Basic Isoforms Research Articles

RETRACTION: Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury

Retraction: M. C. Liu, V. Akle, W. Zheng, J. Kitlen, B. O'Steen, S. F. Larner, J. R. Dave, F. C. Tortella, R. L. Hayes, and K. K. W. Wang, "Extensive Degradation of Myelin Basic Protein Isoforms by Calpain Following Traumatic Brain Injury," Journal of Neurochemistry 98, no. 3 (2006): 700‐712. https://doi.org/10.1111/j.1471‐4159.2006.03882.x.The above article, published online on 19 June 2006, in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor‐in‐Chief, Andrew Lawrence; the International Society for Neurochemistry; and John Wiley and Sons Ltd. A third party reported that they had detected evidence of image manipulation and duplication throughout the published article. An investigation by the publisher and the editors concluded that there was evidence of splicing in Figures 2a, 2b, 3a, 5a, 6a, and 8a. The investigation also found duplications of lanes in Figures 3a, 3d, and 4a. Lastly, the investigation found that the same actin blots in Figure 3 have been used in two other articles by many of the same authors, despite representing different experimental conditions. The authors did not respond to an inquiry by the publisher. The retraction has been agreed to because the results presented in the article can no longer be considered reliable. The authors did not respond to the notice of retraction.

The inhibition of putrescine synthesis affects the in vitro shoot development of Cedrela fissilis Vell. (Meliaceae) by altering endogenous polyamine metabolism and the proteomic profile

Polyamines —(PAs) and proteins have been demonstrated to be fundamental for in vitro shoot development of Cedrela fissilis. We evaluated the influence of 6-benzyladenine (BA) and putrescine (Put) on the growth of shoots, PA metabolism and proteomic profiles of C. fissilis. The longest shoots were obtained under 2.5 µM BA + 2.5 mM Put treatment. The inhibition of Put synthesis by d-arginine (d-arg) reduced the activities of the Put biosynthesis enzymes arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) and the endogenous contents of free Put, resulting in reduced shoot growth. The ODC activity was higher than that of ADC, being ODC the main enzyme in the synthesis of Put in C. fissilis. Inhibition of Put synthesis affected the proteomic profile, reducing the accumulation of the ubiquitin receptor RAD23c, peroxidase 15, ADP-ribosylation factor 1, ADP-ribosylation factor-like protein 8a, profilin-4, profilin-2, glucan endo-1,3-beta-glucosidase, and expansin-like B1 and increasing the accumulation of V-type proton ATPase catalytic subunit A and methionine gamma-lyase, highlighting the relevance of these proteins in promoting the length of shoots. Moreover, the transport protein SEC13 homolog B and the basic isoform glucan endo-1,3-beta-glucosidase, unique proteins in shoots treated with BA + Put, were related to the promotion of shoot growth. Our results show that the modulation of endogenous PAs and proteomic profiles is necessary to regulate in vitro morphogenesis in C. fissilis. Moreover, the ODC enzyme is highly involved in the synthesis of Put during in vitro shoot development and is described for the first time in this species.

Structural and Functional Analysis of CEX Fractions Collected from a Novel Avastin® Biosimilar Candidate and Its Innovator: A Comparative Study.

