Novel serum protein biomarkers indicative of growth hormone doping in healthy human subjects

The detection of recombinant human growth hormone (rhGH) is difficult due to its short half‐life; therefore, novel and robust biomarkers of rhGH abuse are needed. In this study, serum samples derived from subjects treated with rhGH in a randomized, double blind, placebo‐controlled crossover study were analyzed by 2‐DE coupled with MS. Eight healthy male subjects aged 23.2±0.6 years were injected with rhGH (2 mg/day) or saline for 7 days with serum samples drawn at days 0, 3, and 8. Protein intensities were quantified and analyzed for differences between rhGH and placebo treatments. Proteins that showed significant changes were identified and confirmed by Western blotting. These included specific isoforms of α‐1 antitrypsin and transthyretin that increased; and inter‐α‐trypsin inhibitor heavy chain H4, apolipoprotein A‐1, and hemoglobin β chain that decreased. These proteins represent novel biomarkers of short‐term rhGH exposure and may lead to a new method for detecting rhGH doping.     

Lack of effect of antenatal glucocorticoid therapy in the fetal baboon on cerebral cortical glucose transporter proteins

BACKGROUND: Maternal antenatal glucocorticoid therapy is used to accelerate lung maturation of immature babies at risk of preterm delivery. It acutely affects brain activity of the human fetus and reduces the immunoreactivity of neurocytoskeletal and synaptic proteins in the fetal baboon brain. These effects might be based on cerebral energy failure due to a decreased neuronal glucose uptake that has been shown in vitro. METHODS: Glucose uptake into the brain is selectively facilitated by GLUT1 expressed in the blood-brain barrier and GLUT3 expressed in the neuronal membrane. Immunohistochemical distribution of GLUT1 and GLUT3 were examined in the frontal neocortex of the fetal baboon brain at 0.73 gestation (i.e. similar to 28 weeks of human gestation) after maternal betamethasone administration, mimicking the clinical dose regimen. RESULTS: Betamethasone did not alter GLUT1 and GLUT3 immunoreactivity. CONCLUSIONS: The results suggest that inhibition of glucose uptake is not the mechanism for the cerebral effects of antenatal glucocorticoids.     

Mapping of conformational IgE epitopes with peptide-specific monoclonal antibodies reveals simultaneous binding of different IgE antibodies to a surface patch on the major birch pollen allergen, Bet v 1

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients' IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients' IgE binding to Bet v 1 (52-75%) were obtained with mAbs specific for two peptides comprising aa 29-58 (P2) and aa 73-103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.     

Comamonas testosteronan synthase, a bifunctional glycosyltransferase that produces a unique heparosan polysaccharide analog

Glycosaminoglycans (GAGs) are linear hexosamine-containing polysaccharides. These polysaccharides are synthesized by some pathogenic bacteria to form an extracellular coating or capsule. This strategy forms the basis of molecular camouflage since vertebrates possess naturally occurring GAGs that are essential for life. A recent sequence database search identified a putative protein from the opportunistic pathogen Comamonas testosteroni that exhibits similarity with the Pasteurella multocida GAG synthase PmHS1, which is responsible for the synthesis of a heparosan polysaccharide capsule. Initial supportive evidence included glucuronic acid (GlcUA)-containing polysaccharides extracted from C. testosteroni KF-1. We describe here the cloning and analysis of a novel Comamonas GAG synthase, CtTS. The GAG produced by CtTS in vitro consists of the sugars d-GlcUA and N-acetyl-D-glucosamine, but is insensitive to digestion by GAG digesting enzymes, thus has distinct glycosidic linkages from vertebrate GAGs. The backbone structure of the polysaccharide product [-4-D-GlcUA-α1,4-D-GlcNAc-α1-](n) was confirmed by nuclear magnetic resonance. Therefore, this novel GAG, testosteronan, consists of the same sugars as the biomedically relevant GAGs heparosan (N-acetyl-heparosan) and hyaluronan but may have distinct properties useful for future medical applications.     

Seasonal effects of 19 plant species on COD removal in subsurface treatment wetland microcosms

Plants have many well-documented influences in treatment wetlands, but differences in individual species’ effects on year-round and seasonal performance are poorly understood. In this study, we evaluated plant effects on seasonal patterns of organic carbon removal (measured as COD) and sulfate concentration (used as an indicator of rootzone oxidation) in replicated, batch-loaded, greenhouse microcosms simulating subsurface treatment wetlands. Microcosms were planted with monocultures of 19 plant species or left unplanted as controls, dosed every 20 days with synthetic secondary wastewater, and operated over 20 months at temperatures from 4 to 24 °C. Study-long COD removal averaged 70% for controls and 70-97% for individual species. Most species enhanced COD removal significantly and the benefits of plants were greatest at 4-8 °C because COD removal decreased at low temperatures in controls but displayed limited seasonal variation in planted microcosms. Removal was significantly better at 24 °C than 4 °C with two species (Panicum virgatum and Leymus cinereus), significantly poorer with two species (Carex utriculata and Phalaris arundinacea), and did not differ with 15 species. Only one species showed a significant positive correlation between temperature and COD removal (Iris missouriensis, r = 0.67), while two species showed significant negative correlations (better when colder: Carex nebrascensis, r = −0.67; C. utriculata, r = −0.93). High COD removal throughout the study was strongly associated with high SO₄ concentrations at low temperatures, suggesting that plant performance is related to rootzone oxidation and species’ abilities to promote aerobic over anaerobic microbial processes, particularly in winter. Results indicate that improved year-round and cold-season COD removal is common across diverse wetland plant species and novel species can be as good or better than those typically used. Better performing species were largely in the sedge and rush families (Cyperaceae and Juncaceae), while poorer performing species were largely in the grass family (Poaceae).     

