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Biomedical subjects

Steve M Helmke

Publications and source records attributed to Steve M Helmke.

9 recordsLinked to original sources
PubMed2006

Measurement of the NO metabolites, nitrite and nitrate, in human biological fluids by GC-MS.

In this article we critically review the development and application of gas chromatography-mass spectrometry (GC-MS) techniques to the measurement of the nitric oxide (NO) metabolites, nitrite and nitrate, in human biological fluids. Our focus is on the issue of the fitness of any analytical strategy to its intended purpose and the validity of the analytical results generated. The accuracy, precision, recovery, selectivity and sensitivity of the various methods are evaluated and the potential pitfalls, both specific to the methods, and general to the area, are considered. Several examples of the applications of these techniques to clinical investigations of NO physiology are also critically evaluated.

Body Fluids↗
PubMed2006

Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue.

In order to better understand basic mechanisms of tumor development and identify potential new biomarkers, we have performed difference gel electrophoresis (DIGE) and peptide mass fingerprinting on pooled protein extracts from patients with papillary thyroid carcinoma (PTC) compared with matched normal thyroid tissue. Image analysis of DIGE gels comparing PTC and matched normal thyroid tissue protein indicated that 25% of the protein spots were differentially expressed at a 2.5-fold cutoff and 35% at two-fold. Comparison between two different pools of protein from normal thyroid tissues revealed differential protein expression of only 4% at 2.5-fold and 6% at two-fold cutoff. One hundred ninety-two protein spots were identified by MALDI-TOFMS, representing 90 distinct proteins. Excluding albumin, globins and thyroglobulin, imaging software determined 31 proteins to be differentially expressed at the two-fold (or greater) level. Individual gel comparisons (PTC vs. matched normal) from five patients established that 15/31 (48%) of these proteins exhibited statistically significant differential expression. Previously identified molecular markers in this group of proteins include cathepsin B, cytokeratin 19, and galectin-3. Novel differentially expressed proteins include S100A6, moesin, HSP70 (BiP), peroxiredoxin 2, protein phosphatase 2, selenium binding protein 1, vitamin D binding protein, and proteins involved in mitochondrial function. The use of two-dimensional gel electrophoresis (2DGE) revealed a significantly altered protein mass and/or pI in 10%-15% of proteins, suggesting alternatively spliced forms and other posttranslational modification of proteins revealed by this approach. We confirmed S100A6 as a potentially useful biomarker using immunohistochemical analysis (85% sensitivity and 69% specificity for distinguishing benign from malignant thyroid neoplasms). In summary, proteomic analysis of PTC using DIGE and mass spectrometry has confirmed several known biomarkers, uncovered novel potential biomarkers, and provided insights into global pathophysiologic changes in PTC. Many of the differences observed would not have been detected by genomic or other proteomic approaches.

Amino Acid Sequence↗
PubMed2005

Proteomics of the anterior pituitary gland as a model for studying the physiology of a heterogeneous organ.

The anterior pituitary gland (AP) secretes six established hormones that collectively control hundreds of biological and behavioral functions. Because of advances in mass spectrometry (MS), protein labeling, and bioinformatics, it is now possible to characterize, compare, and quantify the AP hormones together with large numbers of nonhormonal AP proteins. For example, by using high-performance liquid chromatography in line with tandem MS we characterized 145 proteins in sub-cellular fractions of the AP of young adult male Golden Syrian hamsters and 115 proteins in subcellular fractions of the AP of young adult male mice. These included hormones, proteins involved in hormone synthesis and release, and housekeeping proteins. We also used difference gel electrophoresis in conjunction with MS and peptide mass fingerprinting to quantify the effects of estrogen on the AP-soluble protein fraction in rats. Ovariectomized rats were administered 50 microg of estradiol valerate subcutaneously and studied 48 hrs later, before the onset of the anticipated surges of gonadotropins in blood. Following DeCyder image analysis, we identified by MS and peptide mass fingerprinting 26 protein spots that were upregulated and 19 protein spots that were downregulated. Estrogen increased levels of acidic isoforms of growth hormone and prolactin, several proteins involved in protein synthesis, folding and secretion, and several metabolic enzymes. Most of the downregulated proteins are involved in RNA or DNA interactions. We followed up on the results with RT-PCR and immunohistochemical techniques to demonstrate that one protein identified by MS in hamster AP, fertility protein SP22, is synthesized in the AP and localized primarily in somatotropes and thyrotropes. These experiments demonstrate the efficacy of our proteomics approach to characterize AP proteins and quantify changes in them. The approaches used to study the AP could serve as a model to investigate other heterogeneous organs.

Animals↗
PubMed2005

Estrogen regulation of the rat anterior pituitary gland proteome.