Avastin® is a humanized recombinant monoclonal antibody used to treat cancer by targeting VEGF-A to inhibit angiogenesis. SIMAB054, an Avastin® biosimilar candidate developed in this study, showed a different charge variant profile than its innovator. Thus, it is fractionated into acidic, main, and basic isoforms and collected physically by Cation Exchange Chromatography (CEX) for a comprehensive structural and functional analysis. The innovator product, fractionated into the same species and collected by the same method, is used as a reference for comparative analysis. Ultra-Performance Liquid Chromatography (UPLC) ESI-QToF was used to analyze the modifications leading to charge heterogeneities at intact protein and peptide levels. The C-terminal lysine clipping and glycosylation profiles of the samples were monitored by intact mAb analysis. The post-translational modifications, including oxidation, deamidation, and N-terminal pyroglutamic acid formation, were determined by peptide mapping analysis in the selected signal peptides. The relative binding affinities of the fractionated charge isoforms against the antigen, VEGF-A, and the neonatal receptor, FcRn, were revealed by Surface Plasmon Resonance (SPR) studies. The results show that all CEX fractions from the innovator product and the SIMAB054 shared the same structural variants, albeit in different ratios. Common glycoforms and post-translational modifications were the same, but at different percentages for some samples. The dissimilarities were mostly originating from the presence of extra C-term Lysin residues, which are prone to enzymatic degradation in the body, and thus they were previously assessed as clinically irrelevant. Another critical finding was the presence of different glyco proteoforms in different charge species, such as increased galactosylation in the acidic and afucosylation in the basic species. SPR characterization of the isolated charge variants further confirmed that basic species found in the CEX analyses of the biosimilar candidate were also present in the innovator product, although at lower amounts. The charge variants’ in vitro antigen- and neonatal receptor-binding activities varied amongst the samples, which could be further investigated in vivo with a larger sample set to reveal the impact on the pharmacokinetics of drug candidates. Minor structural differences may explain antigen-binding differences in the isolated charge variants, which is a key parameter in a comparability exercise. Consequently, such a biosimilar candidate may not comply with high regulatory standards unless the binding differences observed are justified and demonstrated not to have any clinical impact.

O-279 The type of gonadotrophin: Does it improve ART outcome?

Abstract Ovarian stimulation is a vital clinical component of an IVF cycle, with gonadotrophins being the main stimulation drugs. Follicle stimulating hormone (FSH), the key driver of follicle recruitment during an ovarian cycle is a complex glycosylated protein comprised of an α and β subunit with four glycosylation sites. Differences in glycosylation lead to heterogeneity with the FSH isoforms varying in their receptor binding and biological activity. The cyclic physiological variation of FSH isoforms and the FSH threshold/ window concept lead to the selection of a single dominant follicle during a menstrual cycle. Surpassing the FSH threshold and extension of the FSH window with ovarian stimulation using gonadotrophins salvages follicles with higher FSH sensitivity leading to multi-follicular development and retrieval of multiple oocytes. The first IVF birth in 1978 was from a natural cycle and was preceded by several unsuccessful attempts at oocyte retrieval. Ovarian stimulation was therefore incorporated into IVF treatment to overcome the inefficiencies encountered with a natural cycle. The objective of ovarian stimulation during IVF treatment is to obtain several mature oocytes, leading to good quality embryos for immediate replacement and cryopreservation for future use. An ideal stimulation wherein gonadotrophins are the mainstay is one that achieves maximal efficacy, safety and cost-effectiveness. Several gonadotrophin preparations have been available for clinical use over the years since the first use of pregnant mare serum gonadotrophin (PMSG) in 1940 and the first successful birth using human pituitary gonadotrophin (hPG) in 1961. These initial preparations were soon abandoned due to concerns with antigenicity and safety. Subsequent important milestones in the development gonadotrophins for clinical use include the urinary derived products, recombinant FSH preparations, long acting FSH and the biosimilars. Given the selection of gonadotrophins currently available, the apparent question is if they differ? Circulating FSH isoforms change during different stages of the menstrual cycle and through the reproductive stages. More acidic isoforms are predominant during the early follicular phase, with less acidic or basic isoforms predominant in the late follicular phase of the menstrual cycle, and more acidic isoforms during the menopausal stage. Similarly, the different FSH products vary in their properties owing to differences in their production and purification. Urinary derived products are more acidic compared to recombinant preparations and the different recombinant products vary in degree of acidity due to differences in the extent and pattern of glycosylation. The acidic and basic properties of gonadotrophin types influence metabolic stability, half-life and their biological activity. The question then remains if these differences in bioactivity affect clinical outcomes? Clinical effectiveness of the various gonadotrophins types should be addressed in the context of the different pituitary suppression regimens, ovarian response categories and patient variables given the implementation of individualised stimulation protocols. It is also imperative to clearly define successful clinical outcomes incorporating both efficacy and safety. Cumulative live birth rate (CLBR) following fresh and frozen embryo transfers resulting from one episode of ovarian stimulation should be adopted as the measure of the overall efficiency along with OHSS as a measure of immediate safety. There are several studies and systematic reviews comparing the different gonadotrophin types and products for various outcomes including surrogate endpoints, with inconsistent results attributed to differences in the inclusion/ exclusion criteria, heterogeneity across studies and varied quality of the primary studies. Furthermore, there have been sparse studies reporting on CLBR and there is a continuing debate on what would be considered as a relevant clinical difference in efficacy. In addition to efficacy and safety, the ultimate choice of gonadotrophin should be based on regional availability, patient acceptability and cost-effectiveness for a live birth.

