Basic Isoforms Research Articles

Direct speciation of metals in copper-zinc superoxide dismutase isoforms on electrophoresis gels using X-ray absorption near edge structure

X-Ray absorption near edge structure (XANES) can be used for the speciation analysis of inorganic elements bound to proteins, after separation using polyacrylamide gel electrophoresis. In this paper, we report new methodological developments for the speciation of oxidation states in copper–zinc superoxide dismutase (CuZnSOD) isoforms separated in non-denaturing conditions according to their isoelectric point. The nature of CuZnSOD isoforms is still elusive, although differences in their metallation state have been suggested. A non-denaturing protocol for isoelectrofocusing electrophoresis was applied to preserve the metallation state of the proteins. XANES has been performed to study the copper and zinc oxidation states. As expected, zinc is present in its Zn(II) oxidation state in all analysed isoforms. Copper is present in the Cu(II) oxidation state in the main acidic isoform, while it is found in both Cu(II) and Cu(I) states in the main basic isoform. These methodological developments enable the direct speciation analysis of metals in CuZnSOD without any protein purification step that could modify their oxidation state.

Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening

The decrease of strawberry (Fragariaxananassa Duch.) fruit firmness observed during ripening is partly attributed to pectolytic enzymes: polygalacturonases, pectate lyases and pectin methylesterases (PMEs). In this study, PME activity and pectin content and esterification degree were measured in cell walls from ripening fruits. Small green, large green, white, turning, red and over-ripe fruits from the Elsanta cultivar were analyzed. Using the 2F4 antibody directed against the calcium-induced egg box conformation of pectin, we show that calcium-bound acidic pectin was nearly absent from green and white fruits, but increased abruptly at the turning stage, while the total pectin content decreased only slightly as maturation proceeded. Isoelectrofocalisation performed on wall protein extracts revealed the expression of at least six different basic PME isoforms. Maximum PME activity was detected in green fruits and steadily decreased to reach a minimum in senescent fruits. The preliminary role of PMEs and subsequent pectin degradation by pectolytic enzymes is discussed.

Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry

Neurotrauma, as in the case of traumatic brain injury, promotes protease over-activation characterized by the select fragmentation of brain proteins. The resulting polypeptides are indicators of biochemical processes, which can be used to study post-injury dynamics and may also be developed into biomarkers. To this end, we devised a novel mass spectrometry approach to characterize post-injury calpain proteolytic processing of myelin basic protein (MBP), a biomarker of brain injury that denotes white matter damage and recovery. Our approach exceeds conventional immunological assays in its deconvolution of multiple protein isoforms, its absolute quantification of proteolytic fragments and its polypeptide selectivity. We quantified and characterized post-injury proteolytic processing of all MBP isoforms identified in adult rat cortex. Further, the translation of calpain-cleaved MBP into CSF was verified following brain injury. We ascertained that the exon-6 sequence of MBP resulted in a characteristic shift in gel migration for intact and fragmented protein alike. We also found evidence for a second post-TBI cleavage event within exon-2 and for the dimerization of the post-TBI 4.3 kDa fragment. Ultimately, the novel methodology described here can be used to study MBP dynamics and other similar proteolytic events of relevance to brain injury and other CNS processes.

Intermittent hypoxia exposure in a hypobaric chamber and erythropoietin abuse interpretation

The aim of this study was to assess the effect of intermittent hypoxia exposure on direct and indirect methods used to evaluate recombinant human erythropoietin (rhEPO) misuse. Sixteen male triathletes were randomly assigned to either the intermittent hypoxia exposure group (experimental group) or the control normoxic group (control group). The members of the experimental group were exposed to simulated altitude (from 4000 to 5500 m) in a hypobaric chamber for 3 h per day, 5 days a week, for 4 weeks. Blood and urine samples were collected before and after the first and the final exposures, and again 2 weeks after the final exposure. While serum EPO significantly increased after the first [from a mean 8.3 IU · l−1 (s = 3.2) to 16.6 IU · l−1 (s = 4.7)] and final exposures [from 4.6 IU · l−1 (s = 1.4) to 24.8 IU · l−1 (s = 9.3)], haemoglobin, percentage of reticulocytes, and soluble transferrin receptor were not elevated. Second-generation ON/OFF models (indirect rhEPO misuse detection) were insensitive to intermittent hypoxia exposure. The distribution of the urinary EPO isoelectric profiles (direct rhEPO misuse detection) was altered after intermittent hypoxia exposure with a slight shift towards more basic isoforms. However, those shifts never resulted in misinterpretation of results. The intermittent hypoxia exposure protocol studied did not produce any false-positive result for indirect or direct detection of rhEPO misuse in spite of the changes in EPO serum concentrations and urinary EPO isoelectric profiles, respectively.

