Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation

Proteolytic fragments of type II collagen, a major component of joint tissue, have recently been identified as biomarkers for osteoarthritis, a progressive disease associated with cartilage degeneration. A liquid chromatography/tandem mass spectrometry (MS/MS) assay that utilizes online immunoaffinity chromatography and column switching was developed in our laboratory for the neoepitope of type II collagen (NET2C). During method development, peptide collision-induced dissociation (CID) was found to be a significant source of assay variation, which exceeded 10% CV, despite the fact that a stable-isotope-labeled (SIL) internal standard was used to minimize imprecision. This phenomenon was studied in detail using peptides and associated SIL internal standards of varying lengths and amino acid compositions. Variability in peptide CID necessitated the monitoring of multiple MS/MS transitions to obtain acceptable assay precision. The assay was subsequently validated to measure NET2C concentrations in rat urine over the range of 0.1 to 10 ng/mL. The interday accuracy and precision ranged from 3.9 to 13.1 (%CV) and 10.7 to 5.3 (%RE), respectively, across the range of validated concentrations. A specific application of the assay is presented in which the role of estrogen deficiency in the development and progression of osteoarthritis was investigated. In this study, the effect of estrogen on lowering NET2C concentrations in urine in ovariectomized rats was demonstrated.     

The structural basis of cooperative regulation at an alternate genetic switch

Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.     

Mycoplasmas: a distinct cytoskeleton for wall-less bacteria

The bacterial genus Mycoplasma includes a large number of highly genomically-reduced species which in nature are associated with hosts either commensally or pathogenically. Several Mycoplasma species, including Mycoplasma pneumoniae, feature a multifunctional polar structure, the terminal organelle. Essential for colonization of the host and for gliding motility, the terminal organelle is associated with an internal cytoskeleton crucial to its assembly and function. This cytoskeleton is structurally and compositionally novel as compared with the cytoskeletons of other organisms, including other bacteria, is also involved in the cell division process. In this review we discuss the cytoskeletal structures and protein components of the attachment organelle and how they might interact and contribute to its various functions.     

Towards enzyme activated antiprostatic agents

A prodrug conjugate designed to undergo activation by enzymatic prostate specific antigen has been synthesized. The prodrug system undergoes activation with PSA or alpha-chymotrypsin, and shows selective cytotoxicity in a PSA secreting cell line.     

Imatinib ameliorates renal morphological changes in Cyp1a1-Ren2 transgenic rats with inducible ANG II-dependent malignant hypertension

The present study was performed to assess the effects of the platelet-derived growth factor (PDGF) receptor kinase inhibitor imatinib mesylate on the renal morphological changes occurring during the development of malignant hypertension in transgenic rats with inducible expression of the Ren2 gene [TGR(Cyp1a1Ren2)]. Arterial blood pressure was measured by radiotelemetry in male Cyp1a1-Ren2 rats during control conditions and during dietary administration of indole-3-carbinol (I3C; 0.3%) for 14 days to induce malignant hypertension. Rats induced with I3C (n = 5) had higher mean arterial pressures (178 ± 4 vs. 109 ± 2 mmHg, P < 0.001) and increased urinary albumin excretion (Ualb; 13 ± 5 vs. 0.6 ± 0.2 mg/day) compared with noninduced rats (n = 5). Chronic administration of imatinib (60 mg·kg(-1)·day(-1) in drinking water, n = 5) did not alter the magnitude of the hypertension (176 ± 8 mmHg) but prevented the increase in Ualb (1.6 ± 0.3 mg/day). Quantitative analysis of proliferating cell nuclear antigen using immunohistochemistry demonstrated increased proliferating cell number in cortical tubules (38 ± 5 vs. 18 ± 1 cells/mm(2)) and cortical interstitium (40 ± 7 vs. 13 ± 6 cells/mm(2)) of hypertensive rat kidneys. Renal cortical fibrosis evaluated by picrosirius red staining showed increased collagen deposition in kidneys of the hypertensive rats (1.6 ± 0.1 vs. 0.4 ± 0.1% of cortical area). Imatinib attenuated the increase in proliferating cell number in cortical tubules and interstitium (22 ± 5 vs. 38 ± 5 and 22 ± 6 vs. 40 ± 7 cells/mm(2), respectively) and reduced the degree of collagen deposition (0.8 ± 0.2 vs. 1.6 ± 0.1%) in the kidneys of hypertensive rats. These findings demonstrate that the renal pathological changes that occur during the development of malignant hypertension in Cyp1a1-Ren2 rats involve activation of PDGF receptor kinase.     

Electromechanical and structural alterations in the aging rabbit heart and aorta

Aging increases the risk for arrhythmias and sudden cardiac death (SCD). We aimed at elucidating aging-related electrical, functional, and structural changes in the heart and vasculature that account for this heightened arrhythmogenic risk. Young (5-9 mo) and old (3.5-6 yr) female New Zealand White (NZW) rabbits were subjected to in vivo hemodynamic, electrophysiological, and echocardiographic studies as well as ex vivo optical mapping, high-field magnetic resonance imaging (MRI), and histochemical experiments. Aging increased aortic stiffness (baseline pulse wave velocity: young, 3.54 ± 0.36 vs. old, 4.35 ± 0.28 m/s, P < 0.002) and diastolic (end diastolic pressure-volume relations: 3.28 ± 0.5 vs. 4.95 ± 1.5 mmHg/ml, P < 0.05) and systolic (end systolic pressure-volume relations: 20.56 ± 4.2 vs. 33.14 ± 8.4 mmHg/ml, P < 0.01) myocardial elastances in old rabbits. Electrophysiological and optical mapping studies revealed age-related slowing of ventricular and His-Purkinje conduction (His-to-ventricle interval: 23 ± 2.5 vs. 31.9 ± 2.9 ms, P < 0.0001), altered conduction anisotropy, and a greater inducibility of ventricular fibrillation (VF, 3/12 vs. 7/9, P < 0.05) in old rabbits. Histochemical studies confirmed an aging-related increased fibrosis in the ventricles. MRI showed a deterioration of the free-running Purkinje fiber network in ventricular and septal walls in old hearts as well as aging-related alterations of the myofibrillar orientation and myocardial sheet structure that may account for this slowed conduction velocity. Aging leads to parallel stiffening of the aorta and the heart, including an increase in systolic stiffness and contractility and diastolic stiffness. Increasingly, anisotropic conduction velocity due to fibrosis and altered myofibrillar orientation and myocardial sheet structure may contribute to the pathogenesis of VF in old hearts. The aging rabbit model represents a useful tool for elucidating age-related changes that predispose the aging heart to arrhythmias and SCD.