High Molecular Form Research Articles

Determination of the molecular weight of native molecules of γ-hydroxybutyrate dehydrogenase isoforms obtained from maize (Zea mays L.) seedlings by gel chromatography

It is known that under oxygen deficiency the functioning of the tricarboxylic acid cycle is impaired, resulting in the activation of an alternative pathway, the GABA shunt. It maintains the functioning of the citric acid cycle by providing succinic acid. The key phase of this bypass is a reaction catalysed by succinate semialdehyde dehydrogenase (SSADH, EC 1.1.1.16). Under oxygen deficiency, SSADH ceases to function efficiently. This leads to the accumulation of succinic acid semialdehyde in the mitochondrial matrix and its high level adversely affects the plant cell metabolism. γ–hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.61) is an enzyme belonging to the group of oxidoreductases. It transforms γ–hydroxybutyrate into succinic acid semialdehyde, participating in the process of its detoxification, which is important in the maintenance of plant metabolism under oxygen deficiency. To date, unfortunately, there are no data on the biochemical and kinetic features of γ–hydroxybutyrate dehydrogenase. In this regard, our laboratory developed a method for purification of HBDH from green maize leaves, which makes it possible to study the physicochemical properties of this enzyme. During the study, we obtained γ-hydroxybutyrate dehydrogenase from 7-day-old seedlings of Zea mays L. by a five-stage purification. Homogenised plant material with extracted proteins was subjected to two-stage ammonium sulphate fractionation. The catalytic activity was determined spectrophotometrically at λ=340 nm by the amount of reduced NAD+. To remove ammonium salts, we used gel filtration through Sephadex G-25. Proteins were separated according to their charge by DEAE-Sephacel ion exchange chromatography. The enzyme was desorbed using a linear sodium chloride gradient (100-300 mM). The use of gel chromatography through Sephadex G-200 made it possible to determine the molecular mass of the purified enzyme isoforms. The homogeneity of enzyme preparations was confirmed by electrophoresis in polyacrylamide gel with universal AgNO3 staining. We used the tetrazolium method to confirm that the obtained protein preparations were γ-hydroxybutyrate dehydrogenase. As a result, homogeneous preparations of two isoforms of the enzyme (HBDH1 and HBDH2) were obtained. The first isoform of γ-hydroxybutyrate dehydrogenase was 185.7 times purified with a yield of 10% and had a specific activity of 343.6 U/mg of protein. The purification rate of the second isoform was 209 times with a yield of 7.74%. The specific activity of the obtained preparation was 386.7 U/mg of protein. Using gel chromatography through Sephadex G-200, we determined the molecular mass of native molecules of hydroxybutyrate dehydrogenase. We found that in 7-day-old maize seedlings the investigated enzyme was presented in low-molecular and high-molecular forms: for HBDH1 the Mr value was ⁓60.3 kDa, while for HBDH2 the molecular mass of the enzyme was 286 kDa.

Plasma insulin-like growth factor-II (IGF-II) and IGF-II/IGF-I ratio in a chilean case of Doege-Potter Syndrome.

Doege-Potter syndrome is a rare clinical entity characterized by recurrent hypoglycemic events caused by non-pancreatic tumors secreting an incompletely processed high-molecular-weight form of Insulin-like Growth factor-II (IGF-II). To report IGF-II and IGF-I circulating levels in a Chilean case of Doege-Potter syndrome and control individuals, and to identify the high-molecular-weight form of IGF-II. We measured IGF-II and IGF-I plasma levels using enzyme-linked immunoassays (ELISA) in the patient and ten controls. We identified the high-molecular-weight form of IGF-II performed by Western blot. The plasma concentration of IGF-II in the patient was 868.9 ng/mL, which is only slightly > 80th percentile of controls (681,4 ± 212,8 ng/mL; mean ± standard deviation). In contrast, IGF-I plasma concentration in the patient was 17.6 ng/mL, which is notoriously lower than the corresponding levels in controls (109.1 ± 19.1 ng/mL). The IGF-II/IGF-I ratio in the patient was 49.4 (normal value < 10), which is 7.8 times higher compared to the average ratio of controls (6.3 ± 1.5). The high-molecular form of IGF-II presence in samples was confirmed through Western blot. The plasma IGF-II/IGF-I ratio better indicates the Doege-Potter syndrome's metabolic impairment than isolated measurements of circulating IGF-II or IGF-I levels.