Sulfonation and Sialylation of Gonadotropins in Women during the Menstrual Cycle, after Menopause, and with Polycystic Ovarian Syndrome and in Men

More basic isoforms of LH and FSH appear in blood at midcycle and more acidic after menopause. The LH isoforms are more basic in women with polycystic ovarian syndrome (PCOS). These charge alterations may reflect differences in the number of two negatively charged residues on the gonadotropins: sialic acid and sulfonated N-acetylgalactosamine, residues that modulate the half-life of the gonadotropins in blood. The objective of the study was to determine the contributions of sialic acid and sulfonated N-acetylgalactosamine and sialic acid on LH and FSH to the observed alterations in charge. Serum samples were obtained from 59 young women with regular cycles, nine postmenopausal women, 12 women with PCOS, and 40 young men. The number of sulfonated N-acetylgalactosamine and sialic acid residues per LH and FSH molecule in serum and the distributions of molecules with 0-1-2-3-4 sulfonated residues were determined by electrophoretic analyses before and after removal of sialic acid. Considerably decreased sulfonation of LH was found at midcycle and in women with PCOS concomitant with slightly increased sialylation. The sulfonation of LH increased in the luteal phase, and the sialylation was highest after menopause for both hormones. The frequencies of sulfonated LH and FSH isoforms were directly related (P < 0.01) to body mass index in women with PCOS. The observed variations in sialic acid and sulfonated residues on serum gonadotropins are suggested to reflect alterations in the isoform composition of the hormones secreted by the pituitary, resulting in modulations of their biological properties, such as half-life in blood.

Kiwifruit allergy: actinidin is not a major allergen in the United Kingdom

Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy. To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera. Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA. A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.

Effects of Oxidative and Nitrosative Stress on Tetrahymena pyriformis Glyceraldehyde‐3‐Phosphate Dehydrogenase

Previous reports showed that hydrogen peroxide and the NO-generating reagent sodium nitroprusside (SNP)-modulated enzymatic activity of animal glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). These modifications are suggested to have a physiological regulatory role. To gain further insight into this regulatory process the model ciliated protozoan Tetrahymena pyriformis was chosen. Both reagents inhibited growth of T. pyriformis cultures and produced a specific increase of GAPDH protein but only NO seemed to reduce GAPDH activity in cell-free extracts. Both specific activity and pI were found to be altered in the in vivo NO-treated purified enzyme, but no effect was detected by the in vivo H(2)O(2) treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H(2)O(2)-treated cells. In contrast to this, three more acidic isoforms (pIs, 8.6, 8.0 and 7.3) were resolved in purified fractions from SNP-treated cells, suggesting post-translational modification of the enzyme by NO. Nevertheless, a decrease of GAPDH activity by H(2)O(2) and NO, mainly due to a decrease in its V(max) without apparent change in substrate affinity, was observed in vitro in the whole enzyme population. The increase of GAPDH protein level found in vivo suggests a cell response in order to compensate for the inhibitory effect on activity observed in the purified enzyme. This is the first report of NO- and H(2)O(2)-dependent effects on GAPDH of T. pyriformis, and identifies this key protein of central carbon metabolism as a physiological target of oxidative and nitrosative stress in this ciliated protozoan.

Effect of Resveratrol on Antioxidant Enzyme Activities in the Brain of Healthy Rat

We have studied the effect of resveratrol on lipoperoxidation and antioxidant enzyme activity level in the brain of healthy rats. When intraperitoneally administered, resveratrol significantly and dose dependently decreased brain malondialdehyde level. Resveratrol also increased in a dose-dependent way brain superoxide dismutase, catalase and peroxidase activities. Optimal effect on antioxidant enzyme and lipoperoxidation products were obtained with resveratrol concentration of 12.5 mg/kg body wt. Native polyacrylamide gel electrophoresis analysis of antioxidant isoenzymes revealed that resveratrol up regulated at least two acidic superoxide dismutase isoforms called A(1) and A(2), two basic isoforms called B(1) and B(2). Resveratrol also up regulated two catalase isoforms and a broad peroxidase band corresponding to several isoforms. All these findings suggest that resveratrol is able to cross the blood brain barrier and exerts potent antioxidant features. Resveratrol also exerts neuroprotective properties by up regulating several detoxifying enzymes, most of which are iron proteins.

