Alfred L. Yergey

CORTISOL PRODUCTION RATE IN CHILDHOOD AND ADOLESCENCE

We studied the daily cortisol production rate in 33 normal children and adolescents, using a stable isotope-dilution technique employing high-performance liquid chromatography-mass spectrometry. Two indwelling intravenous catheters were inserted and tracer 9,12,12-2H3-cortisol (deuterated cortisol) was infused continuously for 30 hours. After 6 hours of tracer infusion to allow for equilibration, blood was obtained every 20 minutes for 24 hours. The mean (+/- SD) cortisol production rate was 9.5 +/- 2.5 mg/day (6.8 +/- 1.9 mg/m2/day). Cortisol production rate did not vary with sex or pubertal stage. These results suggest that the cortisol production rate in children and adolescents is significantly lower than previously estimated.

De-AMPylation of the Small GTPase Rab1 by the Pathogen Legionella pneumophila

A bacterial pathogen reverses the modification of a host cell protein involved in membrane trafficking. The bacterial pathogen Legionella pneumophila exploits host cell vesicle transport by transiently manipulating the activity of the small guanosine triphosphatase (GTPase) Rab1. The effector protein SidM recruits Rab1 to the Legionella-containing vacuole (LCV), where it activates Rab1 and then AMPylates it by covalently adding adenosine monophosphate (AMP). L. pneumophila GTPase-activating protein LepB inactivates Rab1 before its removal from LCVs. Because LepB cannot bind AMPylated Rab1, the molecular events leading to Rab1 inactivation are unknown. We found that the effector protein SidD from L. pneumophila catalyzed AMP release from Rab1, generating de-AMPylated Rab1 accessible for inactivation by LepB. L. pneumophila mutants lacking SidD were defective for Rab1 removal from LCVs, identifying SidD as the missing link connecting the processes of early Rab1 accumulation and subsequent Rab1 removal during infection.

De Novo Sequencing of Peptides Using MALDI/TOF-TOF

The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated.

Poly(γ-d-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: A potential addition to the anthrax vaccine

Both the protective antigen (PA) and the poly(γ-d-glutamic acid) capsule (γdPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-γdPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic γdPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis γdPGA, Bacillus pumilus γdLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the d or l configuration with active groups at the N or C termini, were bound at 5–32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice. IgG anti-γdPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-γdPGA were elicited by decamers of γdPGA at 10 –20 mol per protein bound to the N- or C-terminal end. High IgG anti-γdPGA levels were elicited by two injections of 2.5 μg of γdPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-γdPGA induced opsonophagocytic killing of B. anthracis tox–, cap+. γdPGA conjugates may enhance the protection conferred by PA alone. γdPGA-rPA conjugates induced both anti-PA and anti-γdPGA.

A newly discovered cholesteryl galactoside from Borrelia burgdorferi

Two major glycolipids, which comprise ≈36% of the total lipid mass from Borrelia burgdorferi, the etiological agent of Lyme disease, were investigated. We determined the fatty acid type, sugar identity, anomeric configuration, and substituent type and position. The structures were identified as cholesteryl 6-O-acyl-β-d-galactopyranoside (B. burgdorferi glycolipid 1, BbGL-I), and 1,2-di-O-acyl-3-O-α-d-galactopyranosyl-sn-glycerol (BbGL-II). The major fatty acids were palmitate and oleate. The structures were corroborated by gas–liquid chromatography MS, matrix-assisted laser desorption/ionization time-of-flight spectroscopy, fast atom bombardment MS, detailed NMR spectrometry, and metabolic labeling. This is a previously undescribed demonstration of a cholesteryl galactoside in bacteria. Lipopolysaccharide was not detected in B. burgdorferi. The two glycolipids have several properties suggesting they may function as lipopolysaccharide: both are main components of the bacterial membrane, surface exposed, and have a three-domain structure. BbGL-I elicited specific antibodies in mice and rabbits, and BbGL-II elicited antibodies that reacted with both glycolipids.