Weighted gene coexpression correlation network analysis reveals a potential molecular regulatory mechanism of anthocyanin accumulation under different storage temperatures in \u2018Friar\u2019 plum

BackgroundFlesh is prone to accumulate more anthocyanin in postharvest ‘Friar’ plum (Prunus salicina Lindl.) fruit stored at an intermediate temperature. However, little is known about the molecular mechanism of anthocyanin accumulation regulated by storage temperature in postharvest plum fruit.ResultsTo reveal the potential molecular regulation mechanism of anthocyanin accumulation in postharvest ‘Friar’ plum fruit stored at different temperatures (0 °C, 10 °C and 25 °C), the fruit quality, metabolite profile and transcriptome of its flesh were investigated. Compared to the plum fruit stored at 0 °C and 25 °C, the fruit stored at 10 °C showed lower fruit firmness after 14 days and reduced the soluble solids content after 21 days of storage. The metabolite analysis indicated that the fruit stored at 10 °C had higher contents of anthocyanins (pelargonidin-3-O-glucoside, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and quercetin-3-O-rutinose), quercetin and sucrose in the flesh. According to the results of weighted gene coexpression correlation network analysis (WGCNA), the turquoise module was positively correlated with the content of anthocyanin components, and flavanone 3-hydroxylase (F3H) and chalcone synthase (CHS) were considered hub genes. Moreover, MYB family transcription factor APL (APL), MYB10 transcription factor (MYB10), ethylene-responsive transcription factor WIN1 (WIN1), basic leucine zipper 43-like (bZIP43) and transcription factor bHLH111-like isoform X2 (bHLH111) were closely related to these hub genes. Further qRT–PCR analysis verified that these transcription factors were specifically more highly expressed in plum flesh stored at 10 °C, and their expression profiles were significantly positively correlated with the structural genes of anthocyanin synthesis as well as the content of anthocyanin components. In addition, the sucrose biosynthesis-associated gene sucrose synthase (SS) was upregulated at 10 °C, which was also closely related to the anthocyanin content of plum fruit stored at 10 °C.ConclusionsThe present results suggest that the transcription factors APL, MYB10, WIN1, bZIP43 and bHLH111 may participate in the accumulation of anthocyanin in ‘Friar’ plum flesh during intermediate storage temperatures by regulating the expression of anthocyanin biosynthetic structural genes. In addition, the SS gene may play a role in anthocyanin accumulation in plum flesh by regulating sucrose biosynthesis.

Two Carbohydrate-Based Natural Extracts Stimulate in vitro Pollen Germination and Pollen Tube Growth of Tomato Under Cold Temperatures.

To date, it is widely accepted by the scientific community that many agricultural regions will experience more extreme temperature fluctuations. These stresses will undoubtedly impact crop production, particularly fruit and seed yields. In fact, pollination is considered as one of the most temperature-sensitive phases of plant development and until now, except for the time-consuming and costly processes of genetic breeding, there is no immediate alternative to address this issue. In this work, we used a multidisciplinary approach using physiological, biochemical, and molecular techniques for studying the effects of two carbohydrate-based natural activators on in vitro tomato pollen germination and pollen tube growth cultured in vitro under cold conditions. Under mild and strong cold temperatures, these two carbohydrate-based compounds significantly enhanced pollen germination and pollen tube growth. The two biostimulants did not induce significant changes in the classical molecular markers implicated in pollen tube growth. Neither the number of callose plugs nor the CALLOSE SYNTHASE genes expression were significantly different between the control and the biostimulated pollen tubes when pollens were cultivated under cold conditions. PECTIN METHYLESTERASE (PME) activities were also similar but a basic PME isoform was not produced or inactive in pollen grown at 8°C. Nevertheless, NADPH oxidase (RBOH) gene expression was correlated with a higher number of viable pollen tubes in biostimulated pollen tubes compared to the control. Our results showed that the two carbohydrate-based products were able to reduce in vitro the effect of cold temperatures on tomato pollen tube growth and at least for one of them to modulate reactive oxygen species production.