Structural insertion/deletion variation in IRF5 is associated with a risk haplotype and defines the precise IRF5 isoforms expressed in systemic lupus erythematosus

To determine whether specific isoforms of IRF5 are transcribed in patients with systemic lupus erythematosus (SLE) who have risk genotypes in the exon 1B donor splice site at single-nucleotide polymorphism (SNP) no. rs2004640. Peripheral blood mononuclear cells were obtained from SLE patients and healthy controls from Argentina, Spain, and Germany and from trio families from Spain and Denmark. A reporter assay was used to investigate the role of SNP no. rs2004640. IRF5 expression in relation to the genotypes of functional SNPs was analyzed using quantitative polymerase chain reaction. Sequencing and genotyping of the IRF5 gene was performed. Sequencing of complementary DNA from individuals with different genotypes showed 4 basic isoforms transcribed from all 5'-untranslated regions (5'-UTRs), suggesting no preferential isoform transcription based on rs2004640 genotypes. Analysis of translation efficiency showed that exon 1A was the most efficient in initiating protein synthesis. We identified a novel polymorphic insertion/deletion that defines the pattern of expression of isoforms of IRF5. The insertion consists of 4 repeats in exon 6 affecting the protein interaction domain. The insertion segregates in the risk haplotype with the high expression allele of a poly(A) site SNP no. rs10954213 and the exon 1B donor splice allele of the 5'-UTR SNP no. rs2004640. The poly(A) polymorphism correlated with levels of IRF5 in cells stimulated with interferon-alpha. The SNP most strongly associated with SLE was SNP no. rs2070197 (P=5.2x10(-11)), which is a proxy of the risk haplotype, but does not appear to be functional. None of the functional variants investigated in this study is strongly associated with SLE, with the exception of the exon 1B donor splice site, and its functional importance appears to be small. Our results suggest that there may be other functional polymorphisms, yet to be identified, in IRF5. We did not observe evidence of epistatic interaction between the functional SNPs.

Functional Specificity of PMCA Isoforms?

In mammals, four different genes encode four PMCA isoforms. PMCA1 and PMCA4 are expressed ubiquitously. PMCA2 and PMCA3 are expressed prevalently in the central nervous systems. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of such elevated number of isoforms is not clear, but it would be plausible to relate it to the cell-specific demands of Ca2+ homeostasis. To characterize functional specificity of PMCA variants we have investigated two aspects: the effects of the overexpression of the different PMCA variants on cellular Ca2+ handling and the existence of possible isoform-specific interactions with partner proteins using a yeast two-hybrid technique. The four basic PMCA isoforms were coexpressed in CHO cells together with the Ca2+-sensitive recombinant photoprotein aequorin. The effects of their overexpression on Ca2+ homeostasis were monitored in the living cells. They had revealed that the ubiquitous isoforms 1 and 4 are less effective in reducing the Ca2+ peaks generated by cell stimulation as compared to the neuron-specific isoforms 2 and 3. To establish whether these differences were related to different and new physiological regulators of the pump, the 90 N-terminal residues of PMCA2 and PMCA4 have been used as baits for the search of molecular partners. Screening of a human brain cDNA library with the PMCA4 bait specified the epsilon-isoform of protein 14-3-3, whereas no 14-3-3 epsilon clone was obtained with the PMCA2 bait. Overexpression of PMCA4/14-3-3 epsilon (but not of PMCA2/14-3-3 epsilon) in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca2+ was impaired. Thus, the interaction with 14-3-3 epsilon inhibited PMCA4 but not PMCA2. The role of PMCA2 has been further characterized by Ca2+ measurements in cells overexpressing different splicing variants. The results indicated that the combination of alternative splicing at two different sites in the pump structure was responsible for different functional characteristics of the pumps.

