Primary structure of an alkaline ribonuclease from bovine liver

A pyrimidine base specific and most basic alkaline RNase named RNase BL4 was isolated from bovine liver as a protein showing a single band on slab gel-electrophoresis. The enzyme is most active at pH 7.5. The enzyme was immunologically distinguishable from the known bovine RNases such as pancreatic RNase (RNase A), seminal RNase, kidney non-secretory RNase (RNase K2), and brain RNase (RNase BRb). The primary structure of this pyrimidine base-specific RNase was determined to be less than EDRMYQRFLRQHVDPDETG- GNDSYCNLMMQRRKMTSHQCKRFNTFIHEDLWNIRSICSTTNIQCKNGQMNCHEGVVRV- TDCRETGSSRAPNCRYRAKASTRRVVIACEGNPEVPVHFDK. It consists of 119 amino acid residues, and is 5 amino acid residues shorter than RNase A. The sequence homology of RNase BL4 with RNase A is 46.2%, and optimal alignment of RNase A and RNase BL4 requires five deletions, one at the 24th position, two at the 75th and 76th positions, and two at the C-terminus in RNase BL4. The RNase BL4 was highly homologous with a porcine liver RNase (RNase PL3, 94.1% homology) studied by Hofsteenge et al. (personal communication from Hofsteenge, J., Matthies, R., and Stones, S.R.).     

Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus.

The crystal structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.5 A resolution by the multiple isomorphous replacement method. The crystal structure was refined by simulated annealing with molecular dynamics. The current crystallographic R-factor is 0.200 in the 10-2.5 A resolution range. The molecular structure which is completely different from the known structures of RNase A and RNase T1 consists of six alpha-helices and seven beta-strands, belonging to the alpha+beta type structure. Two histidine and one glutamic acid residues which were predicted as the most probably functional residues by chemical modification studies are found to be clustered. The steric nature of the active site taken together with the relevant site-directed mutagenesis experiments (Irie et al.) indicates that: (i) the two histidine residues are the general acid and base; and (ii) an aspartic acid residue plays a role of recognizing adenine moiety of the substrate.     

Computational studies on the water‐catalyzed stereoinversion mechanism of glutamic acid residues in peptides and proteins

In contrast with the common belief that all the amino acid residues in higher organisms are l ‐forms, d ‐amino acid residues have been recently detected in various aging tissues. Aspartic acid (Asp) residues are known to be the most prone to stereoinvert via cyclic imide intermediate. Although the glutamic acid (Glu) is similar in chemical structure to Asp, little has been reported to detect d ‐Glu residues in human proteins. In this study, we investigated the mechanism of the Glu‐residue stereoinversion catalyzed by water molecules using B3LYP/6‐31+G(d,p) density functional theory calculations. We propose that the Glu‐residue stereoinversion proceeds via a cyclic imide intermediate, i.e., glutarimide (GI). All calculations were performed by using a model compound in which a Glu residue was capped with acetyl and methylamino groups on the N‐ and C‐termini, respectively. We found that two water molecules catalyze the three steps involved in the GI formation: iminolization, cyclization, and dehydration. The activation energy required for the Glu residue to form a GI intermediate was estimated to be 32.3 kcal mol^?1, which was higher than that of the experimental Asp‐residue stereoinversion. This calculation result suggests that the Glu‐residue stereoinversion is not favored under the physiological condition.     

Effects and mechanisms of prolongevity induced by Lactobacillus gasseri SBT2055 in Caenorhabditis elegans

Lactic‐acid bacteria are widely recognized beneficial host associated groups of the microbiota of humans and animals. Some lactic‐acid bacteria have the ability to extend the lifespan of the model animals. The mechanisms behind the probiotic effects of bacteria are not entirely understood. Recently, we reported the benefit effects of Lactobacillus gasseriSBT2055 (LG2055) on animal and human health, such as preventing influenza A virus, and augmentation of IgA production. Therefore, it was preconceived that LG2055 has the beneficial effects on longevity and/or aging. We examined the effects of LG2055 on lifespan and aging of Caenorhabditis elegans and analyzed the mechanism of prolongevity. Our results demonstrated that LG2055 has the beneficial effects on longevity and anti‐aging of C. elegans. Feeding with LG2055 upregulated the expression of the skn‐1 gene and the target genes of SKN‐1, encoding the antioxidant proteins enhancing antioxidant defense responses. We found that feeding with LG2055 directly activated SKN‐1 activity via p38 MAPK pathway signaling. The oxidative stress response is elicited by mitochondrial dysfunction in aging, and we examined the influence of LG2055 feeding on the membrane potential of mitochondria. Here, the amounts of mitochondria were significantly increased by LG2055 feeding in comparison with the control. Our result suggests that feeding with LG2055 is effective to the extend lifespan in C. elegans by a strengthening of the resistance to oxidative stress and by stimulating the innate immune response signaling including p38MAPK signaling pathway and others.     

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.     

