“Phosphoproteins and phosphopeptides were expressed by E. coli to give yields of 30-200 mg of purified protein per litre with an average degree of phosphorylation at multiple sites of 61-83%. The method employed two compatible cohabiting plasmids having low and high copy number,
expressing a protein kinase and, more abundantly, the substrate (poly)peptide, selleck chemicals respectively. It was used to phosphorylate recombinant beta-casein or osteopontin at multiple casein kinase-2 sites. Two constructs were designed to produce shorter peptides containing one or more clusters of phosphorylation sites resembling the phosphate centres of caseins. In the first, a 53-residue 6-His tagged phosphopeptide was expressed at a 5-fold higher molar yield. The second had multiple tandem repeats of
a tryptic phosphopeptide sequence to give a similar increase in efficiency. Each recombinant phosphopeptide was purified (30100 mg) and small-angle X-ray scattering measurements showed that they, like certain casein and osteopontin phosphopeptides, sequester amorphous calcium phosphate to form calcium phosphate nanoclusters. In principle, the method can provide novel phosphopeptides for the control of biocalcification or be adapted for use with other kinases and cognate proteins or peptides to study the effect of specific phosphorylations on protein structure. Moreover, the insertion of a phosphate centre sequence, possibly with a linker peptide, may allow thermodynamically stable, biocompatible nanoparticles to be BMS202 molecular weight made from virtually any sequence. (C) 2009 Elsevier Inc. All rights reserved.”
“Purpose: Guidelines recommend serum total testosterone measurement as the initial test to evaluate male hypogonadism, reserving free testosterone assessment for men with suspected sex hormone-binding globulin abnormalities or total testosterone near the lower limit of normal. We determined the performance of total testosterone measurement as a test to identify men with normal vs low free testosterone.
Materials and Methods: We examined the electronic
medical records of all 3,672 men evaluated for hypogonadism by a serum testosterone panel, including total testosterone, AZD8055 nmr sex hormone-binding globulin, albumin and calculated free testosterone, from January 1, 1997 through December 31, 2007 in a network that serves veterans in Washington.
Results: The sensitivity and specificity of low total testosterone (less than 280 ng/dl) to rule out and predict low calculated free testosterone was 91.0% and 73.7%, respectively. At thresholds of less than 350 and less than 400 ng/dl the sensitivity of total testosterone for low calculated free testosterone increased to 96.8% and 98.2%, and at thresholds of less than 150 and less than 200 ng/dl specificity increased to 98.9% and 92.6%, respectively.
Conclusions: Total testosterone between 280 and 350 ng/dl is not sensitive enough to reliably exclude hypogonadism.