Detection of unusual high molecular form of albumin in blood serum of COVID-19 patients

Blood sera of 12 severe Covid-19 patients and 14 healthy human donors were subjected to original TCA-extraction/acetone-precipitation followed by SDS-PAAG electrophoresis and mass-spectrometry. 76 kDa protein was detected as one of the differentially expressed proteins in the samples of Covid-19 patients. This 76 kDa protein was identified with mass-spectrometry as human serum albumin. Such molecular form of albumin was absent in blood serum of healthy human donors. The potential ways of generation of the unusual form of human serum albumin and its probable diagnostic value were discussed. Keywords: acetone-precipitation, biomarker proteins, COVID-19, electrophoresis, mass-spectrometry, TCA-extraction, unusual form of human serum albumin

Fibrinogenolytic activity of protease from the culture fluid of &lt;em&gt;Pleurotus ostreatus&lt;/em&gt;

The use of proteases makes it possible to obtain partially hydrolyzed forms of macromolecules with unique properties. The importance of proteases for studying the structure and functions of fibrinogen forces scientists to search for new sources of highly specific proteases. Thus, the aim of this work was to study the content of the Pleurotus ostreatus culture fluid in search of fibrinogen- specific proteases. P. ostreatus was cultured for 14 days at 27°C. The culture fluid was collected and the protein fraction was salted out with NaCl and then dialyzed. Fibrinogen hydrolysis products by P. ostreatus protease were characterized using SDS PAGE under reducing conditions followed by immunoprobing using murine monoclonal antibodies I-5A (anti-Aα505-610) and 2d2a (anti-Bβ26-42). The study of turbidity and platelet aggregation was performed using a Multiskan FC spectrophotometric microplate reader and a SOLAR-2110 aggregometer, respectively. Electron microscopy of fibrils formed by truncated compared with native fibrins was performed using a transmission electron microscope N-600.&#x0D; Analysis of the products of fibrinogen hydrolysis with a fungal protease using SDS-PAGE demonstrated the cleavage of the alpha chain of fibrinogen exclusively with the formation of a truncated form of fibrinogen in which there are no C-terminal portions of αC regions with a molecular weight of 25 kDa. A study of turbidity showed that the polymerization of truncated fibrin is significantly impaired. The rate of lateral association of protofibrils significantly decreased from 1.5 to 2.2 times in the case of truncated fibrinogen compared to the native one depending on the initial concentration of fibrinogen. It was shown that platelet aggregation in the presence of fibrinogen without 25 kDa fragments of αC regions was less effective than in the presence of native fibrinogen. Application of the preparation of the fungal protease allows us to obtain high molecular forms of the fibrinogen molecule with cleaved 25 kDa peptides, which provide new information on the role of these peptides in the fibrinogen functioning.

Fibrin(ogen)olytic and platelet modulating properties of a novel protease from the culture fluid of Pleurotus osreatus