Primary structure of a non-secretory ribonuclease from bovine kidney

The primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRHNCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pancreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).     

Primary structure of a ribonuclease from bovine brain

The primary structure of a pyrimidine base-specific ribonuclease from bovine brain was determined. The sequence determined is (sequence; see text). Although the sequence homology of this RNase with bovine pancreatic RNase A is 78.2%, it consists of 140 amino acid residues, and it is 16 amino acid residues longer than RNase A at the carboxyl-terminal. In addition to an N-glycosylated long carbohydrate chain, the bovine brain RNase has two short O-glycosylated carbohydrate chains at the 129th and the 133rd serine residues. The additional C-terminal tail of the bovine brain RNase has a unique composition: 6 proline, 5 hydrophobic amino acids, and two basic amino acids, arginine and histidine.     

Variations in the light-induced suppression of nocturnal melatonin with special reference to variations in the pupillary light reflex in humans

The purpose of the present study was to elucidate the existence of individual differences of pupil response to light stimulation, and to confirm the reproducibility of this phenomenon. Furthermore, the relationship between the individual differences in nocturnal melatonin suppression induced by lighting and the individual differences of pupillary light response (PLR) was examined. The pupil diameter and salivary melatonin content of 20 male students were measured at the same period of time (00:00-02:30 hr) on different days, accordingly. Illumination (530 nm) produced by a monochromatic light-emitting diode (LED) was employed as the light stimulation: pupil diameter was measured with 4 different levels of illuminance of 1, 3, 30 and 600 lux and melatonin levels were measured at 30 and 600 lux (respective controls were taken at 0 lux). Oral temperature, blood pressure and subjective index of sleepiness were taken in experiments where melatonin levels were measured. Changes of the pupil diameter in response to light were expressed as PLR and light-induced melatonin suppression was expressed as a control-adjusted melatonin suppression score (control-adjusted MSS), which was compared to the melatonin level measured at 0 lux. In the PLR, the coefficients of variation obtained at 30 lux or less were large (51.5, 45.0, 28.4 and 6.2% at 1, 3, 30 and 600 lux, respectively). Correlations of illuminance of any combination at 30 lux or less were statistically significant at less than 1% level (1 vs. 3 lux: r=0.68; 1 vs. 30 lux: r=0.64; 3 vs. 30 lux: r=0.73), which showed the reproducibility of individual differences. The control-adjusted MSS at 600 lux (-1.14+/-1.16) was significantly (p<0.05) lower than that registered at 30 lux (-0.22+/-2.12). PLR values measured at 30 and 600 lux were then correlated with control-adjusted MSS; neither indicated a significant linear relationship. However, the control-adjusted MSS showed around 0 under any of the illuminance conditions in subjects with high PLR. In control-adjusted MSS of low values (i.e., melatonin secretions were easily suppressed), subjects indicated typically low PLR. In subjects with low control-adjusted MSS (n=3), characteristic changes in the autonomic nervous system, such as body temperature and blood pressure, were noted in subjects exposed to low illuminance of 30 lux. The fact that the relationship between PLR and control-adjusted MSS portray a similar pattern even under different luminance conditions suggests that MSS may not be affected in those with high PLR at low illuminance, regardless of the illuminance condition.     

Role of AKT in cyclic strain-induced endothelial cell proliferation and survival

Endothelial cells (ECs) are exposed to repetitive cyclic strain (CS) in vivo by the beating heart. The aim of this study was to assess the influence of CS amplitude and/or frequency on EC proliferation and survival and to determine the role of AKT in CS-induced EC proliferation and survival. Cultured bovine aortic ECs were exposed to 10% strain at a frequency of 60 (60 cpm-10%) or 100 (100 cpm-10%) cycles/min or 15.6% strain at a frequency of 60 cycles/min (60 cpm-15.6%). AKT, glycogen synthase kinase (GSK)-3, BAD, and cleaved caspase-3 were activated by CS in ECs. Increasing the magnitude or frequency of strain resulted in an earlier phosphorylation of GSK-3, although the magnitude of phosphorylation was similar. After CS at 60 cpm-10% for 24 h, the number of nontransfected ECs was significantly increased by 8.5% (P < 0.05). We found that the number of apoptotic ECs was slightly decreased with exposure to CS. ECs transfected with kinase-dead AKT (KA179) as well as plasmids containing a point mutation in the pleckstrin homology domain of AKT (RC25) not only prevented AKT, GSK-3, and BAD phosphorylation but also inhibited the CS-induced increase in cell number as well as the CS-induced protection against apoptosis (both P < 0.05). The ratio of 5'-bromo-2'-deoxyuridine-positive cells was increased when ECs transfected with RC25 and KA179 as well as nontransfected ECs and ECs transfected with Lipofectamine 2000 were exposed to CS. We conclude that AKT is important in enhancing the survival of ECs exposed to CS but is not involved in EC proliferation. apoptosis; glycogen synthase kinase