Progesterone and the distribution of pituitary gonadotropin isoforms in cattle

The objective of the present study was to determine the relative proportion of gonadotropin isoforms in bovine pituitary glands affected by progesterone. Twelve postpubertal heifers (Swiss-Zebu) were assigned to three groups ( n = 4): intact animals in the luteal phase of the estrous cycle (diestrus group); ovariectomized heifers with (OVXP) or without progesterone treatment (OVX). Prior to pituitary gland collection, a blood sample was taken from each animal to determine the circulating progesterone concentration. Pituitary protein extractions processed by chromatofocusing were eluted with a pH gradient ranging from 10.5 to 3.5. The LH and FSH eluent was grouped on the basis of the following three criteria: (1) as either a basic (pH ≥7.5), neutral (pH 7.4–6.5) and acid (pH ≤6.4); (2) according to the pH unit (pH ≥10.5–3.5); (3) on the basis of distinct isoforms 12 peaks of which (A–L) were identified for LH and 11 (I–XI) for FSH. The analysis by range of pH and by pH of elution in the OVX and OVXP groups showed no difference in the LH and FSH isoform ratio, but diestrus cattle differs having a greater ratio ( p < 0.05) of basic LH isoforms (87.5 ± 0.4%) and lesser ratio ( p < 0.05) of acid isoforms (5.4 ± 0.7%). In the diestrus group, the ratio of acid FSH isoform increased (62.1 ± 1.7%), while neutral isoforms decreased (5.7 ± 0.4%, P < 0.05). The analysis by isoform type of LH revealed a greater proportion of isoforms C (pH 9.4) and E (pH 9.0) in the groups with circulating progesterone when compared to the OVX group. The heterogeneity of FSH was quantitatively similar in most isoforms in the three groups, with the exception of the predominant isoform (VIII, pH 4.9) that was more abundant in the diestrus group ( p < 0.05). These results indicate that progesterone with other gonad factors influence the pituitary glicosylation altering the relative proportions of gonadotropin isoforms.

A Causative Link Exists Between Eosinophil Major Basic Protein-2 and Allergen-Induced Airway Hyperresponsiveness

RATIONALE: Eosinophils are a major leukocyte recruited to the lungs in both mouse allergen provocation models and patients with asthma. However, a link between these cells and the pathological changes occurring in the lung remains obscure although eosinophil effector functions have been hypothesized to be mediated, in part, by the release of secreted cationic proteins sequestered in cytoplasmic granules (i.e., the degranulation of activated eosinophils recruited to the lung).METHODS: Gene knockout lines of mice deficient for the two eosinophil major basic proteins (MBP-1, -2) were generated (strain: 129) by homologous recombination in ES cells and used in an acute allergen sensitization/aerosol challenge protocol to define causative links with pulmonary allergic infammation.RESULTS: This study demonstrates that despite observations showing that allergen challenge mouse models display only nominal levels of eosinophil degranulation the loss of the eosinophil granule protein MBP-2 abolished allergen-induced airway hyperresponsiveness (AHR). The loss of AHR in MBP-2-/- mice occurred with out concominant loss of other allergen-induced pulmonary pathologies (e.g., goblet cell metaplasia), suggesting a specific link between this pathophysiologic response and MBP-2 mediated activities. Interestingly, the MBP-2 associated effects on AHR were specific to this major basic protein isoform as the loss of the more abundant granule protein MBP-1 had no effects on pulmonary pathologies, including AHR.CONCLUSIONS: The identification of a causative link between AHR and the release of an eosinophil secondary granule protein (i.e., degranluation) provides a an important link between allergen-induced pathologies and this long suspected eosinophil-mediated activity in the lung. RATIONALE: Eosinophils are a major leukocyte recruited to the lungs in both mouse allergen provocation models and patients with asthma. However, a link between these cells and the pathological changes occurring in the lung remains obscure although eosinophil effector functions have been hypothesized to be mediated, in part, by the release of secreted cationic proteins sequestered in cytoplasmic granules (i.e., the degranulation of activated eosinophils recruited to the lung). METHODS: Gene knockout lines of mice deficient for the two eosinophil major basic proteins (MBP-1, -2) were generated (strain: 129) by homologous recombination in ES cells and used in an acute allergen sensitization/aerosol challenge protocol to define causative links with pulmonary allergic infammation. RESULTS: This study demonstrates that despite observations showing that allergen challenge mouse models display only nominal levels of eosinophil degranulation the loss of the eosinophil granule protein MBP-2 abolished allergen-induced airway hyperresponsiveness (AHR). The loss of AHR in MBP-2-/- mice occurred with out concominant loss of other allergen-induced pulmonary pathologies (e.g., goblet cell metaplasia), suggesting a specific link between this pathophysiologic response and MBP-2 mediated activities. Interestingly, the MBP-2 associated effects on AHR were specific to this major basic protein isoform as the loss of the more abundant granule protein MBP-1 had no effects on pulmonary pathologies, including AHR. CONCLUSIONS: The identification of a causative link between AHR and the release of an eosinophil secondary granule protein (i.e., degranluation) provides a an important link between allergen-induced pathologies and this long suspected eosinophil-mediated activity in the lung.

Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit.

Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa 'Hayward' (green-fleshed kiwifruit) and A. chinensis 'Hort16A' and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in 'Hort16A', the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.

Antioxidative enzymes during germination of two lines of Serbian spruce [Picea omorika (Panc.) Purkyně

Two lines of Picea omorika (Panc.) Purkyn? were compared with respect to germination percentage as well as specific activity and isoenzyme pattern of catalase, superoxide dismutase, and peroxidase (POD) during germination. Line A had a higher germination percentage and higher enzyme activities in dry seeds and seedlings compared to line C. Peroxidase activity was not detected in dry seeds, but measured up to 10 U/g and 28 U/g on the 7th day of germination in lines C and A, respectively. The most abundant POD basic isoform in seedlings of both lines (pI 8.2) was not found previously in needles of adult Serbian spruce trees of the same lines. .

Gastrokine 1 expression in patients with and without Helicobacter pylori infection

Background To understand the molecular changes underlying Helicobacter pylori-related gastric diseases is mandatory to prevent gastric cancer. Proteomic technology is providing a rapid expansion of the basic knowledge, particularly in the discovery of new biomarkers involved in the tumourigenesis. Aim To characterise changes in protein expression level of the gastric mucosa in H. pylori-infected patients. Methods The population enrolled comprised 41 dyspeptic patients. Proteins extracted from gastric mucosal specimens were analysed by 2-dimensional electrophoresis, sequenced by MALDI-TOF and identified by Edman's degradation. Results Twenty-one out of 41 patients had H. pylori infection of whom 17 had anti-CagA IgG antibodies. Several proteins were identified, of which Rho guanosine diphosphatase dissociation inhibitor α and heat shock protein 27 increased and glutathione transferase and antrum mucosa protein-18 decreased in H. pylori-positive in respect to H. pylori-negative patients. Interestingly, antrum mucosa protein-18, currently referred as gastrokine-1, showed two isoforms differing in the first N-terminal amino acid residue. Both gastrokine-1 isoforms were observed in the H. pylori-negative group whereas a lower expression or even absence of the gastrokine-1 basic isoform was found in a subgroup (7/21) of H. pylori-positive patients with moderate–severe gastritis. Conclusion Our study demonstrated the presence of gastrokine-1 isoforms of which the basic isoform was reduced in a subset of patients with H. pylori infection.

O.043bis Cleft distraction versus orthognathic surgery – Which one causes more distress?

The plasma membrane Ca2+ ATPase (PMCA pump) is a member of the superfamily of P-type pumps. It has 10 transmembrane helices and 2 cytosolic loops, one of which contains the catalytic center. Its most distinctive feature is a C-terminal tail that contains most of the regulatory sites including that for calmodulin. The pump is also regulated by acidic phospholipids, kinases, a dimerization process, and numerous protein interactors. In mammals, four genes code for the four basic isoforms. Isoform complexity is increased by alternative splicing of primary transcripts. Pumps 2 and 3 are expressed preferentially in the nervous system. The pumps coexist with more powerful systems that clear Ca2+ from the bulk cytosol: their role is thus the regulation of Ca2+ in selected subplasma membrane microdomains, where a number of important Ca2+-dependent enzymes interact with them. Malfunctions of the pump lead to disease phenotypes that affect the nervous system preferentially.