The use of proteases makes it possible to obtain partially hydrolyzed forms of macromolecules with unique properties. The importance of proteases for studying the structure and functions of fibrinogen forces scientists to search for new sources of highly specific proteases. Thus, the aim of this work was to study the content of the Pleurotus ostreatus culture fluid in search of fibrinogen-specific proteases. P. ostreatus was cultured for 14 days at 27 ° C. The culture fluid was collected and the protein fraction was salted out with NaCl and then dialyzed. Fibrinogen hydrolysis products by P. ostreatus protease were characterized using SDS PAGE under reducing conditions followed by immunoprobing using murine monoclonal antibodies I-5A (anti-Aα505-610) and 2d2a (anti-Bβ26-42). The study of turbidity and platelet aggregation was performed using a Multiskan FC spectrophotometric microplate reader and a SOLAR-2110 aggregometer, respectively. Electron microscopy of fibrils formed by truncated compared with native fibrins was performed using a transmission electron microscope N-600.Analysis of the products of fibrinogen hydrolysis with a fungal protease using SDS-PAGE demonstrated the cleavage of the alpha chain of fibrinogen exclusively with the formation of a truncated form of fibrinogen in which there are no C-terminal portions of αC regions with a molecular weight of 25 kDa. A study of turbidity showed that the polymerization of truncated fibrin is significantly impaired. The rate of lateral association of protofibrils significantly decreased from 1.5 to 2.2 times in the case of truncated fibrinogen compared to the native one depending on the initial concentration of fibrinogen. It was shown that platelet aggregation in the presence of fibrinogen without 25 kDa fragments of αC regions was less effective than in the presence of native fibrinogen. Application of the preparation of the fungal protease allows us to obtain high molecular forms of the fibrinogen molecule with cleaved 25 kDa peptides, which provide new information on the role of these peptides in the fibrinogen functioning.

Phospho-PTM proteomic discovery of novel EPO- modulated kinases and phosphatases, including PTPN18 as a positive regulator of EPOR/JAK2 Signaling

The formation of erythroid progenitor cells depends sharply upon erythropoietin (EPO), its cell surface receptor (erythropoietin receptor, EPOR), and Janus kinase 2 (JAK2). Clinically, recombinant human EPO (rhEPO) additionally is an important anti-anemia agent for chronic kidney disease (CKD), myelodysplastic syndrome (MDS) and chemotherapy, but induces hypertension, and can exert certain pro-tumorigenic effects. Cellular signals transduced by EPOR/JAK2 complexes, and the nature of EPO-modulated signal transduction factors, therefore are of significant interest. By employing phospho-tyrosine post-translational modification (p-Y PTM) proteomics and human EPO- dependent UT7epo cells, we have identified 22 novel kinases and phosphatases as novel EPO targets, together with their specific sites of p-Y modification. New kinases modified due to EPO include membrane palmitoylated protein 1 (MPP1) and guanylate kinase 1 (GUK1) guanylate kinases, together with the cytoskeleton remodeling kinases, pseudopodium enriched atypical kinase 1 (PEAK1) and AP2 associated kinase 1 (AAK1). Novel EPO- modified phosphatases include protein tyrosine phosphatase receptor type A (PTPRA), phosphohistidine phosphatase 1 (PHPT1), tensin 2 (TENC1), ubiquitin associated and SH3 domain containing B (UBASH3B) and protein tyrosine phosphatase non-receptor type 18 (PTPN18). Based on PTPN18's high expression in hematopoietic progenitors, its novel connection to JAK kinase signaling, and a unique EPO- regulated PTPN18-pY389 motif which is modulated by JAK2 inhibitors, PTPN18's actions in UT7epo cells were investigated. Upon ectopic expression, wt-PTPN18 promoted EPO dose-dependent cell proliferation, and survival. Mechanistically, PTPN18 sustained the EPO- induced activation of not only mitogen-activated protein kinases 1 and 3 (ERK1/2), AKT serine/threonine kinase 1–3 (AKT), and signal transducer and activator of transcription 5A and 5B (STAT5), but also JAK2. Each effect further proved to depend upon PTPN18's EPO- modulated (p)Y389 site. In analyses of the EPOR and the associated adaptor protein RHEX (regulator of hemoglobinization and erythroid cell expansion), wt-PTPN18 increased high molecular weight EPOR forms, while sharply inhibiting the EPO-induced phosphorylation of RHEX-pY141. Each effect likewise depended upon PTPN18-Y389. PTPN18 thus promotes signals for EPO-dependent hematopoietic cell growth, and may represent a new druggable target for myeloproliferative neoplasms.

Tox4 modulates cell fate reprogramming.