Thermospray liquid chromatography/mass spectrometry for the analysis of L-carnitine and its short-chain acyl derivatives

A method utilizing thermospray high-performance liquid chromatography/mass spectrometry for the separation and direct analysis of carnitine, acetylcarnitine, and propionylcarnitine is described. On-column analysis of mixtures of the acylcarnitines with their corresponding stable, isotope-labeled analogs at nanomolar concentrations has indicated that isotope dilution assays can be applied towards the analysis of carnitine and short-chain acylcarnitines present in biological samples.

Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases

Ubiquitin-like protein/sentrin-specific proteases (Ulp/SENPs) mediate both processing and deconjugation of small ubiquitin-like modifier proteins (SUMOs). Here, we show that Ulp/SENP family members SENP3 and SENP5 localize within the granular component of the nucleolus, a subnucleolar compartment that contains B23/nucleophosmin. B23/nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/nucleophosmin binds SENP3 and SENP5 in Xenopus laevis egg extracts and that it is essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells. After either codepletion of SENP3 and SENP5 or depletion of B23/nucleophosmin, we observed accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/nucleophosmin function in vivo.

Quantifying proteins by mass spectrometry: The selectivity of SRM is only part of the problem

Precise and accurate protein quantification is critical to many areas of proteomics. Antibody‐based approaches are costly and time‐consuming to develop, consequently, there is considerable interest in alternative quantitative methods that are versatile and can be implemented without the considerable delays associated with antibody development and characterization. Approaches based on MS have therefore attracted considerable attention and are now frequently touted as the most practical and powerful of all options. Nevertheless, there are serious limitations associated with quantifying a protein based on tandem mass analysis of one or two peptides generated by either chemical or enzymatic cleavage. In an accompanying Viewpoint article, Molloy and coworkers point out that selectivity is not necessarily guaranteed despite the power of SRM. Here we address an additional concern that can also compromise specificity. In complex mammalian systems, multiple proteins can serve as precursors of a single peptide and consequently, depending on the peptide(s) selected, protein levels may be significantly under‐ or overestimated.

Measurement of True Calcium Absorption in Premature Infants Using Intravenous 46Ca and Oral 44Ca

ABSTRACT: We have developed a method for measuring true fractional calcium absorption (α) in premature infants using two stable isotopes of calcium and tested it in seven studies in seven infants (birth weight 1543 ± 65 g, gestation 32.8 ± 7 wk). A total of 7.5 μg/kg 46Ca was given as a single intravenous bolus. Immediately thereafter 1.25 mg/kg of 44CA was given in a single gavage feeding of standard infant formula (Enfamil). A metabolic isolette was used to obtain 4-h collections of urine for 24 h total. 46Ca and 44Ca were measured in urine by thermal ionization mass spectroscopy and expressed as the ratio to naturally occurring 48Ca. The differences in the 46Ca/48Ca and 44Ca/48Ca ratios from natural levels (Δ% excess 46Ca and Δ% excess 44Ca) were calculated. Percent absorption (α) equals a constant times cumulative Δ% excess 44Ca/A% excess 46Ca. The calculation of α is independent of urine volume or concentration. The Δ% excess 46Ca, showed the expected multiexponential decline as a function of time, and Δ% excess 44Ca usually peaked during a 4− to 8-h urine collection. Calculations of α using increasingly long sampling times showed that a plateau had been reached by 12 h. α values calculated after 16–24 h in the seven infants at 2 wk of age were 41, 48, 45, 46, 25, 55, and 51%. Repeat studies at 3 wk of age were 46, 60, and 54%. These values are somewhat higher than net percent calcium absorption values reported for standard formula and thus appear very appropriate. This methodology will be very valuable in studying factors that may affect true calcium absorption in premature infants.

Stable Isotopic Measurement of Endogenous Fecal Calcium Excretion in Children

Using a stable isotopic technique in which 42Ca was administered via a bolus injection, we measured endogenous fecal calcium excretion, Vf, in five healthy children, aged 3-14 years. The Vf averaged 1.4 +/- 0.4 mg/kg/day, and was lower than urinary Ca excretion (Vu) in four of the five children. These results for Vf are consistent with previously reported results for Vf in healthy adults and much lower than those reported in premature infants. These results may be useful in understanding developmental changes in Ca metabolism and in interpreting dual tracer Ca isotope studies in children.