Caprine luteinizing hormone isoforms during the follicular phase and anestrus

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 μg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater ( p < 0.05) in A (12.0 ± 0.8%) as compared with F (5 ± 2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during A, and 3 ± 1% during F ( p < 0.05). Predominant isoforms in A eluted in the pH range 9.99–9.0 (42 ± 3%) as compared to 7 ± 3% ( p < 0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99–8.0, representing 55 ± 8%, while in A the proportion was 11 ± 2% ( p < 0.01). Isoforms eluted at the pH range 7.9–7 represented a significantly greater proportion during A (5.0 ± 0.6%) as compared with F (3 ± 1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.

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

Axonal injury is one of the key features of traumatic brain injury (TBI), yet little is known about the integrity of the myelin sheath. We report that the 21.5 and 18.5-kDa myelin basic protein (MBP) isoforms degrade into N-terminal fragments (of 10 and 8 kDa) in the ipsilateral hippocampus and cortex between 2 h and 3 days after controlled cortical impact (in a rat model of TBI), but exhibit no degradation contralaterally. Using N-terminal microsequencing and mass spectrometry, we identified a novel in vivo MBP cleavage site between Phe114 and Lys115. A MBP C-terminal fragment-specific antibody was then raised and shown to specifically detect MBP fragments in affected brain regions following TBI. In vitro naive brain lysate and purified MBP digestion showed that MBP is sensitive to calpain, producing the characteristic MBP fragments observed in TBI. We hypothesize that TBI-mediated axonal injury causes secondary structural damage to the adjacent myelin membrane, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, which might further promote axonal vulnerability.

Intact Glycoform Characterization of Erythropoietin-α and Erythropoietin-β by CZE-ESI-TOF-MS

Glycosylation of recombinant human erythropoietin (rhEPO) is a post-translational process which depends on the type of cell in which rhEPO is synthesized, but also on the cell culture conditions and the final purification steps. These glycosylation modifications alter the biological activity, solubility and lifetime of rhEPO in blood. Thus, a rapid and simple method for the elucidation of the carbohydrate microheterogeneity of rhEPO is needed in order to evaluate a certain manufacturing process or assure the quality of the final product. Based on a recently developed method [1], the accurate mass determination of the intact glycoforms from two types of commercial rhEPO (epoetin-α and epoetin-β) by capillary electrophoresis-electrospray-time of flight-mass spectrometry is presented. The sample treatment consists of a fast and simple preconcentration step of the ready-to-use drug achieved by a centrifugal filter device. Characterization of the carbohydrate composition of each single glycoform is performed, in agreement with the results in glycan and glycopeptide analysis reported by other authors. The main differences between the carbohydrate structures of both epoetins are shown: the existence of two additional basic sialic acid isoforms for epoetin-β and the higher degree of acetylation for epoetin-α. The agreement of the main glycoforms for both epoetins is shown by molecular mass agreement. The high accuracy and reproducibility of the mass measurements with a standard deviation below 1 Da is proved by repeated analysis of European Pharmacopoeia rhEPO. Summarizing, the presented method enables the fast and reliable characterization of intact rhEPO in pharmaceutical products.

Oxidative Damage of DJ-1 Is Linked to Sporadic Parkinson and Alzheimer Diseases

Mutations in DJ-1 cause an autosomal recessive, early onset familial form of Parkinson disease (PD). However, little is presently known about the role of DJ-1 in the more common sporadic form of PD and in other age-related neurodegenerative diseases, such as Alzheimer disease (AD). Here we report that DJ-1 is oxidatively damaged in the brains of patients with idiopathic PD and AD. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified 10 different DJ-1 isoforms, of which the acidic isoforms (pI 5.5 and 5.7) of DJ-1 monomer and the basic isoforms (pI 8.0 and 8.4) of SDS-resistant DJ-1 dimer are selectively accumulated in PD and AD frontal cortex tissues compared with age-matched controls. Quantitative Western blot analysis shows that the total level of DJ-1 protein is significantly increased in PD and AD brains. Mass spectrometry analyses reveal that DJ-1 is not only susceptible to cysteine oxidation but also to previously unsuspected methionine oxidation. Furthermore, we show that DJ-1 protein is irreversibly oxidized by carbonylation as well as by methionine oxidation to methionine sulfone in PD and AD. Our study provides new insights into the oxidative modifications of DJ-1 and indicates association of oxidative damage to DJ-1 with sporadic PD and AD.