia and prostate cancer.

BACKGROUND: Early diagnosis of prostate cancer (CaP) can be addressed by studying prostatic intraepithelial neoplasia (PIN) as precancer (high-grade PIN or HGPIN). This article attempts to analyze the diagnostic role of telomerase as an early marker of carcinogenesis. METHODS: Complex urological patient evaluation and assessment of telomerase activity. RESULTS: Out of 92 patients 44% were diagnosed with CaP, 49% with low-grade PIN (LGPIN) in association with benign prostatic hyperplasia (BPH), and 7% with HGPIN in association with BPH. Active telomerase (AT) in prostate biopsy specimens was detected in 98% of patients with CaP, in 33% of patients with HGPIN, and in 20% of patients with LGPIN. In the event of simultaneous detection of AT and PIN in initial prostate biopsy specimens, further monitoring for 0.5-4.0 years revealed CaP development in 50-56% of cases. Further follow-up of patients with PIN and absent telomerase activity in initial biopsy specimens did not demonstrate the development of CaP. The PSA level was significantly higher in patients with active telomerase in the prostate tissue than in telomerase negative patients. CONCLUSIONS: Telomerase activity in the prostate tissue increases the risk of CaP development in patients with PIN. Detection of telomerase activity in prostate biopsy specimens from patients with PIN enables selection of a group of patients with high risk of CaP development and reduction of the number of prostate biopsies performed in other patients.

Targeted delivery of doxorubicin: drug delivery system based on PAMAM dendrimers.

Polyamidoamine (PAMAM) dendrimers of the second generation (G2) are branched polymers containing 16 surface amino groups that allow them to be used as universal carriers on creating systems for drug delivery. G2 labeled with fluorescein isothiocyanate (FITC) efficiently bound with the surface of tumor cells at 4°C and was absorbed by the cells at 37°C. The covalent binding to G2-FITC of a vector protein, a recombinant fragment of the human alpha-fetoprotein receptor-binding domain (rAFP3D), increased the binding and endocytosis efficiency more than threefold. Covalent conjugates of G2 with doxorubicin (Dox) obtained by acid-labile linking of cis-aconitic anhydride (CAA) without the vector protein (G2-Dox) and with the vector protein rAFP3D (rAFP3D-G2-Dox) were accumulated by the tumor cells with high efficiency. However, a selective effect was observed only in rAFP3D-G2-Dox, which also demonstrated high cytotoxic activity against the human ovarian adenocarcinoma SKOV3 cells and low cytotoxicity against human peripheral blood lymphocytes. Based on these results, rAFP3D-G2 conjugate is promising for selective delivery of antitumor drugs.

New protein vector ApE1 for targeted delivery of anticancer drugs.

A new chimeric gene ApE1 encoding the receptor-binding domain of the human alpha-fetoprotein fused to a sequence of 22 glutamic acid residues was constructed. A new bacterial producer strain E. coli SHExT7 ApE1 was selected for ApE1 production in a soluble state. A simplified method was developed to purify ApE1 from bacterial biomass. It was shown that the new vector protein selectively interacts with AFP receptors on the tumor cell surface and can be efficiently accumulated in tumor cells. In addition, ApE1 was shown to be stable in storage and during its chemical modification. An increased number of carboxyl groups in the molecule allows the production of cytotoxic compound conjugates with higher drug-loading capacity and enhanced tumor targeting potential.

[The distribution of iodine-125 labeled alpha-fetoprotein in the animal organism and its accumulation in the tumor].

The distribution of iodine-125 labeled human alpha-fetoprotein in mice was studied after its intravenous injection. The maximal accumulation of alpha-fetoprotein in different tissues and organs of animals was observed mainly 5 hours after injection. Then the protein was gradually eliminated from the body. In the liver, intestine and blood of intact animals 125I-alpha-fetoprotein persists for at least three days. Accumulation of alpha-fetoprotein in various tissues and organs may determine the different biological effects of this protein. In the mice with transplanted lymphatic leukemia cells P388 the high level of alpha-fetoprotein accumulation was detected in the tumor tissue, reaching 6% of the injected amount per 1 g of tissue. This allows considering the radionuclide-labeled alpha-fetoprotein as a promising medical radionuclide marker for the radiological detection of malignant tumors.

Quantitation of soybean allergens using tandem mass spectrometry.

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 µg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

Optical properties of metamaterials based on asymmetric double-wire structures.

We performed theoretical and experimental investigations of the magnetic properties of metamaterials based on asymmetric double-wire structures. Using the multipole model for the description of metamaterials, we investigated the influence of the geometrical asymmetry of the structure on the macroscopic effective parameters. The results show that the larger wire in the system dominates the dynamics of the structure and defines the orientation and the strength of the microscopic currents. As a result the magnetization of the structure can be significantly enhanced for certain asymmetric configurations of the double-wire structure.