ABSTRACTReprogramming to induced pluripotency induces the switch of somatic cell identity to induced pluripotent stem cells (iPSCs). However, the mediators and mechanisms of reprogramming remain largely unclear. To elucidate the mediators and mechanisms of reprogramming, we used a siRNA-mediated knockdown approach for selected candidate genes during the conversion of somatic cells into iPSCs. We identified Tox4 as a novel factor that modulates cell fate through an assay that determined the efficiency of iPSC reprogramming. We found that Tox4 is needed early in reprogramming to efficiently generate early reprogramming intermediates, irrespective of the reprogramming conditions used. Tox4 enables proper exogenous reprogramming factor expression, and the closing and opening of putative somatic and pluripotency enhancers early during reprogramming, respectively. We show that the TOX4 protein assembles into a high molecular form. Moreover, Tox4 is also required for the efficient conversion of fibroblasts towards the neuronal fate, suggesting a broader role of Tox4 in modulating cell fate. Our study reveals Tox4 as a novel transcriptional modulator of cell fate that mediates reprogramming from the somatic state to the pluripotent and neuronal fate.This article has an associated First Person interview with the first author of the paper.

Serum prolactin binding in various forms of hyperprolactinemia

Binding of 125I-labeled prolactin in the sera of women with different forms of hyperprolactinemia was studied. Antiprolactin autoantibodies were detected in the blood of 24% of women with idiopathic hyperprolactinemia. In contrast to idiopathic hyperprolactinemia, there were no autoantibodies to prolactin in pregnant women with physiological hyperprolactinemia and in hyperprolactinemia of tumor origin. These data indicate that the presence of these antibodies in circulating blood can be one cause of idiopathic hyperprolactinemia. Moreover, the prevalence of high-molecular form of immunoreactive prolactin in the blood can be due to binding of an appreciable amount of prolactin monomer to antiprolactin autoantibodies.

Abstract 4396: Gli, not Androgen Receptor, is the primary driver of prostate cell growth

Abstract Introduction Hedgehog (Hh) signaling regulates the activity of Gli transcription factors. Gli controls genes needed for development, cell growth and cell motility. Canonical Hh signaling through Smoothened [Smo] suppresses the proteolysis of Gli2 and Gli3, maintaining them in high molecular weight active forms. Previously, we showed that active androgen receptors ([AR] liganded AR full length and AR-V7) recognize and binds Gli3 its Protein Processing Domain. This binding overrides the need for Smo action and provides a means for stabilization and activation of Gli3 in prostate cancer (PCa) cells without Hh. Studies on the effects of AR binding to Gli2 are more challenging because it is expressed at much lower levels than Gli3. Here, we report that AR also affects Gli2 protein stability and show that Gli2, like Gli3, is a driver of PCa cell growth that is overexpressed in castration resistant disease. Method Immunohistochemistry (IHC) using Gli2- and Gli3-specific antibodies was used to assess expression of Gli2 and Gli3 in human PCa tumor microarrays. Western blot were used to identify and quantify levels of active Gli2 and Gli3 in androgen-treated PCa cells (LNCaP, LNCaP-AI, LAPC4). Proximity ligation assays (PLA) was used to visualize in situ and quantify AR-Gli2 complexes in PCa cells. Gli2- and Gli3-specific siRNAs were used to knock down Gli expression in AR+ (LNCaP-AI, LAPC4, 22Rv1) and AR- (PC3) PCa, cells followed by the Gli reporter assay and the cell growth assay. Results IHC outcomes showed that nuclear Gli2 and Gli3 protein expression was significantly higher in castration resistant tumors compared to primary disease. Androgens increased expression of active Gli2 and Gli3 protein expression in PCa cells and stimulated Gli reporter activity. PLA showed the presence of intranuclear complexes of Gli2/Gli3-AR-Full-length in androgen-treated LNCaP and Gli2-AR-V7 complexes in CWR22rv1 cells. Knockdown of Gli2 or Gli3 present significant reduction in Gli transcriptional activity as well as inhibition on growth of both AR+ and AR- PCa cells that were tested. Conclusion AR activity affects Gli2 processing, as it does for Gli3. We can visualize and quantify AR-Gli2/Gli3 complexes in situ in PCa cells. Gli2 suppression, as well as Gli3 suppression, has a profound effect on PCa cell growth but combined suppression has the strongest effects in certain PCa cells. Finally, Gli2 and Gli3 protein expression is elevated in castration resistant disease. Citation Format: Jane Foo, Na Li, Shabnam Massah, Mannan Nouri, Mike Wang, Ralph Buttyan. Gli, not Androgen Receptor, is the primary driver of prostate cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4396.

