Myocardial subproteomic analysis of a constitutively active Rac1-expressing transgenic mouse with lethal myocardial hypertrophy

A two-dimensional gel electrophoresis (2-DE)-based proteomic approach was used to study a transgenic mouse model of acerbated dilated cardiomyopathy in which the small monomeric GTPase, Rac1, was constitutively expressed exclusively in the myocardium. A subfractionation procedure allowed for the focused analysis of both cytoplasmic and myofilament protein-enriched extracts of ventricular tissue from Rac1 transgenic and age-matched nontransgenic (NTG) mice. The majority of these mice displayed severe hypertrophy (heart-to-body weight ratios >2-fold greater in the Rac1 mice) and died from overt heart failure between days 14 and 17. Comparative 2-DE analysis (pH 3-10, 12% SDS-PAGE) derived from Rac1 (n = 4) and NTG (n = 4) groups revealed differences in mean protein spot intensities. Twelve proteins from the cytoplasmic protein-enriched extract met our criteria for robustness and spot resolution and were identified. These proteins represent a broad distribution of cellular functions with only some previously implicated in myocardial hypertrophy. The myofilament subproteome displayed no change in posttranslational modification, but further analysis by one-dimensional Western blot showed increased quantities of myofilament proteins in the Rac1 mouse ventricles. Additionally, three proteins with different functionality that were altered in the cytoplasmic protein-enriched subproteome, tubulin beta-chain, manganese superoxide dismutase, and malate dehydrogenase, were analyzed at days 7, 9, and 11 to assess their role in the development of the dilated cardiomyopathic phenotype. The quantity of all three proteins peaked at day 9, suggesting an early response in cardiac hypertrophic failure.     

Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol

Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardioprotection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apoptosis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and preconditioning- related proteins were identified that were all upregulated by resveratrol: MAPKK, two different alphaB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxiredoxin- 2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -alphaB-crystallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofilament regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins.     

Right and left ventricular function after chronic pulmonary artery banding in rats assessed with biventricular pressure-volume loops

In many patients with congenital heart disease, the right ventricle (RV) is subjected to abnormal loading conditions. To better understand the state of compensated RV hypertrophy, which could eventually progress to decompensation, we studied the effects of RV pressure overload in rats. In the present study, we report the biventricular adaptation to 6 wk of pulmonary artery banding (PAB). PAB resulted in an RV pressure overload to approximately 60% of systemic level and a twofold increase in RV mass (P < 0.01). Systemic hemodynamic parameters were not altered, and overt signs of heart failure were absent. Load-independent measures of ventricular function (end-systolic pressure-volume relation, preload recruitable stroke work relation, maximum first time derivative of pressure divided by end-diastolic volume), assessed by means of pressure-volume (PV) loops, demonstrated a two- to threefold increase in RV contractility under baseline conditions in PAB rats. RV contractility increased in response to dobutamine stimulation (2.5 microg.kg(-1).min(-1)) both in PAB and sham-operated rats in a similar fashion, indicating preserved RV contractile reserve in PAB rats. Left ventricular (LV) contractility at baseline was unaffected in PAB rats, although LV volume in PAB rats was slightly decreased. LV contractility increased in response to dobutamine (2.5 microg.kg(-1).min(-1)), both in PAB and sham rats, whereas the response to a higher dose of dobutamine (5 microg.kg(-1).min(-1)) was blunted in PAB rats. RV pressure overload (6 wk) in rats resulted in a state of compensated RV hypertrophy with preserved RV contractile reserve, whereas LV contractile state at baseline was not affected. Furthermore, this study demonstrates the feasibility of performing biventricular PV-loop measurements in rats.     

