Structure-dependent nonenzymatic deamidation of glutaminyl and asparaginyl pentapeptides.

Nonenzymatic deamidation rates for 52 glutaminyl and 52 asparaginyl pentapeptides in pH 7.4, 37.0 degrees C. 0.15 m Tris-HCl buffer have been determined by direct injection mass spectrometry. These and the previously reported 306 asparginyl rates have been combined in a self-consistent model for peptide deamidation. This model depends quantitatively upon peptide structure and involves succinimide, glutarimide and hydrolysis mechanisms. The experimental values and suitable interpolated values have been combined to provide deamidation rate values in pH 7.4, 37.0 degrees C. 0.15 m Tris-HCl buffer for the entire set of 648 single-amide permutations of ordinary amino acid residues in GlyXxxAsnYyyGly and GlyXxxGlnYyyGly. Thus, knowledge about sequence-dependent deamidation in peptides is extended to include very long deamidation half-times in the range of 2-50 years.

Prediction of primary structure deamidation rates of asparaginyl and glutaminyl peptides through steric and catalytic effects.

The primary sequence dependence of deamidation has been quantitatively explained on the basis of a simple steric and catalytic model. Application to the known deamidation rates of peptides produces a table of coefficients that permits calculation of the known deamidation rates and prediction of deamidation rates for peptide sequences that have not yet been measured. This work permits a better understanding of deamidation, provides a prediction procedure for protein engineering, and facilitates improved computation of peptide and protein primary, secondary, tertiary, and quaternary structure deamidation rates.

Deamidation of human proteins.

Deamidation of asparaginyl and glutaminyl residues causes time-dependent changes in charge and conformation of peptides and proteins. Quantitative and experimentally verified predictive calculations of the deamidation rates of 1,371 asparaginyl residues in a representative collection of 126 human proteins have been performed. These rates suggest that deamidation is a biologically relevant phenomenon in a remarkably large percentage of human proteins.

Mass spectrometric evaluation of synthetic peptides as primary structure models for peptide and protein deamidation.

Solid-phase peptide synthesis and deamidation measurements using a novel mass spectrometric technique were carried out for 94 model asparaginyl peptides from 3 to 13 residues in length. Deamidation rates of these peptides in pH 7.4, 37.0 degrees C, 0.15 M Tris-HCl buffer were measured and evaluated. It was found that they validate the use of pentapeptide models as surrogates for the primary sequence dependence of peptide and protein deamidation rates and the discovery by difference of secondary, tertiary and quaternary structure effects. Deamidation of the pentapeptide models, compared with that of longer peptides of more intricate structure, is discussed, and the application of this technique to deamidation measurement of intact proteins is demonstrated.

Prediction of protein deamidation rates from primary and three-dimensional structure.

A method for the quantitative estimation of instability with respect to deamidation of the asparaginyl (Asn) residues in proteins is described. The procedure involves the observation of several simple aspects of the three-dimensional environment of each Asn residue in the protein and a calculation that includes these observations, the primary amino acid residue sequence, and the previously reported complete set of sequence-dependent rates of deamidation for Asn pentapeptides. This method is demonstrated and evaluated for 23 proteins in which 31 unstable and 167 stable Asn residues have been reported and for 7 unstable and 63 stable Asn residues that have been reported in 61 human hemoglobin variants. The relative importance of primary structure and three-dimensional structure in Asn deamidation is estimated.

Molecular clocks.

A convenient and precise mass spectrometric method for measurement of the deamidation rates of glutaminyl and asparaginyl residues in peptides and proteins has been developed; the rates of deamidation of 306 asparaginyl sequences in model peptides at pH 7.4, 37.0 degrees C, 0.15 M Tris.HCl buffer have been determined; a library of 913 amide-containing peptides for use by other investigators in similar studies has been established; and, by means of simultaneous deamidation rate measurements of rabbit muscle aldolase and appropriate model peptides in the same solutions, the use of this method for quantitative measurement of the relative effects of primary, secondary, tertiary, and quaternary protein structure on deamidation rates has been demonstrated. The measured rates are discussed with respect to the hypothesis that glutaminyl and asparaginyl residues serve, through deamidation, as molecular timers of biological events.

Characterization of cyclooxygenase in laryngeal papilloma by molecular techniques.

OBJECTIVES: Demonstrate the induction of cyclooxygenase-2 (COX-2) in laryngeal papilloma Discuss the possible causal role of COX-2 in papilloma formation. Consider the potential for treatment of papilloma using selective COX-2 inhibitors. STUDY DESIGN: Molecular biological analysis of COX-1 and COX-2 in laryngeal papilloma. METHODS: Tissue samples from five patients with recurrent respiratory papillomatosis (RRP) were analyzed by in situ hybridization, immunohistochemical staining, and reverse transcription polymerase chain reaction (RT-PCR) techniques. RESULTS: In situ hybridization to COX-2 mRNA showed strong autoradiographic signal surrounding fibrovascular cores. COX-1 autoradiographic signal was low intensity or nondetectable. Normal buccal mucosa biopsies showed low-density or nondetectable autoradiographic signal for both COX-1 and COX-2 mRNAs. In situ hybridization results were corroborated by RT-PCR studies. Levels of COX-2 mRNA were 13-fold more than those in normal mucosa. Immunohistochemical staining for COX-1 and COX-2 showed a similar pattern to that seen with in situ hybridization in both normal and papilloma tissues. CONCLUSIONS: There is an elevation of COX-2 expression in papilloma tissues. This may represent a causal role of COX-2 in the formation and proliferation of laryngeal papilloma. There may also be a role for selective COX-2 inhibition for the treatment of

Haematology and blood biochemistry during the course of psoroptic scabies in sheep.