Molecular size-dependent specificity of hyaluronan on functional properties, morphology and matrix composition of mammary cancer cells

High levels of hyaluronan (ΗΑ), a major extracellular matrix (ECM) glycosaminoglycan, have been correlated with poor clinical outcome in several malignancies, including breast cancer. The high and low molecular weight HΑ forms exert diverse biological functions. Depending on their molecular size, ΗΑ forms either promote or attenuate signaling cascades that regulate cancer progression. In order to evaluate the effects of different ΗΑ forms on breast cancer cells' behavior, ΗΑ fragments of defined molecular size were synthesized. Breast cancer cells of different estrogen receptor (ER) status – the low metastatic, ERα-positive MCF-7 epithelial cells and the highly aggressive, ERβ-positive MDA-MB-231 mesenchymal cells – were evaluated following treatment with HA fragments. Scanning electron microscopy revealed that HA fragments critically affect the morphology of breast cancer cells in a molecular-size dependent mode. Moreover, the ΗΑ fragments affect cell functional properties, the expression of major ECM mediators and epithelial-to-mesenchymal transition (ΕΜΤ) markers. Notably, treatment with 200 kDa ΗΑ increased the expression levels of the epithelial marker Ε-cadherin and reduced the expression levels of HA synthase 2 and mesenchymal markers, like fibronectin and snail2/slug. These novel data suggest that the effects of HA in breast cancer cells depend on the molecular size and the ER status. An in-depth understanding on the mechanistic basis of these effects may contribute on the development of novel therapeutic strategies for the pharmacological targeting of aggressive breast cancer.

High-Titre Neutralizing Antibodies to H1N1 Influenza Virus after Mouse Immunization with Yeast Expressed H1 Antigen: A Promising Influenza Vaccine Candidate.

H1N1 influenza virus is still regarded as a serious pandemic threat. The most effective method of protection against influenza virus and the way to reduce the risk of epidemic or pandemic spread is vaccination. Influenza vaccine manufactured in a traditional way, though well developed, has some drawbacks and limitations which have stimulated interest in developing alternative approaches. In this study, we demonstrate that the recombinant H1 vaccine based on the hydrophilic haemagglutinin (HA) domain and produced in the yeast system elicited high titres of serum haemagglutination-inhibiting antibodies in mice. Transmission electron microscopy showed that H1 antigen oligomerizes into functional higher molecular forms similar to rosette-like structures. Analysis of the N-linked glycans using mass spectrometry revealed that the H1 protein is glycosylated at the same sites as the native HA. The recombinant antigen was secreted into a culture medium reaching approximately 10 mg/l. These results suggest that H1 produced in Pichia pastoris can be considered as the vaccine candidate against H1N1 virus.

Molecular Markers of Early Diagnosis of Alzheimer Disease: Prospects for Research in Peripheral Tissues

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of elderly and old-aged people. Cerebrospinal fluid (CSF) and peripheral tissues—lymphocytes and platelets, buccal and olfactory epithelium, and skin fibroblasts—are used for intravital diagnostics of the expression of signaling molecules (AD markers). There are several changes in the production of hyperphosphorylated τ-protein form, BACE1, and peptide Aβ42 in CSF in the case of AD, but CSF sampling can have a number of side effects. Biopsies of the epithelium and portions of blood are a less traumatic method of obtaining tissue samples for AD diagnosis. An increase in the expression of the hyperphosphorylated form of τ-protein is shown in the blood lymphocytes of AD patients. An increase in the content of high molecular forms of phosphorylated τ protein and amyloid precursor protein (APP) was also detected in the platelets of AD patients. Changes in the amount of two miRNA families, miR-132 and miR-134, were detected in blood cells 1–5 years before the manifestation of clinical signs of AD. An increase in the bound-calcium concentration, Aβ40 and Aβ42 peptide synthesis, and τ protein was observed in AD skin fibroblasts. In olfactory and buccal epithelia, an increase in the expression of hyperphosphorylated τ-protein form and Aβ peptide was detected in AD patients. The detection of AD markers in peripheral tissues available for biopsy is highly important for intravital diagnostics, prevention, and targeted treatment of AD.

