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Journal Article↗
Biomedical subjects
Robert H Williams
Publications and source records attributed to Robert H Williams.
Quantitative measurement of deamidation in lens betaB2-crystallin and peptides by direct electrospray injection and fragmentation in a Fourier transform mass spectrometer.
PURPOSE: Deamidation of lens crystallins and specific deamidation sites have been suggested to be associated with aging and cataracts. However, these studies have been hindered by the lack of suitable quantitative methods of measurement of protein deamidation. We demonstrate herein a method to quantitatively measure deamidation of proteins and peptides without prior sample preparation or separation in order to directly compare the amidated and deamidated forms. We have tested the hypothesis that the 19 mDa mass defect that distinguishes deamidated peptides and proteins from the ordinary natural isotopic species can be utilized for quantitative measurement of their rate and extent of deamidation. The measurement technique used was ion cyclotron resonance Fourier transform mass spectrometry (FTMS), alone with no prior sample preparation or separation. The amidated and deamidated species were recombinantly expressed human eye lens betaB2-crystallins and the peptides GlyIleAsnAlaGly and GlyAsnAsnAsnGly. FTMS measurements of lens proteins from a 1-month-old human donor were also carried out. METHODS: Wild type and mutant human eye lens betaB2-crystallins with Gln162 replaced by Glu162 were produced in bacteria, and GlyIleAsnAlaGly and GlyAsnAsnAsnGly were synthesized by Merrifield solid-phase peptide synthesis. The peptides were deamidated in pH 7.4, 37.00 degrees C, 0.15 M Tris-HCl aqueous solution for 18 successive time intervals before analysis. Mutant and wildtype betaB2-crystallin solutions at various compositional percentages were mixed and analyzed. The peptides were introduced by electrospray ionization and immediately analyzed in the ion cyclotron resonance (ICR) Fourier transform mass analyzer. Two mass defect analysis procedures were demonstrated for the proteins. In the first, betaB2-crystallin was introduced into the mass spectrometer by electrospray ionization and the +29 isotopic group was selectively introduced into the ICR mass analyzer, where 14 residue and 18 residue laser-induced fragments were separated and the extent of deamidation determined by mass defect analysis. In the second, betaB2-crystallin was introduced into the mass spectrometer by electrospray ionization and the entire sample was fragmented by collision ionization before introduction into the ICR mass analyzer, where 14 residue fragments were separated and the extent of deamidation determined by mass defect analysis. RESULTS: The betaB2-crystallin mass spectra showed a good quantitative dependence upon extent of deamidation. Direct injection by electrospray ionization followed by ion selection and laser fragmentation or by collision fragmentation produced fragments of amidated and deamidated betaB2-crystallin that were appropriate for FTMS quantitative analysis. The two peptides exhibited the expected four deamidation rate curves with acceptable precision. CONCLUSIONS: Mass defect FTMS quantitative analysis of protein deamidation, as reported for the first time herein and illustrated with betaB2-crystallin, should prove quite useful. This procedure omits gel separation, chromatography, enzymatic digestion, derivatization, and other procedures that currently add cost and time while degrading quantitative comparison of the amidated and deamidated forms. Mass defect FTMS is also well suited to quantitative deamidation rate studies of peptides. The substantial potential significance of this technique is evident, as example, for lens crystallins where it makes possible quantitative studies of age and disease-dependent deamidation that have heretofore been very difficult. This technique should allow convenient and reliable identification and quantitative measurement of specific deamidation sites that may play a role in aging and cataracts.
Amides↗
Platelet-derived NO slows thrombus growth on a collagen type III surface.
Nitric oxide (NO) is a free radical that plays an important role in modulating platelet adhesion and aggregation. Platelets are a source of vascular NO, but since erythrocytes avidly scavenge NO, the functional significance of platelet-derived NO is not clear. Our purpose was to determine if NO from platelets affects platelet thrombus formation in the presence of anticoagulated whole blood in an in vitro parallel plate flow system. We studied platelet adhesion and aggregation on a collagen type III surface in the presence of physiologically relevant fluid mechanical shear stress. We found that certain receptor mediated agonists (insulin and isoproterenol) caused a concentration dependent reduction in thrombus formation at a shear rate of 1000 s-1. This effect was mediated by NO since it was abolished in the presence of the NO inhibitor L-nitro-arginine-methyl-ester (L-NAME). As expected, at venous levels of shear rate (100 s-1) neither of the agonists had any effect on thrombus formation since platelet adhesion does not depend on activation at these low levels of shear. Interestingly, at a shear rate of 2000 s-1 the addition of L-NAME caused an increase in platelet coverage suggesting that shear, by itself, induces NO production by platelets. This is the first demonstration of shear stress causing platelets to produce an inhibitor of platelet activation. These results demonstrate that the development of a platelet thrombus is regulated in a complex way and that platelets produce functionally significant amounts of NO even in the presence of whole blood.