Estrogen is known to affect the regulation of all six of the established anterior pituitary gland (AP) hormones, but little is known of the specifics of its regulation of the AP hormones, their isoforms, and nonhormonal AP proteins. We used difference gel electrophoresis in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting to quantify the effects of estrogen on the AP-soluble protein fraction in rats. Two-month-old rats were ovariectomized and used at 6 months of age. They were injected subcutaneously with sesame oil vehicle or 50 mug estradiol valerate in vehicle and studied 48 hrs later, approximately 3 hrs before the time of the anticipated onset of the estrogen-induced surges of gonadotropins in blood. The APs were pooled, and the soluble protein fraction was examined in replicate analyses. After DeCyder software analysis, we identified 26 protein spots that had a 1.5-fold or greater average increase in the experimental group relative to the controls. Nineteen showed a 1.5-fold or greater decrease. Estrogen increased levels of the more acidic isoforms of growth hormone and prolactin and of proteins involved in protein synthesis, folding, and secretion (e.g., eukaryotic translation elongation factor 2, ERp57, ERp29, Hsc70-ps1, calreticulin, coatomer delta subunit, and secretogranin II) and of some metabolic enzymes (e.g., arginosuccinate synthetase, enolase 1, creatine kinase B, phosphoglycerate mutase, malate dehydrogenase, pyruvate kinase, and aldolase A). The majority of the downregulated proteins were involved in RNA or DNA interactions (e.g., five heterogeneous nuclear ribonucleoproteins, DEAD-box proteins 17 and 48, ssDNA binding protein PUR-alpha, PTB-associated splicing factor, and Pigpen protein), but isovaleryl coenzyme A dehydrogenase, mitochondrial aldehyde dehydrogenase, stathmin 1, vinculin, radixin, and secretogranin III were also reduced. Our results indicate that estrogen acts in vivo within 48 hrs to modulate levels of a significant number of AP proteins.

Animals↗
PubMed2004

Simultaneous quantification of human cardiac alpha- and beta-myosin heavy chain proteins by MALDI-TOF mass spectrometry.

We have developed a novel method for quantifying protein isoforms, in both relative and absolute terms, based on MALDI-TOF mass spectrometry. The utility of the approach is demonstrated by quantifying the alpha and beta protein isoforms of myosin heavy chain (MyHC) in human atrial tissue. Alpha-MyHC (726-741) and beta-MyHC (724-739) were identified as isoform-specific tryptic peptides. A calibration curve was constructed by plotting ion current ratios against molar ratios of the two peptides prepared synthetically. MyHC was digested by trypsin and the ion current ratio determined for the two tryptic peptides. The ion current ratio was converted to the peptide ratio and hence the isoform ratio by reference to the standard curve. The accuracy of the method was confirmed by a comparison between these results and those determined by an established method of MyHC isoform ratio determination. So that the molar ratio could be converted to absolute values, a third peptide, an analogue of the two peptides being measured, was synthesized for use as an internal standard (IS). The measured ion current ratios of synthetic alpha-MyHC (726-741), beta-MyHC (724-739), and IS peptides were used to generate standard curves. A known quantity of the IS was added to the MyHC digests. The measured ion current ratios were converted to the actual quantities of the isoform-specific peptides and hence the actual quantity of each protein isoform by reference to the standard curves. This method is of general applicability, especially when isoform quantification is required.

Humans↗
PubMed2004

The Ku protein complex interacts with YY1, is up-regulated in human heart failure, and represses alpha myosin heavy-chain gene expression.

Human heart failure is accompanied by repression of genes such as alpha myosin heavy chain (alphaMyHC) and SERCA2A and the induction of fetal genes such as betaMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human alphaMyHC promoter. We have now identified a region of the alphaMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human alphaMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases alphaMyHC mRNA expression and increases skeletal alpha-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the alphaMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human alphaMyHC promoter during heart failure.

Antigens, Nuclear↗
PubMed2003

Analysis of myosin heavy chain functionality in the heart.

Comparison of mammalian cardiac alpha- and beta-myosin heavy chain isoforms reveals 93% identity. To date, genetic methodologies have effected only minor switches in the mammalian cardiac myosin isoforms. Using cardiac-specific transgenesis, we have now obtained major myosin isoform shifts and/or replacements. Clusters of non-identical amino acids are found in functionally important regions, i.e. the surface loops 1 and 2, suggesting that these structures may regulate isoform-specific characteristics. Loop 1 alters filament sliding velocity, whereas Loop 2 modulates actin-activated ATPase rate in Dictyostelium myosin, but this remains untested in mammalian cardiac myosins. Alpha --> beta isoform switches were engineered into mouse hearts via transgenesis. To assess the structural basis of isoform diversity, chimeric myosins in which the sequences of either Loop 1+Loop 2 or Loop 2 of alpha-myosin were exchanged for those of beta-myosin were expressed in vivo. 2-fold differences in filament sliding velocity and ATPase activity were found between the two isoforms. Filament sliding velocity of the Loop 1+Loop 2 chimera and the ATPase activities of both loop chimeras were not significantly different compared with alpha-myosin. In mouse cardiac isoforms, myosin functionality does not depend on Loop 1 or Loop 2 sequences and must lie partially in other non-homologous residues.

Amino Acid Sequence↗
PubMedDate not supplied

Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled-coil template.

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.

3T3 Cells↗