Immunohistochemical analysis of human skeletal muscle AMP deaminase deficiency. Evidence of a correlation between the muscle HPRG content and the level of the residual AMP deaminase activity

We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability

The Prognostic Value of Histidine-Rich Glycoprotein RNA in Breast Tissue Using Unmodified Gold Nanoparticles Assay

The aim of is this study is to explore the role of tissue histidine-rich glycoprotein (HRG) RNA as a promising clinically useful biomarker for breast cancer patients prognosis using nanogold assay. Expression of the HRG RNA was assessed by gold nanoparticles and conventional RT-PCR after purification by magnetic nanoparticles in breast tissue samples. The study included 120 patients, 60 of which were histologically proven breast carcinoma cases, 30 had benign breast lesions and 30 were healthy individuals who had undergone reductive plastic surgery. ER, PR and HER2 status were also investigated. The prognostic significance of tissue HRG RNA expression in breast cancer was explored. The magnetic nanoparticles coated with specific thiol modified oligonucleotide probe were used successfully in purification of HRG RNA from breast tissue total RNAs with satisfactory yield. The developed HRG AuNPs assay had a sensitivity and a specificity of 90 %, and a detection limit of 1.5 nmol/l. The concordance rate between the HRG AuNPs assay with RT-PCR after RNA purification using magnetic nanoparticles was 93.3 %. The median follow-up period was 60 months. Among traditional prognostic biomarkers, HRG was a significant independent prognostic marker in relapse-free survival (RFS). HRG RNA is an independent prognostic marker for breast cancer and can be detected using gold NPs assay, which is rapid, sensitive, specific, inexpensive to extend the value for breast cancer prognosis. © 2014 Springer Science+Business Media

Role of troponin T and AMP deaminase in the modulation of skeletal muscle contraction

In fast muscle, isoforms of troponin T (TnT) contain an N-terminal hypervariable region that does not bind any protein of the thin filament. The N-terminal domain of TnT is removed by calpain during stress conditions and so could modulate the role of TnT in the regulation of contraction by affecting the TnT-binding affinity for tropomyosin (Tm) depending on the sequence and charge within the domain. During skeletal muscle contraction, the myokinase reaction is displaced by AMP deaminase (AMPD), an allosteric metalloenzyme, toward the formation of ATP. An unrestrained AMPD activity follows the proteolytic cleavage of the enzyme in vivo that releases a 97 aa N-terminal fragment, removing the inhibition exerted by the binding of ATP to a zinc site in the N-terminal region. Rabbit fast TnT or its phosphorylated 50-aa residue N-terminal peptide restores in AMPD the inhibition by ATP, removed in vitro by the release of a 95 aa N-terminal fragment by trypsin. Since the N-terminal region of fast rabbit TnT contains a putative zinc-binding motif, it can be inferred that TnT mimics the regulatory action exerted in native AMPD by the N-terminal domain that holds the enzyme in a less active conformation due to the presence of a zinc ion connecting the N-terminal and C-terminal regions. Together with evidence that AMPD is localized on the myofibril, the data reported in this review on the interactions between AMPD and TnT strongly suggest that these proteins mutually combine to fine-tune the regulation of muscle contraction in fast muscle