γ-Tubulin has a conserved intrinsic property of self-polymerization into double stranded filaments and fibrillar networks

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.

Differential metabolic effects of constant moderate versus high intensity interval training in high-fat fed mice: possible role of muscle adiponectin.

Exercise regimens may have differing effects in the presence of obesity. In addition to being fat derived, adiponectin has recently been described as a myokine that regulates insulin sensitivity, which may link to exercise‐related metabolic benefits in obesity. Whether skeletal muscle adiponectin varies in different exercise modalities is unclear. This study investigated the comparative effects of 10 weeks of endurance constant‐moderate intensity exercise (END) with high intensity interval training (HIIT), on metabolic outcomes, including muscle adiponectin in a mouse model of diet‐induced obesity. Ten‐week‐old male C57BL/6 mice were fed a high‐fat diet (HFD) (45% FAT) or standard CHOW diet ab libitum and underwent one of three training regimes: (1) no exercise, (2) END, or (3) HIIT (8 bouts of 2.5 min with eight periods of rest of 2.5 min) for 10 weeks (3 × 40 min sessions/week). Chow‐fed mice acted as controls. Compared with HFD alone, both training programs similarly protected against body weight gain (HFD = 45 ± 2; END = 37 ± 2; HIIT = 36 ± 2 g), preserved lean/fat tissue mass ratio (HFD = 0.64 ± 0.09; END = 0.34 ± 0.13; HIIT = 0.33 ± 0.13), and improved blood glucose excursion during an insulin tolerance test (HFD = 411 ± 54; END = 350 ± 57; HIIT = 320 ± 66 arbitrary units [AU]). Alterations in fasting glycemia, insulinemia, and AST/ALT ratios were prevented only by END. END, but not HIIT increased skeletal muscle adiponectin mRNA (14‐fold; P < 0.05) and increased protein content of high molecular weight (HMW) adiponectin (3.3‐fold), whereas HIIT induced a milder increase (2.4‐fold). Compared with HFD, neither END nor HIIT altered circulating low (LMW) or high (HMW) molecular weight adiponectin forms. Furthermore, only END prevented the HFD downregulation of PGC1α (P < 0.05) mRNA levels downstream of muscle adiponectin. These data show that different training programs affect muscle adiponectin to differing degrees. Together these results suggest that END is a more effective regimen to prevent HFD‐induced metabolic disturbances in mice.

Phenolic compounds increase the transcription of mouse intestinal maltase-glucoamylase and sucrase-isomaltase.

Diverse natural phenolic compounds show inhibition activity of intestinal α-glucosidases, which may constitute the molecular basis for their ability to control systemic glycemia. Additionally, phenolics can modify mRNA expression for proteins involved in nutritional, metabolic or immune processes. To explore the possibility that phenolics can regulate the mRNA expression, enzymatic activity, and protein synthesis/processing of intestinal Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI), small intestinal explants from Balb/c mice were cultured for 24 h in the presence or absence of gallic acid, caffeic acid, and (+)-catechin at 0.1, 0.5, and 1 mM. We measured the levels of MGAM and SI mRNA expression by qRT-PCR, maltase and sucrase activities by a standard colorimetric method and the molecular size distribution of MGAM and SI proteins by western blotting. mRNA expression for MGAM was induced by the three phenolic compounds at 0.1 mM. mRNA expression for SI was induced by caffeic and gallic acids, but not by (+)-catechin. Caffeic acid was the most effective inducer of mRNA expression of these enzymes. Total maltase and sucrase activities were not affected by treatment with phenolics. The proportion of high molecular size forms of MGAM was significantly increased by two of the three phenolic compounds, but little effect was observed on SI proteins. Thus, changes in the protein synthesis/processing, affecting the proportions of the different molecular forms of MGAM, may account for the lack of correlation between mRNA expression and enzymatic activity.

