Sodium/hydrogen exchanger NHA2 is critical for insulin secretion in β-cells.

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human β-cells, as well as β-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular Ca(2+) homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in β-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary β-cells, suggesting defective endo-exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the β-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters.

The effect of a period of intensive exercise on the isoform test to detect growth hormone doping in sports.

OBJECTIVE: The major objective of this study was to investigate the effects of several days of intense exercise on growth hormone (hGH) testing using the World Anti-Doping Agencies hGH isoform differential immunoassays. Additionally the effects of circadian variation and exercise type on the isoform ratios were also investigated. STUDY DESIGN: 15 male athletes performed a simulated nine day cycling stage race. Blood samples were collected twice daily over a period of 15days (stage race+three days before and after). hGH isoforms were analysed by the official WADA immunoassays (CMZ Assay GmbH). RESULTS: All measured isoform ratios were far below the WADA decision limits for an adverse analytical finding. Changes in the isoform ratios could not be clearly connected to circadian variation, exercise duration or intensity. CONCLUSIONS: The present study demonstrates that the hGH isoform ratios are not significantly affected by exercise or circadian variation. We demonstrated that heavy, long term exercise does not interfere with the decision limits for an adverse analytical finding.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo.

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

EH-myomesin splice isoform is a novel marker for dilated cardiomyopathy.

The M-band is the prominent cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. To investigate M-band alterations in heart disease, we analyzed the expression of its main components, proteins of the myomesin family, in mouse and human cardiomyopathy. Cardiac function was assessed by echocardiography and compared to the expression pattern of myomesins evaluated with RT-PCR, Western blot, and immunofluorescent analysis. Disease progression in transgenic mouse models for dilated cardiomyopathy (DCM) was accompanied by specific M-band alterations. The dominant splice isoform in the embryonic heart, EH-myomesin, was strongly up-regulated in the failing heart and correlated with a decrease in cardiac function (R = -0.86). In addition, we have analyzed the expressions of myomesins in human myocardial biopsies (N = 40) obtained from DCM patients, DCM patients supported by a left ventricular assist device (LVAD), hypertrophic cardiomyopathy (HCM) patients and controls. Quantitative RT-PCR revealed that the EH-myomesin isoform was up-regulated 41-fold (P < 0.001) in the DCM patients compared to control patients. In DCM hearts supported by a LVAD and HCM hearts, the EH-myomesin expression was comparable to controls. Immunofluorescent analyses indicate that EH-myomesin was enhanced in a cell-specific manner, leading to a higher heterogeneity of the myocytes' cytoskeleton through the myocardial wall. We suggest that the up-regulation of EH-myomesin denotes an adaptive remodeling of the sarcomere cytoskeleton in the dilated heart and might serve as a marker for DCM in mouse and human myocardium.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.

Statistical Analysis and Numerics of Heat Exchanger Models

The identifiability of the parameters of a heat exchanger model without phase change was studied in this Master’s thesis using synthetically made data. A fast, two-step Markov chain Monte Carlo method (MCMC) was tested with a couple of case studies and a heat exchanger model. The two-step MCMC-method worked well and decreased the computation time compared to the traditional MCMC-method. The effect of measurement accuracy of certain control variables to the identifiability of parameters was also studied. The accuracy used did not seem to have a remarkable effect to the identifiability of parameters. The use of the posterior distribution of parameters in different heat exchanger geometries was studied. It would be computationally most efficient to use the same posterior distribution among different geometries in the optimisation of heat exchanger networks. According to the results, this was possible in the case when the frontal surface areas were the same among different geometries. In the other cases the same posterior distribution can be used for optimisation too, but that will give a wider predictive distribution as a result. For condensing surface heat exchangers the numerical stability of the simulation model was studied. As a result, a stable algorithm was developed.

Limestone Reactions in a Fluidized Bed Heat Exchanger of an Oxy-fuel Circulating Fluidized Bed Boiler

Oxy-fuel combustion in a circulating fluidized bed (CFB) boiler appears to be a promising option for capturing CO2 in power plants. Oxy-fuel combustion is based on burning of fuel in the mixture of oxygen and re-circulated flue gas instead of air. Limestone (CaCO3) is typically used for capturing of SO2 in CFB boilers where limestone calcines to calcium oxide (CaO). Because of high CO2 concentration in oxy-fuel combustion, calcination reaction may be hindered or carbonation, the reverse reaction of calcination, may occur. Carbonation of CaO particles can cause problems especially in the circulation loop of a CFB boiler where temperature level is lower than in the furnace. The aim of the thesis was to examine carbonation of CaO in a fluidized bed heat exchanger of a CFB boiler featuring oxy-fuel combustion. The calculations and analyzing were based on measurement data from an oxy-fuel pilot plant and on 0-dimensional (0D) gas balance of a fluidized bed heat exchanger. Additionally, the objective was to develop a 1-dimensional (1D) model of a fluidized bed heat exchanger by searching a suitable pre-exponential factor for a carbonation rate constant. On the basis of gas measurement data and the 0D gas balance, it was found that the amount of fluidization gas decreased as it flew through the fluidized bed heat exchanger. Most likely the reason for this was carbonation of CaO. It was discovered that temperature has a promoting effect on the reaction rate of carbonation. With the 1D model, a suitable pre-exponential factor for the equation of carbonation rate constant was found. However, during measurements there were several uncertainties, and in the calculations plenty of assumptions were made. Besides, the temperature level in the fluidized bed heat exchanger was relatively low during the measurements. Carbonation should be considered when fluidized bed heat exchangers and the capacity of related fans are designed for a CFB boiler with oxy-fuel combustion.