Detection of recombinant erythropoietin biosimilar Jimaixin™ after administration in healthy subjects.

Jimaixin™ (Jintan Ltd, China) is a biosimilar of recombinant erythropoietin (rEPO) now authorized for therapeutic application in China. With a risk of abuse by athletes, a clear evaluation of its detection using the electrophoretic methods in use in antidoping laboratories was necessary. In a previous work, we showed that Jimaixin™ electrophoretic profile presented slight changes compared with the original drug (first generation rEPO) and that a spike of Jimaixin™ in urine and serum was well identified by SDS-PAGE but with less performance by IEF-PAGE unless a neuraminidase treatment was applied first. The aims of this research were to perform an intravenous administration of Jimaixin™ on three healthy subjects (one microdose [10IU/kg] and three therapeutic doses [50 IU/kg]) and to evaluate the detection in urine and blood up to 7days post administration. Analysis of the samples showed that Jimaixin™ detection was complicated by IEF-PAGE due to the loss of the most distinctive basic isoforms. In addition, a neuraminidase treatment did not improve detection (contrary to the observations from spike experiments). On the contrary, Jimaixin™ was very efficiently detected in blood and urine by SDS-PAGE: up to 40 h after a microdose and up to 7days after the therapeutic doses. The effect of Jimaixin™ on hematological parameters was limited to a clear but transitory increase of the reticulocytes. These data give new elements to better survey a potential misuse of Jimaixin™ by athletes.

Tobacco hairy root’s peroxidases are rhythmically controlled by phenol exposure

Plants like almost all living organisms, have developed a biological clock or circadian clock (CC) capable of synchronizing and adjusting various metabolic and physiological processes at certain times of the day and in a period of 24 h. This endogenous timekeeping is able to predict the environmental changes providing adaptive advantages against stressful conditions. Therefore, the aim of this work was to analyze the possible link between metabolism of xenobiotic compounds (MXC) and the CC. Synchronized Nicotiana tabacum hairy roots (HRs) were used as a validated plant model system, and peroxidases (PODs), key enzymes of the phase I in the MCX, were evaluated after phenol treatment. Two POD genes were selected and their temporal expression profiles as well as the total POD activity were analyzed in order to find circadian oscillations either under control conditions or phenol treatment. It was demonstrated that these PODs genes showed oscillatory profiles with an ultradian period (period length shorter than the circadian period), and preserving the same phases and expression peaks still under phenol treatment. The total PODs activity showed also a marked oscillatory behavior mainly in phenol-treated HRs with the highest levels at ZT23. Untreated HRs showed decrease and increase in the intensity of some basic isoforms at light and dark phase, respectively, while in phenol- treated HRs, an increase in the intensity of almost all isoforms was observed, mainly during the dark phase, being coincident with the high PODs activity detected at ZT23. The periodic analysis determined an ultradian period either in total POD activity or in the POD activity of isoform VI, being 18.7 and 15.3 h, respectively. Curiously, in phenol treated HRs, the period length of total POD activity was longer than in untreated HRs, suggesting that phenol could induce a marked oscillatory behavior in the POD activity with better performance during the dark phase, which explain the higher phenol removal efficiencies at ZT23. These findings showed novel information about the performance of PODs, which would be rhythmically controlled at biochemical level, by phenol exposure.

Computational Methods and Online Resources for Identification of piRNA-Related Molecules.

piRNAs are a class of small non-coding RNA molecules, which interact with the PIWI family and have many important and diverse biological functions. The present review is aimed to provide guidelines and contribute to piRNA research. We focused on the four types of identification models on piRNA-related molecules, including piRNA, piRNA cluster, piRNA target, and disease-related piRNA. We evaluated the types of tools for the identification of piRNAs based on five aspects: datasets, features, classifiers, performance, and usability. We found the precision of 2lpiRNApred was the highest in datasets of model organisms, piRNN had a better performance of datasets of non-model organisms, and 2L-piRNA had the fastest recognition speed of all tools. In addition, we presented an overview of piRNA databases. The databases were divided into six categories: basic annotation, comprehensive annotation, isoform, cluster, target, and disease. We found that piRNA data of non-model organisms, piRNA target data, and piRNA-disease-associated data should be strengthened. Our review might assist researchers in selecting appropriate tools or datasets for their studies, reveal potential problems and shed light on future bioinformatics studies.