Identification of the IGF-1 processing product human Ec/rodent Eb peptide in various tissues: Evidence for its differential regulation after exercise-induced muscle damage in humans

ObjectiveInsulin-like growth factor-1 (IGF-1) is a pleiotropic factor expressed in various tissues and plays a critical role in skeletal muscle physiology. Alternative splicing of the IGF-1 gene gives rise to different precursor polypeptides (isoforms) which could undergo post-translational cleavage, generating the common mature IGF-1 peptide and different carboxyl terminal extension (E-) peptides, with the fate of the latter being, so far, unknown. The objective if this study was to identify the IGF-1Ec forms or processing product(s), other than mature IGF-1, generated in different human and rodent tissues and particularly in human skeletal muscle after exercise-induced damage. DesignProtein lysates from a wide range of human and rodent tissues were immunoblotted with a rabbit anti-human Ec polyclonal antibody raised against the last 24 amino acids of the C-terminal of the Ec peptide. This antibody can recognize the Ec peptide, both as part of IGF-1Ec and alone, and also the corresponding rodent forms, due to the high homology that the human Ec shares with the rodent Eb. ResultsWe were able to confirm, for the first time, that the human Ec peptide and its rodent homologous Eb peptide are produced simultaneously with their precursor protein (pro-IGF-1Ec/Eb) in vivo, in a wide range of tissues (e.g. muscle, liver, heart). Proprotein convertase furin digestion of human muscle and liver protein lysates confirmed that the higher molecular form, pro-IGF-1Ec, can be cleaved to produce the free Ec peptide. Furthermore, initial evidence is provided that Ec peptide is differentially regulated during the process of muscle regeneration after exercise-induced damage in humans. ConclusionsThe findings of this study possibly imply that the post-translational modification of the IGF-1Ec pro-peptide may regulate the bioavailability and activity of the processing product(s).

Prion-like Aggregation of Mitochondrial Antiviral Signaling Protein in Lupus Patients Is Associated With Increased Levels of Type I Interferon.

Increased levels of type I interferon (IFN) and type I IFN-regulated genes are found in patients with systemic lupus erythematosus (SLE) and may be central to its pathogenesis. Mitochondrial antiviral signaling protein (MAVS) is a key regulator of type I IFN that undergoes a dramatic prion-like aggregation and self propagates the activation signal from viral RNA to amplify downstream IFN production. We undertook this study to determine whether such MAVS aggregates might play a role in the sustained increased production of type I IFN in SLE. Peripheral blood mononuclear cells were isolated and mitochondrial extracts were prepared. MAVS aggregation was detected by semidenatured agarose gel electrophoresis and confirmed by immunofluorescence staining. MAVS-associated signaling proteins were analyzed by Western blotting. MAVS aggregation-associated gene expression signature was analyzed by microarray. In blood cells from 22 of 67 SLE patients, essentially all MAVS was in a high molecular weight aggregated form. None of 6 rheumatoid arthritis patients and only 3 of 33 healthy controls had abnormal MAVS. Compared to MAVS aggregate-negative patients, MAVS aggregate-positive SLE patients had significantly higher serum levels of IFNβ and significantly increased levels of autoantibodies against Sm and U1 RNP. Gene array data revealed a characteristic gene expression pattern in these patients, with altered expression of genes involved in IFN signaling and membrane trafficking. Persistent MAVS aggregates may lead to increased type I IFN production and result in unmitigated signals leading to autoimmunity.