Functional and phenotypic relevance of differentially expressed proteins in calcineurin mutants of Caenorhabditis elegans

Calcineurin is a heterodimeric serine/threonine protein phosphatase, important for many cellular processes such as T-cell regulation, cardiac hypertrophy and kidney development. We previously reported the characterization of Caenorhabditis elegans calcineurin mutants as providing a simple but excellent genetic model system for studying in vivo functions of calcineurin. Calcineurin loss-of-function mutants, cnb-1(lf), and gain-of-function mutants, tax-6(gf), show certain opposite phenotypes as well as some similar phenotypes. In order to explain the phenotypic similarity observed in both loss-of-function and gain-of-function mutants, we examined the proteins that followed similar trends in both mutants relative to wild-type worms by using 2-DE. Interestingly, VHA-13, HSP-6 and phosphoenolpyruvate carboxykinase are down-regulated in both mutants. A total of 96 differentially regulated proteins were identified by MALDI-TOF/MS. Among these, 42 proteins are up-regulated and 54 proteins are down-regulated in calcineurin mutants. Furthermore, knock-down of about 30% of the genes, which are down-regulated in calcineurin mutants, showed some of the phenotypes of calcineurin-null mutants. This analysis suggests the functional relevance of these proteins to calcineurin activity in C. elegans.     

Cytoskeletal role in protection of the failing heart by β-adrenergic blockade

Formation of a dense microtubule network that impedes cardiac contraction and intracellular transport occurs in severe pressure overload hypertrophy. This process is highly dynamic, since microtubule depolymerization causes striking improvement in contractile function. A molecular etiology for this cytoskeletal alteration has been defined in terms of type 1 and type 2A phosphatase-dependent site-specific dephosphorylation of the predominant myocardial microtubule-associated protein (MAP)4, which then decorates and stabilizes microtubules. This persistent phosphatase activation is dependent upon ongoing upstream activity of p21-activated kinase-1, or Pak1. Because cardiac β-adrenergic activity is markedly and continuously increased in decompensated hypertrophy, and because β-adrenergic activation of cardiac Pak1 and phosphatases has been demonstrated, we asked here whether the highly maladaptive cardiac microtubule phenotype seen in pathological hypertrophy is based on β-adrenergic overdrive and thus could be reversed by β-adrenergic blockade. The data in this study, which were designed to answer this question, show that such is the case; that is, β(1)- (but not β(2)-) adrenergic input activates this pathway, which consists of Pak1 activation, increased phosphatase activity, MAP4 dephosphorylation, and thus the stabilization of a dense microtubule network. These data were gathered in a feline model of severe right ventricular (RV) pressure overload hypertrophy in response to tight pulmonary artery banding (PAB) in which a stable, twofold increase in RV mass is reached by 2 wk after pressure overloading. After 2 wk of hypertrophy induction, these PAB cats during the following 2 wk either had no further treatment or had β-adrenergic blockade. The pathological microtubule phenotype and the severe RV cellular contractile dysfunction otherwise seen in this model of RV hypertrophy (PAB No Treatment) was reversed in the treated (PAB β-Blockade) cats. Thus these data provide both a specific etiology and a specific remedy for the abnormal microtubule network found in some forms of pathological cardiac hypertrophy.     

Proteomic characterization of acid stress response in Synechocystis sp. PCC 6803

A comparative proteomic analysis using 2-DE coupled with MALDI-MS and LC-MS/MS was performed in Synechocystis sp. PCC 6803 to identify protein candidates involved in acid stress response in cyanobacteria. Comparison of soluble proteins from the cytoplasmic fraction of cells grown on media set at pH 7.5 and 5.5 using 2-DE identified four proteins, which showed significant changes in the abundance. Surprisingly, several general stress proteins, either the heat shock family proteins or chaperonins, did not show perceptible fold changes in response to acidity. Compared to the cytoplasmic proteome, the periplasmic proteome showed remarkable changes as a function of external pH. Protein expression profiling at different external pH, i.e., 9.0, 7.5, 6.0 and 5.5, allowed classifying the periplasmic proteins depending on their preferential expression patterns towards acidity or alkalinity. Among the acid- and base-induced proteins, oxalate decarboxylase and carbonic anhydrase were already known for their role in pH homeostasis. Several unknown proteins from the periplasm, that showed significant changes in response to pH, provide ideal targets for further studies in understanding pH stress response in cyanobacteria. This study also identified 14 novel proteins, hitherto unknown from the periplasmic space of Synechocystis.     