Optimizing the Performance of a Heat Exchanger Network

This research is the continuation and a joint work with a master thesis that has been done in this department recently by Hemamali Chathurangani Yashika Jayathunga. The mathematical system of the equations in the designed Heat Exchanger Network synthesis has been extended by adding a number of equipment; such as heat exchangers, mixers and dividers. The solutions of the system is obtained and the optimal setting of the valves (Each divider contains a valve) is calculated by introducing grid-based optimization. Finding the best position of the valves will lead to maximization of the transferred heat in the hot stream and minimization of the pressure drop in the cold stream. The aim of the following thesis will be achieved by practicing the cost optimization to model an optimized network.

The Na+/H+ exchanger Nhx1 of Saccharomyces cerevisiae is essential to limit drug toxicity

Nhx1 est un antiport vacuolaire de Na+/H+ chez la levure Saccharomyces cerevisiae. Nhx1 joue un rôle important dans le maintien de l’homéostasie ionique du cytoplasme de la cellule. En effet, la mutation du gène NHX1 chez la levure nhx1Δ entraîne une perte de l’homéostasie cellulaire quand les cellules sont cultivées dans un milieu de faible osmolarité. Ce travail rapporte pour la première fois, et contrairement à la cellule parentale, que la mutation du gène NHX1 a pour effet une sensibilité du mutant nhx1Δ à une variété des drogues et des agents cationiques et anioniques lorsque les cellules sont cultivées dans un milieu riche. En outre, dans ces conditions de culture, aucune sensibilité n’a été observée chez le mutant nhx1Δ quand les cellules sont traitées avec différentes concentrations de sel. Nous avons aussi démontré que la sensibilité du mutant nhx1Δ aux différents agents ainsi que la sécrétion de l’enzyme carboxypeptidase Y observé chez ce mutant n’ont pas été restauré lorsque les cellules sont cultivées dans des milieux avec différents pH ou avec différentes concentrations de sel. Enfin, une analyse génétique a révélé que le mutant nhx1Δ montre un phénotype distinct d’autres mutants qui ont un défaut dans le trafic entre le compartiment pré-vacuolaire et l’appareil de Golgi quand ces cellules sont traitées avec différents agents. Cette analyse prouve que la sensibilité de nhx1Δ aux différents agents n’est pas liée au trafic entre le compartiment pré-vacuolaire et l’appareil de Golgi.

A new isoform of anti-lipopolysaccharide factor identified from the blue swimmer crab, Portunus pelagicus: Molecular characteristics and phylogeny

Anti-lipopolysaccharide factors are small proteins that bind and neutralize lipopolysaccharide and exhibit potent antimicrobial activities. This study presents the molecular characterization and phylogenetic analysis of the first ALF isoform (Pp-ALF1; JQ745295) identified from the hemocytes of Portunus pelagicus. The full length cDNA of Pp-ALF1 consisted of 880 base pairs encoding 293 amino acids with an ORF of 123 amino acids and contains a putative signal peptide of 24 amino acids. Pp-ALF1 possessed a predicted molecular weight (MW) of 13.86 kDa and theoretical isoelectric point (pI) of 8.49. Two highly conserved cysteine residues and putative LPS binding domain were observed in Pp-ALF1. Peptide model of Pp-ALF1 consisted of two α-helices crowded against a four-strand β-sheet. Comparison of amino acid sequences and neighbor joining tree showed that Pp-ALF1 has a maximum similarity (46%) to ALF present in Portunus trituberculatus followed by 39% similarity to ALF of Eriocheir sinensis and 38% similarity to ALFs of Scylla paramamosain and Scylla serrata. Pp-ALF1 is found to be a new isoform of ALF family and its characteristic similarity with other known ALFs signifies its role in protection against invading pathogens.

A Novel Isoform of the Hepatic Antimicrobial Peptide, Hepcidin (Hepc-CB1), from a Deep-Sea Fish, the Spinyjaw Greeneye Chlorophthalmus bicornis (Norman, 1939): Molecular Characterisation and Phylogeny

Hepcidin is cysteine-rich short peptide of innate immune system of fishes, equipped to perform prevention and proliferation of invading pathogens like bacteria and viruses by limiting iron availability and activating intracellular cascades. Hepcidins are diverse in teleost fishes, due to the varied aquatic environments including exposure to pathogens, oxygenation and iron concentration. In the present study, we report a 87-amino acid (aa) preprohepcidin (Hepc-CB1) with a signal peptide of 24 aa, a prodomain of 39 aa and a bioactive mature peptide of 24 aa from the gill mRNA transcripts of the deep-sea fish spinyjaw greeneye, Chlorophthalmus bicornis. Molecular characterisation and phylogenetic analysis categorised the peptide to HAMP2-like group with a mature peptide of 2.53 kDa; a net positive charge (?3) and capacity to form b-hairpin-like structure configured by 8 conserved cysteines. The present work provides new insight into the mass gene duplication events and adaptive evolution of hepcidin isoforms with respect to environmental influences and positive Darwinian selection. This work reports a novel hepcidin isoform under the group HAMP2 from a nonacanthopterygian deep-sea fish, C. bicornis

Molecular Characterisation and Phylogenetic Analysis of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Zc-hepc1), from the Coral Fish Moorish idol, Zanclus cornutus (Linnaeus,1758)

Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of ?1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a b-hairpin-like structure with two antiparallel b-sheets. This is the first report of an AMP from the coral fish Z. cornutus.

The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat ( RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT ( HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 ( AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract - but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.