Single and multilayer metamaterials fabricated by nanoimprint lithography.

We demonstrate for the first time a fast and easy nanoimprint lithography (NIL) based stacking process of negative index structures like fishnet and Swiss-cross metamaterials. The process takes a few seconds, is cheap and produces three-dimensional (3D) negative index materials (NIMs) on a large area which is suitable for mass production. It can be performed on all common substrates even on flexible plastic foils. This work is therefore an important step toward novel and breakthrough applications of NIMs such as cloaking devices, perfect lenses and magnification of objects using NIM prisms. The optical properties of the fabricated samples were measured by means of transmission and reflection spectroscopy. From the measured data we retrieved the effective refractive index which is shown to be negative for a wavelength around 1.8 µm for the fishnet metamaterial while the Swiss-cross metamaterial samples show a distinct resonance at wavelength around 1.4 µm.

[High-efficient renaturation of immobilized recombinant C-terminal fragment of human alpha-fetoprotein].

C-terminal fragment of a human oncofetal alpha-fetoprotein (AFP) may be used in targeted cytostatics delivery to malignant cells of many tumors. AFP fragment (from 404 to 595 amino acids residues of a full-sized protein) was cloned and produced in Escherichia coli cells, BL21 strain (DE3) in the form of inclusion bodies. To obtain a functionally active protein, is it necessary to renature the protein. The renaturation procedure of the AFP third domain (rAFP3D) is considerably complicated by the fact that the protein is hydrophobic and contains a large number of S-S bonds. A renaturation technique of rAFP3D immobilized on silicic metal chelate resin has been developed. The yield of renatured C-terminal fragment was no less than 60% with purity on the order of 98%. The developed technique has been applied for the first time for hydrophobic protein with a large number of S-S bonds. The approach can be applied for efficient renaturation of other hydrophobic proteins with a large number of disulfide bonds for scientific and practical purposes.

A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity.

The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase-client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase-substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH(292)SMSDPGSTYR derived from the pyruvate dehydrogenase E1alpha subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.

Evolution of seed allergen quantification--from antibodies to mass spectrometry.

Development of accurate, high-throughput approaches for protein allergen quantification is important for the seed industry as a means to monitor natural variability in expression and ensure introduced transgenes do not collaterally alter the expression of any known allergen. Analytical approaches for protein quantification have undergone a renaissance in recent years with the emergence of soft-ionization approaches and advanced mass spectrometers capable of achieving low attomolar sensitivity. These advances coupled with bioinformatic tools to mine mass spectral data are collectively referred to as proteomics, and allow for the large-scale study of proteins with high precision and quantitative accuracy. In this review, we discuss differential and quantitative proteomics workflows that proceed from discovery profiling to targeted, quantitative analysis of specific proteins using stable isotopically-labeled, synthetic peptide doping standards. These synthetic peptide standards, also referred to as AQUA peptides, are synthetic mimics to proteotypic peptides and allow for absolute quantification of proteins in complex biological mixtures. The approaches discussed herein are ideal for the analysis of prominently expressed proteins such as protein allergens from plant seed, as no gels or sample pre-fractionation is required. We discuss these new techniques in the context of traditional, antibody-based technologies for allergen detection and quantification.

[Purification and characterization of recombinant human alpha-fetoprotein fragment, corresponding to the C-terminal structural domain].

Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.

Diagnosis and treatment protocols of cutaneous melanoma: latest approach 2010.

Cutaneous melanoma is the most aggressive skin malignancies with increasing rate of incidence in the latest decades. New imaging technique plays an important role in melanoma management: dermoscopy and computer dermoscopy, ultrasound, MRI, CT, PET and PET/CT. Due to the dermoscopy and lesion diagnosis in early stages the increasing number of curative melanoma are registered. Sentinel lymph node biopsy became a compulsory phase for patients with tumor thickness > 1 mm. Serological biomarkers proved to be a necessary investigation for melanoma diagnosis, follow-up and treatment response. Current TNM melanoma staging is based on AJCC classification since 2001 witch includes new elements like histopathologic ulceration in stage I and II and lymph node micro- and macrometastases in stage III. Treatment protocols include surgical tumor excision with only 1-2 cm safety margins and radical lymphadenectomy is performed after positive sentinel lymph node biopsy. The adjuvant treatment in advanced stages including chemotherapy, unspecific immunotherapy and interferon offers poor results regarding free disease terms rate of survival. The advanced therapeutic procedure like golden nanospheres and gene therapy are recently studied and represent an alternative for future treatment of melanoma. Follow-up protocols have a great importance for detection of the melanoma recurrences and include clinical, serological and imaging evaluation. Despite all new knowledge and technological support the advanced stage melanoma management still remain an unsolved problem.

Type of pH sensitive linker reveals different time-dependent intracellular localization, in vitro and in vivo efficiency in alpha-fetoprotein receptor targeted doxorubicin conjugate.