Evaluation of erythropoietin biosimilars Epotin™, Hemax® and Jimaixin™ by electrophoretic methods used for doping control analysis and specific N-glycan analysis revealed structural differences from original epoetin alfa drug Eprex®

Recombinant human erythropoietin (rEPO) biosimilars are copies of epoetin drugs developed after the first patents ended. However differences in the process of production can result in small structural differences when compared to the reference product. Differences in N-glycosylation profiles are of particular importance for rEPOs, because they can drastically impact the half-life in circulation and activity. Changes of structure can also impact electrophoretic profiles that are used to reveal the presence of a rEPO in a doping control sample. In this study three not well characterized biosimilars were evaluated (Jimaixin™ authorized in China, and Hemax® and Epotin™ authorized in Algeria). As these products could be used for doping, first their EPO profiles were determined using the antidoping methods (electrophoretic separation by the charge (isolectric focusing, IEF-PAGE) or the molecular weight (SDS-PAGE) and specific EPO immunodetection). Compared to the original epoetin alfa Eprex®, it revealed more basic isoforms for Epotin™ and Jimaixin™ after IEF-PAGE and a slightly lower molecular weight after SDS-PAGE in particular for Hemax®. To better understand the reason for these differences, EPO specific N-glycans were evaluated using two complementary approaches: MALDI-TOF mass spectrometry (MS) and hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. All three biosimilars presented a significant decrease in the major glycan forms of Eprex® along with an increase in less complex forms. Jimaixin™ and Epotin™ presented also a lower amount of fully sialylated forms. HILIC method also showed that O-acetylation level of sialic acid residues might vary from one rEPO to the other.

Peroxidase zymograms obtained by agarose native gel electrophoresis have unmet resolution and completeness

A recently published method to separate protein isoforms by means of a flat-bed agarose native gel was adapted for identifying simultaneously both acid and basic isoforms of plant peroxidases. These were evidenced by in situ activity staining using alternative substrates for which the isoforms showed specific preference. Such approach allowed the detection of a significantly higher number of horseradish peroxidases than the conventional methods based on sample separation by acrylamide gel and the single bands were clearer to observe. Samples recovered from different plant species and with variable level of purity were successfully analyzed for their peroxidase (POX) isoform pattern. We expect that the innovative electrophoretic methodology illustrated in this work will strongly improve the output of experiments that aim at relating the activity and expression variation of specific enzyme biomarkers with physiological and pathological conditions.

Potential Neurodevelopmental Effects of Pediatric Intensive Care Sedation and Analgesia: Repetitive Benzodiazepine and Opioid Exposure Alters Expression of Glial and Synaptic Proteins in Juvenile Rats.

Objectives:Sedatives are suspected contributors to neurologic dysfunction in PICU patients, to whom they are administered during sensitive neurodevelopment. Relevant preclinical modeling has largely used comparatively brief anesthesia in infant age-approximate animals, with insufficient study of repetitive combined drug administration during childhood. We hypothesized that childhood neurodevelopment is selectively vulnerable to repeated treatment with benzodiazepine and opioid. We report a preclinical model of combined midazolam and morphine in early childhood age-approximate rats.Design:Animal model.Setting:Basic science laboratory.Subjects:Male and female Long-Evans rats.Interventions:Injections of morphine + midazolam were administered twice daily from postnatal days 18–22, tapering on postnatal days 23 and 24. Control groups included saline, morphine, or midazolam. To screen for acute neurodevelopmental effects, brain homogenates were analyzed by western blot for synaptophysin, drebrin, glial fibrillary acidic protein, S100 calcium-binding protein B, ionized calcium-binding adaptor molecule 1, and myelin basic proteins. Data analysis used Kruskal-Wallis with Dunn posttest, with a p value of less than 0.05 significance.Measurements and Main Results:Morphine + midazolam and morphine animals gained less weight than saline or midazolam (p ≤ 0.01). Compared with saline, morphine + midazolam expressed significantly higher drebrin levels (p = 0.01), with numerically but not statistically decreased glial fibrillary acidic protein. Similarly, morphine animals exhibited less glial fibrillary acidic protein and more S100 calcium-binding protein B and synaptophysin. Midazolam animals expressed significantly more S100 calcium-binding protein B (p < 0.001) and 17–18.5 kDa myelin basic protein splicing isoform (p = 0.01), with numerically increased synaptophysin, ionized calcium-binding adaptor molecule 1, and 21.5 kDa myelin basic protein, and decreased glial fibrillary acidic protein.Conclusions:Analysis of brain tissue in this novel rodent model of repetitive morphine and midazolam administration showed effects on synaptic, astrocytic, microglial, and myelin proteins. These findings warrant further investigation because they may have implications for critically ill children requiring sedation and analgesia.