Expression of stress proteins (HSP-70 and HSP-90) in the rabbit urinary bladder subjected to partial outlet obstruction

Partial obstruction of the rabbit bladder outlet induces a rapid hypertrophy characterized by increased bladder mass, increased smooth muscle content, and increased collagen deposition. In addition, partial outlet obstruction induces decreased contractile responses to both field stimulation and postsynaptic receptor stimulation. Although the morphological and contractile responses to partial outlet obstruction have been well characterized, there is little information on the cellular and molecular mechanisms of these changes. In a previous study, we demonstrated that one of the earliest genes to be expressed following partial outlet obstruction in rabbits was the gene expressing stress protein-70 (HSP-70). In order to further define the genetic and molecular basis of these responses, the expression of stress gene products HSP-70 and HSP-90 in rabbit urinary bladder subjected to partial outlet obstruction has been quantitatively evaluated by Western blot coupled with laser densitometry using anti-HSP-70 and-90 monoclonal antibodies. The data show that stress gene products HSP-70 and HSP-90 are constitutively expressed in control rabbit bladder tissue and transiently increased following partial outlet obstruction. Increased content of HSP-70 was detected at 6 hr after obstruction and reached a maximum (2.7-fold over the control level) at 24 hr. Increased HSP-90 was also detected at 6 hr but reached a maximum (4.5-fold over the control level) at 12 hr. By 7 day post-obstruction, the content of these two proteins returned to the control levels. This study suggests that alterations of stress gene expression resulting in increased HSP-70 and 90 may play an important role in the response of the bladder to partial outlet obstruction.     

Differential protein expression in hypertrophic heart with and without hypertension in spontaneously hypertensive rats

Although cardiac hypertrophy in hypertension has been well recognized, the molecular mechanisms for the development of hypertrophy are still largely unknown. In this study, the protein expression profiles of left ventricular myocardia in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at different ages were analyzed using 2-DE in combination with MALDI-TOF/TOF MS/MS. The results showed that 20 proteins were modulated in the hypertrophic myocardium. Out of these modulated proteins, 13 proteins presented significant changes in SHR at an early stage prior to the development of sustained hypertension, while the changes of the other 7 protein expressions occurred only at a late stage in SHR when the blood pressure was significantly elevated, and were largely reversible by treatment with rennin-angiotensin-aldosterone system inhibitors losartan or enalapril. These data demonstrate that the changes in energy metabolism in the hypertrophied heart favor an increase in glycolysis and a decrease in oxidation of fatty acid and glucose, which occur at an early stage in SHR without hypertension. Our results also provide evidence to support the hypothesis that oxidative stress plays an important role in the development of hypertensive cardiac hypertrophy.     

High heterogeneity within the ribosomal proteins of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 80S ribosome

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms.Patrick Giavalisco, Daniel Wilson are contributed equally to this work.     

Proteomic analysis of bovine conceptus fluids during early pregnancy

A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy.     

Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and alpha-B-crystallin

We performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as alpha-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and alpha-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.     

H2O2 activation of HSP25/27 protects desmin from calpain proteolysis in rat ventricular myocytes

Ischemia-reperfusion-induced Ca(2+) overload results in activation of calpain-1 in the heart. Calpain-dependent proteolysis contributes to myocardial dysfunction and cell death. Previously, preischemic treatment with low doses of H(2)O(2) was shown to improve postischemic function and reduce myocardial infarct size. Our aim was to determine the mechanism by which H(2)O(2) protects the heart. We hypothesized that H(2)O(2) causes the activation of p38 MAPK which initiates translocation of heat shock protein 25/27 (HSP25/27) to the myofilament Z disk. We further hypothesized that HSP25/27 shields structural proteins, particularly desmin, from calpain-induced proteolysis. To address this hypothesis, we first determined that an ischemia-reperfusion-induced decrease in desmin content could be blocked by H(2)O(2) pretreatment of hearts from rats. We next determined that ventricular myocytes that underwent Ca(2+) overload also demonstrated a calpain-dependent disruption of desmin that could be reduced by H(2)O(2)/p38 MAPK activation. Furthermore, myocytes acutely treated with H(2)O(2) exhibited a decrease in cleavage of desmin upon exposure to exogenous calpain-1 compared with myocytes not pretreated with H(2)O(2). The H(2)O(2)-induced attenuation of desmin degradation by calpain-1 was blocked by inhibition of p38 MAPK. In a final series of experiments, we demonstrated that cardiac myofilaments exposed to recombinant phosphorylated HSP27, but not nonphosphorylated HSP27, had a significant reduction in the calpain-induced degradation of desmin compared with non-HSP27-treated myofilaments. These findings are consistent with the hypothesis that H(2)O(2)-induced activation of p38 MAPK and subsequent HSP25/27 translocation attenuates desmin degradation brought about by calpain-1 activation in ischemia-reperfused hearts.     

