Collapse of homochirality of amino acids in proteins from various tissues during aging.

Prior to the emergence of life, it is believed that only L-amino acids were selected for formation of proteins, and that D-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, recently the occurrence of proteins containing D-beta-aspartyl (Asp) residues from various tissues of elderly subjects has been reported. Here, we discuss the presence of D-beta-Asp-containing proteins in the lens, ciliary body, drusen, and sclera of the eye, skin, cardiac muscle, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscles of the stomach, and small and large intestines. Since the D-beta-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. UV Rays and oxidative stress can accelerate the formation of the D-beta-Asp residue in proteins.

Differential rate constants of racemization of aspartyl and asparaginyl residues in human alpha A-crystallin mutants.

Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized to d-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.

[A case of primary pulmonary nocardiosis with multiple pulmonary nodules successfully treated with moxifloxacin].

A 18-year-old man was admitted with fever. His chest radiograph and CT scan showed consolidation shadow in the right middle lobe and multiple nodules in both lungs. He was treated with meropenem and minocycline. After this antibiotic therapy, the consolidation shadow disappeared and the multiple nodules were slightly reduced in their size. Since filamentous bacteria suspicious of Nocardia grew transiently in the initial sputum culture, we started to treat him with oral sulfametoxazole-trimethoprim. However, because agranulocytosis was caused by sulfametoxazole-trimethoprim therapy, we had to change the anti-bacterial therapy to minocycline. Minocycline was not effective, and the nodules enlarged. For accurate diagnosis, we employed video-assisted thoracic surgery (VATS) to investigate the histological and bacterial analyses of the pulmonary nodules. Histological findings of the pulmonary nodule obtained by VATS revealed granuloma with central necrosis associated with neutrophilic micro-abscess. Filamentous gram-positive bacteria in pulmonary nodule tissue was stained positively with both Grocott and Ziehl-Neelsen staining. Taking these findings together, we diagnosed primary pulmonary nocardiosis. Three months after initiating moxifloxacin, the size of the multiple pulmonary nodules was markedly reduced. Our experience with this case suggests that moxifloxacin can be recommended for the treatment of pulmonary nocardiosis.

Differential susceptibility of alpha A- and alpha B-crystallin to gamma-ray irradiation.

Alpha-crystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin.

Increase in random component of heart rate variability coinciding with developmental and degenerative stages of life.

OBJECTIVE: To adapt to a new environment or situation, biological systems explore the most convenient state while moving between attractors by the force of random fluctuation. From this concept, the random component in physiological signals is assumed to increase during developmental and degenerative stages of life. To examine this hypothesis, we measured the age-dependent changes in the random component of heart rate variability (HRV) in 24-h electrocardiography (ECG) big data and in patients with a developmental disorder (DD). APPROACH: We measured separately regulated and random components of HRV with autoregressive (AR) model fitting, by which the ratio of random component as the fractional variance of AR residual time series. From the ALLSTAR database of about 304 000 ambulatory 24-h ECGs, we randomly extracted the data of 1930 men and 1987 women uniformly for all ages from 0 to 100 years old (100 cases per 5-year strata for each sex). Data were also obtained from male pediatric patients with DD (age 10-15 years). MAIN RESULTS: While the variance of the regulated component of HRV increased from age 0 to 20, decreased with age until 40, and reached a plateau in both sexes, the ratio of the random component was high at birth, decreased with age until 35 in men and 30 in women, and increased again after 75 in men and 85 in women (P < 0.0001 for all). In patients with a DD, the ratio of the random component was significantly lower than that in age-and-sex matched subjects in the database. SIGNIFICANCE: We found that the ratio of the random component of HRV is increased during developmental and degenerative stages of life and that it may be reduced in DD patients during their development.

Kinetic study of racemization of aspartyl residues in recombinant human alphaA-crystallin.

Asp58 and Asp151 in human lens alphaA-crystallin invert and isomerize to d-beta-aspartyl residues with age. Here, we report that the racemization rate constants (k) of Asp58 and Asp151 residues in human recombinant alphaA-crystallin at 37 degrees C are 3.72 +/- 0.8 x 10(-4) and 10.7 +/- 0.7 x 10(-4)/day, respectively. The activation energy of racemization of Asp58 and Asp151 in the protein was 27.0 +/- 0.5 kcal/mol and 21.0 +/- 0.5 kcal/mol, respectively. The time required for the D/L ratio of Asp58 and Asp151 to approximate to 1.0 (D/L ratio of Asp = 0.99) at 37 degrees C was estimated as 20.9 +/- 3.7 and 6.80 +/- 0.4 years, respectively. Thus, Asp151 is more susceptible to racemization than Asp58, consistent with data from short model peptides. However, the racemization rates of both Asp58 and Asp151 residues in the protein were twice as rapid as in model peptides. These results indicate that the racemization of Asp residues in alphaA-crystallin may be influenced not only by the primary structure but also by the higher order structure around Asp residues in the protein.