Calponin-3 deficiency augments contractile activity, plasticity, fibrogenic response and Yap/Taz transcriptional activation in lens epithelial cells and explants

The transparent ocular lens plays a crucial role in vision by focusing light on to the retina with loss of lens transparency leading to impairment of vision. While maintenance of epithelial phenotype is recognized to be essential for lens development and function, knowledge of the identity of different molecular mechanisms regulating lens epithelial characteristics remains incomplete. This study reports that CNN-3, the acidic isoform of calponin, an actin binding contractile protein, is expressed preferentially and abundantly relative to the basic and neutral isoforms of calponin in the ocular lens, and distributes predominantly to the epithelium in both mouse and human lenses. Expression and MEKK1-mediated threonine 288 phosphorylation of CNN-3 is induced by extracellular cues including TGF-β2 and lysophosphatidic acid. Importantly, siRNA-induced deficiency of CNN3 in lens epithelial cell cultures and explants results in actin stress fiber reorganization, stimulation of focal adhesion formation, Yap activation, increases in the levels of α-smooth muscle actin, connective tissue growth factor and fibronectin, and decreases in E-cadherin expression. These results reveal that CNN3 plays a crucial role in regulating lens epithelial contractile activity and provide supporting evidence that CNN-3 deficiency is associated with the induction of epithelial plasticity, fibrogenic activity and mechanosensitive Yap/Taz transcriptional activation.

CSF SerpinA1 in Creutzfeldt-Jakob disease and frontotemporal lobar degeneration.

ObjectiveSerpinA1 (alpha‐1 antitrypsin) is an acute inflammatory protein, which seems to play a role in neurodegeneration and neuroinflammation. In Alzheimer’s disease and synucleinopathies, SerpinA1 is overexpressed in the brain and the cerebrospinal fluid (CSF) showing abnormal patterns of its charge isoforms. To date, no comprehensive studies explored SerpinA1 CSF isoforms in Creutzfeldt–Jakob disease (CJD) and frontotemporal lobar degeneration (FTLD).MethodsUsing a capillary isoelectric focusing immunoassay, we analyzed CSF SerpinA1 isoforms in control cases (n = 31) and patients with a definite or probable diagnosis of CJD (n=77) or FTLD (n = 30), belonging to several disease subtypes.ResultsThe overall SerpinA1 signal was significantly higher than in controls in CJD subtypes linked to abnormal prion protein (PrPSc) type 1, such as sporadic CJD (sCJD) MM(V)1, and in FTLD‐TDP. Moreover, CJD linked to PrPSc type 1 and FTLD‐TAU groups showed a significant relative increase of acidic and basic isoforms in comparison with controls, thereby forming two distinct SerpinA1 isoform profiles.InterpretationCJD linked to PrPSc type 1 and FTLD show a differential upregulation and post‐translational modifications of CSF SerpinA1. Further studies are needed to clarify whether these findings may reflect a common, albeit disease‐specific, pathogenetic mechanism related to neurodegeneration.