The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo

Recombinant adeno-associated viral (rAAV) vectors containing oversized genomes provide transgene expression despite low efficiency packaging of complete genomes. Here, we characterized the properties of oversized rAAV2/8 vectors (up to 5.4 kb) encoding human factor VIII (FVIII) under the transcriptional control of three liver promoters. All vectors provided sustained production of active FVIII in mice for 7 months and contained comparable levels of vector genomes and complete expression cassettes in liver. Therefore, for the 5.4 kb genome size range, a strong expression cassette was more important for FVIII production than the vector genome size. To evaluate the potency of slightly oversized vectors, a 5.1 kb AAVrh8R/FVIII vector was compared to a 4.6 kb (wild-type size) vector with an identical expression cassette (but containing a smaller C1-domain deleted FVIII) for 3 months in mice. The 5.1 kb vector had twofold to threefold lower levels of plasma FVIII protein and liver vector genomes than that obtained with the 4.6 kb vector. Vector genomes for both vectors persisted equally and existed primarily as high molecular weight concatemeric circular forms in liver. Taken together, these results indicate that the slightly oversized vectors containing heterogeneously packaged vector genomes generated a functional transgene product but exhibited a twofold to threefold lower in vivo potency.

Abstract 12951: Human C-Type Natriuretic Peptide-53: A Distinct Molecular Form With in vitro and in vivo Biological Actions Through cGMP Generation

Introduction: ANP and BNP are cardiac hormones that have a natriuretic and diuretic role in heart failure (HF) through guanylyl cyclase receptor A (GC-A) and cGMP generation. C-type natriuretic peptide (CNP) is part of this hormone family and is produced in the heart and endothelium. The biologically active form, CNP-22, is known to have potent anti-remodeling properties through the activation of GC-B and cGMP, but lacks natriuretic actions. Recently we have identified that another larger CNP form, CNP-53, is elevated in the plasma of acute decompensated HF patients, however it unclear if CNP-53 possesses biological actions. Hypothesis: We hypothesized that CNP-53 would be biologically active in vivo and in vitro, and would stimulate GC-B specific cGMP production. Methods: HEK 293 cells over-expressing GC-A and GC-B and human cardiac fibroblasts (hCFs), in which GC-B are in abundance, were stimulated with CNP-53 for 10 minutes at a dose of (10-8M). Two groups of anesthetized rats (n=8) received a 75-minute infusion of normal saline vehicle (V) or CNP-53 (1 ug/kg/min) and then mean arterial pressure (MAP), sodium excretion and urinary and plasma cGMP were assessed. Results: CNP-53 significantly activated cGMP in GC-B HEK cells (75±9 vs. 0.4±0.1 pmol/well, p&lt;0.05) and in hCFs (0.17±0.03 vs. 0.00±0.00 pmol/well, p&lt;0.05) compared to no treatment. In contrast CNP-53 failed to generate cGMP in GC-A HEK cells. In vivo, CNP-53 decreased MAP compared to vehicle (V: 101±2, CNP-53: 94±2 mmHg, p&lt;0.05). CNP-53 had a greater natriuretic effect compared vehicle (V: 0.86±0.11, CNP-53: 1.39±0.21 uEq/min, p&lt;0.05). Urinary cGMP excretion (V: 33±6, CNP-53: 177±28 pmol/min, p&lt;0.05) and plasma cGMP generation was also significantly higher with CNP-53 compared to vehicle (V: 9±2, CNP-53: 91±3 pmol/ml, p&lt;0.05). Conclusions: CNP-53, a higher molecular form of CNP that was reported to be increased human HF plasma, is bioactive with GC-B specific cGMP generation in vitro. Moreover, infusion of CNP-53 generates significant urinary and plasma cGMP and has natriuretic actions with minimal hypotensive effects in vivo. This study advances our understanding of CNP biology and provides new insights into potential CNP therapeutic opportunities targeting the progression of HF.

Biochemical Processes at the Stage of Withering during Black Tea Production

We determined the molecular weight and some properties of multiple forms of phenol oxidase from tea leaves and four other perennial plants. It was shown that multiple high- and low-molecular forms of phenol oxidase differed in substrate specificity. Low-molecular forms of the enzyme mostly demonstrated hydroxylase activity, while high-molecular forms showed catechol oxidase activity. It was revealed that the withering stage of black tea production is accompanied by the formation of only high-molecular forms of phenol oxidase, which possess catechol oxidase activity crucial for the procurement of oxidative reactions and the quality of the product.