Haematology and blood biochemistry were monitored during the development and regression of psoroptic scabies in sheep. There were rises in numbers of neutrophils and eosinophils and in the fibrinogen concentrations, with lowered haemoglobin concentrations any lymphocyte numbers. The only biochemical parameters that were affected were the estimated serum globulin concentration, which rose during the disease process, and the serum albumin concentration, which fell. All of these changes reverted to normal soon after treatment.

Suppression of squamous cell carcinoma in hairless mice by dietary nutrient variation.

In experiments involving the induction of squamous cell carcinoma in 1846 hairless mice that were maintained on a wide variety of diets, it was found that those diets with the least optimum balance of nutrients had the greatest inhibitory effect on growth of cancer. Rate of onset and severity of tumors was caused to vary over a 20-fold range by means of dietary balance alone. These experiments suggest that dietary variation in general and intentional malnutrition in particular should be given special attention in the control of existing cancer in humans.

A prospective evaluation of successful terminal ileum intubation during routine colonoscopy.

This study prospectively evaluated a dedicated effort to intubate the terminal ileum in an unselected population of patients undergoing colonoscopy in conjunction with our training program. The influence of the visual appearance of the terminal ileal mucosa on patient management was assessed subjectively. The cecum was reached in 270/295 (91%) patients, with successful terminal ileal intubation being accomplished in 213/270 (79%) examinations. The time required to intubate the terminal ileum (x = 3.4 minutes; range, 30 seconds to 10 minutes) was not significantly different (p < 0.05) between the several levels of training in our fellowship program. The terminal ileum was considered to be visually abnormal in 4 cases, but only 1 of these was abnormal on histologic examination. In this unselected population, routine ileoscopy was unrewarding for finding an incidental positive diagnosis; however, brief attempts at terminal ileal intubation may be useful to improve or maintain endoscopic skills, especially in a training setting.

Cultural inroads for health service to the African American elderly.

Cultural factors determine how African American elderly perceive and use health care programs. This paper looks at some of these factors and make suggestions and recommendations to health care workers.

Distribution of glutamine and asparagine residues and their near neighbors in peptides and proteins.

In a statistical study of neighboring residues in 1465 peptides and proteins comprising 450,431 residues, it was found that the preferences for residues neighboring to glutamine and asparagine residues are consistent with the hypothesis that the rates of deamidation of these residues are of biological significance. Some dipeptide and tripeptide structures have special usefulness and some are especially undesirable. More such structures exist for amide residues than for other residues, and their specific types are those most relevant to the deamidation of amide residues under biological conditions.

Quantitative measurement of human physiological age by profiling of body fluids and pattern recognition.

Quantitative correlations with human age are demonstrated for 60 substances from a group of 200 substances measured in the urine of 235 men. Simplified pattern recognition calculations are used to combine these correlations into patterns of human age and to demonstrate their utility for the quantitative measurement of human physiological age and aging rate. The empirical use of these techniques for the extension of human life-span and diminution of human suffering from degenerative diseases is discussed. Current experimental limitations of this method are demonstrated and evaluated. The application of these techniques can form the basis for a significant advance in the quality of human life.

Identification of compounds in mouse urine vapor by gas chromatography and mass spectrometry.

Eighty compounds in normal mouse urine vapor are identified, 61 of which have not previously been reported in normal mouse urine. Two of the ten most strongly age-correlated substances in the mouse urine vapor are confirmed as 3-hydroxy-3-methylbutene and trans-3-hepten-2-one.

Molecular clocks, molecular profiles, and optimum diets: three approaches to the problem of aging.

It has been hypothesized that the deamidation of glutaminyl and asparaginyl residues serves as a molecular clock for many biological processes including protein turnover, development, and aging. At present, this hypothesis has passed some experimental tests which are necessary but not sufficient for its acceptance. The current state of evidence about deamidation as a molecular clock is discussed. In addition, since the molecular biology of aging, especially in humans, is only partly understood, it is of value to develop quantitative, empirical measures of physiological human age and to use these measures to evaluate alternative human living conditions, especially easily adopted alternatives like variations in diet. This may allow some decrease in the suffering and loss from human aging until such time as molecular biology provides superior and more intellectually satisfying answers. An empirical system which consists of quantitative measurement of several hundred human chemical constituents followed by computerized pattern recognition is described. It is hoped that this system will eventually become an aid in the minimization of the rate of human aging through changes in diet and other factors.

Degradation of encephalitogenic protein in aerobic ascorbic acid solutions.

Aerobic ascorbic acid solutions are capable of extensively cleaving the peptide chain of the myelin basic protein. Cleavage occurred most readily with C-terminal to aspartic acid, serine, threonine, glutamic acid and leucine residues.