PURIFICACIÓN Y ALGUNAS PROPIEDADES BIOQUÍMICAS Y MOLECULARES DE UNA NUEVA FOSFOLIPASA A2 NO MIOTÓXICA DEL VENENO DE LA SERPIENTE Bothrops atrox

Las fosfolipasas A2 (PLA2 ) del veneno de las serpientes, son enzimas con una variedad de efectos biológicos, debido a sus diferentes isoformas y algunas pudiendo ser miotoxinas. El objetivo de la investigación fue purificar, caracterizar y evaluar la actividad miotóxica de una isoforma de PLA2 ácida (BaPer-PLA2a). Se purificó por DEAE Sephadex-A50, Sephadex-G75 y un sistema automatizado de presión media-NGC. La BaPer-PLA2a tuvo una actividad específica de 34,1 U/mg y un peso molecular de ~14,5 kDa por PAGE-SDS en condiciones no reductoras. Del veneno se obtuvo el ARN total, para la síntesis de ADNc y un amplificado de ~480 pb. Se dedujo de la secuencia de ADNc una proteína madura de 124 aminoácidos con un punto isoeléctrico (4,41), siendo una isoforma ácida, asimismo presentó una estructura primaria con regiones conservadas y los residuos His48, Asp49 y Tyr52 identificados en el centro catalítico. Adicionalmente, el modelo teórico estructural posee una identidad mayor al 70 % con otras PLA2 ácidas. Finalmente, la BaPer-PLA2a no presenta actividad miotóxica, sin embargo, al combinarla con la isoforma de PLA2 básica incrementó la actividad miotoxina de esta última en 21,58 %.

Extended Stability of Reconstituted and Diluted SB3 (Trastuzumab Biosimilar) Assessed by Physicochemical and Biological Properties.

IntroductionStability information for the trastuzumab biosimilar SB3 is limited to 48 h at 2–8 °C for the reconstituted solution and 24 h at up to 30 °C for diluted solutions. Extended physicochemical stability and biological activity were assessed to evaluate the advanced preparation of reconstituted and diluted SB3.MethodsUnder controlled and aseptic conditions, the stability of reconstituted and diluted SB3 was evaluated using several assessments and according to the UK’s National Health Service guidance. Reconstituted SB3 was stored at 25 ± 2 °C with 60 ± 5% relative humidity for 3 days, and subsequently diluted SB3 (0.32–4 mg/mL) was stored in an infusion bag in the absence of light at 25 ± 2 °C with 60 ± 5% relative humidity for 28 days and 5 ± 3 °C for 28 days, respectively. Physicochemical stability (appearance, pH, protein concentration, size exclusion high-performance liquid chromatography, non-reducing capillary electrophoresis–sodium dodecyl sulfate, imaged capillary isoelectric focusing), biological activity (competitive inhibition binding assay to human epidermal growth factor receptor 2 by fluorescence resonance energy transfer, anti-proliferation assay), and properties with a potential safety impact (subvisible particulates, submicronic aggregation by dynamic light scattering) were determined.ResultsNo physicochemical instability signs or biological activity changes were observed for either reconstituted or diluted SB3 up to 28 days; all stability acceptance criteria were met. No major change was noted in the proportion of molecular weight variants (high molecular weight impurity, total purity) or relative percentages of acidic, main, and basic charge isoforms of the protein. No increases in particulates or aggregates in terms of a potential safety impact were noted.ConclusionThe physicochemical stability and biological activity of reconstituted and diluted SB3 are maintained for extended time periods beyond those denoted in the product labeling, which allows for advanced SB3 preparation and may reduce drug wastage and preparation time.FundingSamsung Bioepis Co., Ltd.

Isoforms of Group 1 Allergens from a Tropical/Subtropical Para Grass (Urochloa mutica) Display Different Levels of IgE Reactivity and Cross-reactivity.

Group 1 grass pollen allergens, or beta-expansins, are the most important major allergens from tropical/subtropical grasses. This study aimed to investigate the sequence similarity, and immunoreactivity of group 1 allergens from Para grass (Urochloa mutica). Three isoforms (Uro m 1.01, Uro m 1.02, and Uro m 1.03) were cloned from cDNA of Para grass pollen. The acidic-neutral isoforms rUro m 1.01 and rUro m 1.02 could effectively inhibited beta-expansins in pollen extract of Bermuda and Johnson grasses, suggesting that these isoforms could be major cross-reacting allergens among these grasses. In contrast, the basic isoform rUro m 1.03 had limited IgE reactivity. Thus, group 1 allergens both acidic-neutral and basic isoforms could have markedly different IgE reactivity.