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M(r) proteins and was complemented by the 2-DE/MS method, which showed a preference for low M(r) proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied.     

Characterization of HSP-70 cognate proteins from wheat

Summary Animal and plant cells contain a family of constitutively expressed HSP-70 cognate proteins that are localized in different subcellular locations and are presumed to play a role in protein folding and transport. Utilizing antibodies raised against the yeast endoplasmicreticulum-localized HSP-70 cognate termed BiP/GRP-78, as well as antibodies raised against the Escherichia coli HSP-70 protein DnaK, we have identified and characterized a large family of closely related proteins in wheat. One protein band of 78 kDa that is apparently closely related to yeast BiP was localized in the endoplasmic reticulum. This band cross-reacted with the yeast BiP but not with the DnaK-specific antibodies. The yeast BiP antibodies also recognized a cytoplasmic protein of 70 kDa that is probably related to the HSC-70 cognate proteins. These two proteins were further confirmed as HSP-70 cognates by their ability to bind to an ATP-agarose column. Probing of proteins from purified wheat mitochondrial preparations with the yeast BiP and DnaK-specific antibodies showed that this organelle contained a family of HSP-70-related proteins. The yeast BiP antibodies recognized two mitochondrial proteins of 60 and 58 kDa, but failed to detect any protein in the size rang of 70 to 80 kDa. However, the presence of immunologically distinct proteins of 90 and 78 kDa, as well as of lower molecular weight from this family in the mitochondria, was shown by probing with the DnaK-specific antibodies. A new protein of 30 kDa, cross-reacting with anti-yeast BiP antibodies, was detected only in developing seeds, close to their maturity. The evolution of HSP-70 cognate proteins in wheat as shown in this study is discussed.     

Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and α-B-crystallin

We performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as α-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and α-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.     

Protein profiling the effects of in vitro hyperoxic exposure on fetal rabbit lung

The aim of this study was to test the hypothesis that acute in vitro exposure of prematurely delivered fetal rabbit lungs to hyperoxic conditions will induce the expression of an adaptive cassette of proteins that mediates antioxidant and inflammatory processes. To test this hypothesis, ex situ fetal rabbit lung explants were prepared from New Zealand white rabbits delivered by cesarean section on day 29 of gestation and incubated under air (21% O2; 5% CO2) or hyperoxic (95% O2; 5% CO2) atmospheres. Total tissue protein was extracted following incubation and subjected to 2-DE. Using this technique, 1500-2000 protein spots were resolved per gel. Treatment-dependent, differentially expressed proteins were identified by image analysis (Melanie II) and MALDI-TOF MS and MALDI-MS/MS. The analysis identified 12 protein spots that were differentially expressed by 1.5-fold or more (p<0.05) by exposure to hyperoxic conditions. Six of these differentially expressed proteins were identified as vimentin, annexin I, inorganic pyrophosphatase, prohibitin, an N-terminal fragment of ATP synthase and heat shock protein 27. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation.     

Heat shock proteins of adult and embryonic human ocular lenses.

We investigated the presence and distribution of heat shock proteins, HSP-70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449-10453], HSP-40, HSc-70, HSP-27, and alphabeta-crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS-PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP-27 and alphabeta-crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP-27. The presence of HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age-related cataract.     

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.