Despite the presence of a variety of modern anticancer drugs at the market, doxorubicin (Dox) is still widely used in antineoplastic therapy, although its administration causes severe side effects. To enhance specific activity of such molecules, various approaches have been exploited: targeted moieties like monoclonal antibodies, onco-specific proteins and peptides are utilized as specific vector molecules; environment sensitive linkers are exploited to facilitate transported drug release at the target point etc. Acid-labile linkers are frequently used in synthesis due to the ability to be cleaved inside specific cellular compartments with acidic environment, avoiding possible recycling mechanisms. Two types of conjugates containing different acid-labile linkers have been synthesized. In vitro efficiency of doxorubicin conjugates with recombinant receptor-binding domain of human alpha-fetoprotein (3dAFPpG) synthesized with use of cis-aconitic anhydride (CAA) and linker based on succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 3-(2-pyridyldithio)propionic acid hydrazide (PDPH) was compared. The 3dAFPpG-SPDP-PDPH-Dox revealed a comparable with unmodified doxorubicin cytotoxic effect against the Dox sensitive MCF7 cell line and greater cytotoxicity against the anthracycline resistant MCF7Adr cells. Meanwhile the 3dAFPpG-CAA-Dox cytotoxic effect was significantly lower, although doxorubicin's pH-dependent release profiles and intracellular accumulation rates were similar. These differences in cytotoxic activity were arguably explained by the dissimilarities in intracellular doxorubicin localization, which may originate from thiol reductase activity in lysosomes and late endosomes.

[Telomeric oligonucleotide complexes with PGEk protein vector: internalization by target cells and antiproliferative properties].

The recombinant protein PGEk, containing residual of the human epidermal factor (hEGF) bearing DNA binding sequence, retains ability of hEGF to bind with hEGF receptor and to induce cell proliferation was shown. On an example of PGEk complexes with telomeric mimic-oligodeoxyribonucleotide d(TTAGGG)4 and with its thio-analogue we had found such systems can be effectively and selectively internalized by hEGF receptors super expressing cells. The association of this process with a protein/oligonucleotide ratio in complexes was investigated. The intracellular localization of oligonucleotides was explored. We had shown that PGEk not only promotes intensive delivery of oligonucleotides, but also protects them from degradation by nucleases. The oligonucleotides in composition of complexes have considerably more expressed cytotoxic activity in comparison with free oligonucleotides.

[Prostate-specific antigen and telomerase in prostatic cancers].

A correlation between the blood level of prostate-specific antigen (PSA) and the prostatic tissue activity of telomerase was analyzed in prostate cancer (PC), low- and high-grade prostatic intraepithelial neoplasia (LG PIN, HG PIN) and in benign prostatic hyperplasia (BPH). The study was based on the results of a comprehensive examination of 92 patients from the Clinic of Urology, I. M. Sechenov Moscow Medical Academy. 44% of the patients were diagnosed as having PC; 49% had LG PIN and BPH; 7% had HG PIN and BPH. Active telomerase in the prostate biopsy specimens was found in 98% of the patients with PC, in 33% of those with HG PIN, and in 20% of those with LG PIN. When active telomerase was detected in the prostate biopsy specimens of patients with urological cancer (PC, PIN, BPH, the blood content of total PSA) was statistically significantly higher than that in the absence of this enzyme.

Variation in Seed Allergen Content From Three Varieties of Soybean Cultivated in Nine Different Locations in Iowa, Illinois, and Indiana.

Soybean (Glycine max) is an important food stock, and also considered an allergenic food with at least eight well characterized allergens. However, it is a less prevalent allergen source than many other foods and is rarely life-threatening. Soybean is incorporated into commonly consumed foods, and therefore, the allergens pose a potential concern for individuals already sensitized. The protein profile of soybean can be affected by several factors including genetic and environmental. To investigate how soybean allergen content may be affected by genetics and/or environment, nine soy allergens were quantified from three commercial soybean varieties grown at nine locations in three states within a single climate zone in North America; Iowa, Illinois, and Indiana, United States. Quantitation was achieved using liquid chromatography-selected reaction monitoring (LC-SRM) tandem mass spectrometry with AQUA peptide standards specific to the nine target allergens. Quantitation of allergen concentration indicated that both genetics and location affected specific allergen content. Seven of the nine allergens were significantly influenced by genetics, with the exceptions of glycinin G4 and KTI 3. The allergens P34, Gly m Bd 28k, glycinin G3, and KTI 1 showed statistically significant impact from location as well, but at a lower threshold of significance compared with genetics (cultivar/variety). This dataset contributes to our understanding of the natural variation of endogenous allergens, as it represents a sampling of soybeans grown in a controlled, distributed plot design under agronomic conditions common for commercial soybean food and feed production. The aim was to build upon our recent understanding of how allergens are expressed as part of the overall soybean proteome.