A V1143F mutation in the neuronal-enriched isoform 2 of the PMCA pump is linked with ataxia

The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca2+ oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca2+ homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca2+ signaling in neurons demands the continuous activity of Ca2+ pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane Ca2+ATPases (PMCA pumps) play a key role in the regulation of Ca2+ handling in selected sub-plasma membrane microdomains. Among the four basic PMCA pump isoforms existing in mammals, isoforms 2 and 3 are particularly enriched in the nervous system. In humans, genetic mutations in the PMCA2 gene in association with cadherin 23 mutations have been linked to hearing loss phenotypes, while those occurring in the PMCA3 gene were associated with X-linked congenital cerebellar ataxias. Here we describe a novel missense mutation (V1143F) in the calmodulin binding domain (CaM-BD) of the PMCA2 protein. The mutant pump was present in a patient showing congenital cerebellar ataxia but no overt signs of deafness, in line with the absence of mutations in the cadherin 23 gene. Biochemical and molecular dynamics studies on the mutated PMCA2 have revealed that the V1143F substitution alters the binding of calmodulin to the CaM-BD leading to impaired Ca2+ ejection.

The PMCA pumps in genetically determined neuronal pathologies.

Ca2+ signals regulate most aspects of animal cell life. They are of particular importance to the nervous system, in which they regulate specific functions, from neuronal development to synaptic plasticity. The homeostasis of cell Ca2+ must thus be very precisely regulated: in all cells Ca2+ pumps transport it from the cytosol to the extracellular medium (the Plasma Membrane Ca2+ ATPases, hereafter referred to as PMCA pumps) or to the lumen of intracellular organelles (the Sarco/Endoplasmatic Reticulum Ca2+ ATPase and the Secretory Pathway Ca2+ ATPase, hereafter referred to as SERCA and SPCA pumps, respectively). In neurons and other excitable cells a powerful plasma membrane Na+/Ca2+ exchanger (NCX) also exports Ca2+ from cells. Quantitatively, the PMCA pumps are of minor importance to the bulk regulation of neuronal Ca2+. However, they are important in the regulation of Ca2+ in specific sub-plasma membrane microdomains which contain a number of enzymes that are relevant to neuronal function. The PMCA pumps (of which 4 basic isoforms are expressed in animal cells) are P-type ATPases that are characterized by a long C-terminal cytosolic tail which is the site of interaction with most of the regulatory factors of the pump, the most important being calmodulin. In resting neurons, at low intracellular Ca2+the C-terminal tail of the PMCA interacts with the main body of the protein keeping it in an autoinhibited state. Local Ca2+ increase activates calmodulin that removes the C-terminal tail from the inhibitory sites. Dysregulation of the Ca2+ signals are incompatible with healthy neuronal life. A number of genetic mutations of PMCA pumps are associated with pathological phenotypes, those of the neuron-specific PMCA 2 and PMCA 3 being the best characterized. PMCA 2 mutations are associated with deafness and PMCA 3 mutations are linked to cerebellar ataxias. Biochemical analysis of the mutated pumps overexpressed in model cells have revealed their decreased ability to export Ca2+. The defect in the bulk cytosolic Ca2+ homeostasis is minor, in keeping with the role of the PMCA pumps in the local control of Ca2+ in specialized plasma membrane microdomains.

Separation of peroxidases from Miscanthus x giganteus, their partial characterisation and application for degradation of dyes

Due to wide applicative potential of peroxidases (POXs), the search for novel sources and forms, possibly with better characteristics and performances, is justified. In this study, POXs from Miscanthus x giganteus rhizomes grown in chernozem-like soil and mine tailings were examined. Higher activity of POXs in samples originating from the metal-contaminated soil was found. The quantity of acidic isoforms was much greater than basic. The rates of reactions catalysed by acidic POX isoforms decreased slightly at 50 °C, whereas stability of basic isoforms was affected at 40 °C. Concentrations of Zn, Mn and Fe were higher in rhizomes grown in mine tailings, and negatively correlated with the concentration of proteins. Basic POX isoforms effectively degraded CBB R250, while Amidoblack 10b was predominantly degraded by acidic isoforms. Thus, Miscanthus x giganteus can be used as a source of POXs which can be applied for dye decomposition and, possibly, waste water management.