T Lymphocyte Density and Distribution in Human Colorectal Mucosa,and Inefficiency of Current Cell Isolation Protocols

Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3⁺ lymphocytes, including CD4⁺ and CD8⁺ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r²=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3⁺ T lymphocytes with 37,400 ± 2,801 cells/mm³ and 33,700 ± 4,324 cell/mm³, respectively. Sigmoid mucosa contained 57% CD3⁺CD4⁺ and 40% CD3⁺CD8⁺ T lymphocytes which calculates to 21,300 ± 1,476/mm³ and 15,000 ± 275/mm³ T lymphocytes, respectively. Rectal mucosa had 57% CD3⁺CD4⁺ and 42% CD3⁺CD8⁺ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm³, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm³ and 2,708 ± 245/mm³ CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3⁺, CD4⁺ and CD8⁺ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing.     

Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavβ₂

Ahnak1 has been implicated in protein kinase A (PKA)-mediated control of cardiac L-type Ca(2+) channels (Cav1.2) through its interaction with the Cavβ(2) regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavβ(2) in the T-tubule system. In previous studies Cavβ(2) attachment sites which impacted the channel's PKA regulation have been located to ahnak1's proximal C-terminus (ahnak1(4889-5535), ahnak1(5462-5535)). In this study, we mapped the ahnak1-interacting regions in Cavβ(2) and investigated whether Cavβ(2) phosphorylation affects its binding behavior. In vitro binding assays with Cavβ(2) truncation mutants and ahnak1(4889-5535) revealed that the core region of Cavβ(2) consisting of Src-homology 3 (SH3), HOOK, and guanylate kinase (GK) domains was important for ahnak1 interaction while the C- and N-terminal regions were dispensable. Furthermore, Ser-296 in the GK domain of Cavβ(2) was identified as novel PKA phosphorylation site by mass spectrometry. Surface plasmon resonance (SPR) binding analysis showed that Ser-296 phosphorylation did not affect the high affinity interaction (K(D)≈35 nM) between Cavβ(2) and the α(1C) I-II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak1(5462-5535) revealed that PKA phosphorylation of Cavβ(2) significantly increased the binding affinity and, in parallel, it reduced the binding capacity. Intriguingly, the phosphorylation mimic substitution Glu-296 fully reproduced both effects, increased the affinity by ≈2.4-fold and reduced the capacity by ≈60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavβ(2) phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1's modulator function on Cav1.2 channel activity.     

Improved analysis of bacterial CGH data beyond the log-ratio paradigm

Background

While the theory of enzyme kinetics is fundamental to analyzing and simulating biochemical systems, the derivation of rate equations for complex mechanisms for enzyme-catalyzed reactions is cumbersome and error prone. Therefore, a number of algorithms and related computer programs have been developed to assist in such derivations. Yet although a number of algorithms, programs, and software packages are reported in the literature, one or more significant limitation is associated with each of these tools. Furthermore, none is freely available for download and use by the community.

Results

We have implemented an algorithm based on the schematic method of King and Altman (KA) that employs the topological theory of linear graphs for systematic generation of valid reaction patterns in a GUI-based stand-alone computer program called KAPattern. The underlying algorithm allows for the assumption steady-state, rapid equilibrium-binding, and/or irreversibility for individual steps in catalytic mechanisms. The program can automatically generate MathML and MATLAB output files that users can easily incorporate into simulation programs.

Conclusion

A computer program, called KAPattern, for generating rate equations for complex enzyme system is a freely available and can be accessed at http://www.biocoda.org.     

Esterase-16 (ES-16) of the rat (Rattus norvegicus): Identification and genetic characterization

Esterase-16, an esterase present in lung and other tissues of the laboratory rat, has been characterized by its biochemical properties (electrophoretic mobility, substrate pattern, sensitivity to inhibitors) and genetic variation in 107 inbred strains and substrains including 14 RI strains. It was classified as a carboxylesterase (EC 3.1.1.1). The phenotype ES-16A (BN/Han and 63 other strains) was defined as a narrow electrophoretic band migrating between ES-1A and ES-13A, ES-16B (LEW/Han and 42 other strains) exhibited the same electrophoretic mobility as ES-16A but was distinguished by its extremely weak activity. Segregation of ES-16 in RI strains and backcrosses indicated linkage to linkage group V (LGV). The Es-16 locus was tentatively placed into esterase cluster 2 and homology with Es-7 of the house mouse is proposed.