Journal Article↗
Comparative analysis of a liquid-based Pap test and concurrent HPV DNA assay of residual samples. A study of 608 cases.
OBJECTIVE: To retrospectively study the HPV DNA assay of residual samples from the ThinPrep Pap Test (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) PreserveCyt (Cytyc) vial as a quality improvement (QI) indicator for management of patients with abnormal cervical cytology. STUDY DESIGN: Six hundred eight residual sample vials of liquid-based Pap-Test specimens were selected for the study based on Pap-test results from October 1998 to March 2001. The specimen vials were forwarded to the reference laboratory (American Medical Laboratories, Chantilly, Virginia, U.S.A.) for HPV DNA assay using the Hybrid Capture System method (Digene Corporation, Gaithersburg, Maryland, U.S.A.). At the time of HPV DNA assay, the residual samples were between 8 days to 10 months old, and each vial contained 4 mL. Of the 608 study cases, 76 were WNL, 115 contained BCC, 172 contained ASCUS, 179 were LSIL and 66 were HSIL. In this study, the 191 WNL and BCC cases were designated as the disease-free control group. The HPV DNA typing results were reported as low-risk, high/intermediate-risk or HPV DNA "not detected" HPV types. The HPV DNA testing results were compared to the Pap-Test diagnoses and statistical analysis performed. RESULTS: The following information reflects the percentage of HPV DNA-positive cases based on the Pap-Test diagnoses: 16.2% in WNL and BCC, 51.1% in ASCUS, 94.4% in LSIL and 98.4% in HSIL. Sensitivity (95.5%), specificity (83.7%), false negative value (4.4%), false positive value (16.2%) and predictive value of a positive (88.3%) and negative (93.5%) Pap-Test were calculated on the basis of HPV DNA testing results for 436 cases that were diagnosed as either SIL or negative (WNL and BCC). ASCUS (172) Pap-Test cases were considered borderline--disease positive and excluded from statistical analysis. CONCLUSION: The HPV DNA assay of residual samples from ThinPrep Pap-Test liquid-based specimens is an objective adjunct to the gynecologic cytology QI protocol and is the gold standard reference test for triaging women with equivocal cytologic diagnoses. The great value of HPV DNA testing is its high sensitivity (95.5%), specificity (83.7%) and negative predictive value (93.5%). HPV DNA testing results can be used as a tool to better determine the need for referrals for colposcopic biopsy, especially for patients with an ASCUS diagnosis. The residual Pap-Test specimens are stable and reproducible for HPV DNA typing. A working flow chart for our gynecologic cytology QI program was produced from the Pap-Test and HPV DNA assay results. This offer presents the added benefit of minimizing the problem of sample variation. The prevalence of HPV infection was 16.2% in this study.
Adult↗
Total homocysteine, diet, and lipid profiles in type 1 and type 2 diabetic and nondiabetic adolescents.
BACKGROUND AND RESEARCH OBJECTIVE: Limited research is available on the possible differences in the cardiovascular risk factors of total homocysteine (tHcy), dietary energy, and lipids among adolescents with type 1 diabetes mellitus (DM), type 2 DM, or healthy controls. This study's primary aim was to compare the dietary energy and the intake of macronutrients and micronutrients of folate, and vitamins B6 and B12, as well as lipids and tHcy for adolescents with type 1 DM, type 2 DM, and healthy non-DM controls. SUBJECTS AND METHODS: This secondary analysis of the merging of 2 datasets included the following adolescents: 50 with type 1 DM, 14 with type 2 DM, and 53 controls. Mean ages for those with type 1 versus type 2 DM were 15.2 +/- 1.9 versus 16.1 +/- 1.9 years, respectively. Mean age for the controls was 16.5 +/- 1.0 years. Variables included fasting tHcy and lipids, and 24-hour dietary recalls for macronutrients and micronutrients. Hemoglobin A1c was obtained for those with DM. Statistical analyses included one-way analyses of variance, Pearson correlations, and stepwise regression. RESULTS AND CONCLUSIONS: Adolescents with type 1 DM had the lowest tHcy values (P <.05), which were reflective of the limited extant research with this population. Lipid profiles and dietary energy did not differ significantly among the 3 groups. Hemoglobin A1c was related to total cholesterol and triglycerides in those with type 1 DM, confirming the importance of promoting better metabolic control in lipid management for these youth. Future research should continue to explore the validity of tHcy and lipids as predictors of CV risks for youth with type 1 and type 2 DM.
Adolescent↗