Functional tyrosine residue in the active center of human dipeptidyl peptidase III

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.     

Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin

Background and Aims

In the genus Anemone two small groups of taxa occur with the highest ploidy levels 2n = 6x = 48, belonging to the closely related clades: the montane/alpine Baldensis clade and the more temperate Multifida clade. To understand the formation of polyploids within these groups, the evolution of allohexaploid A. baldensis (AABBDD, 2n = 6x = 48) from Europe and allotetraploid Anemone multifida (BBDD, 2n = 4x = 32) from America was analysed.

Methods

Internal transcribed spacer and non-transcribed spacer sequences were used as molecular markers for phylogenetic analyses. Cytogenetic studies, including genomic in situ hybridization with genomic DNA of potential parental species as probe, fluorescence in situ hybridization with 5S and 18S rDNA as probes and 18S rDNA restriction analyses, were used to identify the parental origin of chromosomes and to study genomic changes following polyploidization.

Key Results

This study shows that A. multifida (BBDD, 2n= 4x = 32) and A. baldensis (AABBDD, 2n = 6x = 48) are allopolyploids originating from the crosses of diploid members of the Multifida (donor of the A and B subgenomes) and Baldensis groups (donor of the D subgenome). The A and B subgenomes are closely related to the genomes of A. sylvestris, A. virginiana and A. cylindrica, indicating that these species or their progeny might be the ancestral donors of the B subgenome of A. multifida and A and B subgenomes of A. baldensis. Both polyploids have undergone genomic changes such as interchromosomal translocation affecting B and D subgenomes and changes at rDNA sites. Anemone multifida has lost the 35S rDNA loci characteristic of the maternal donor (B subgenome) and maintained only the rDNA loci of the paternal donor (D subgenome).

Conclusions

It is proposed that A. multifida and A. baldensis probably had a common ancestor and their evolution was facilitated by vegetation changes during the Quaternary, resulting in their present disjunctive distribution.     

Environment-related variations of the composition of the essential oils of rosemary (Rosmarinus officinalis L.) in the Balkan Penninsula

Composition of the essential oils of Rosmarinus officinalis of ten populations from the Balkan Peninsula were determined by GC/FID and GC/MS. The main constituents were 1,8-cineole, camphor, α-pinene, and borneol. Multivariate statistical analysis (UPGMA cluster analysis and principal-component analysis (PCA)) revealed two major types of rosemary oil, i.e., 1,8-cineole and camphor-type, and two intermediate types, i.e., camphor/1,8-cineole/borneol type and 1,8-cineole/camphor type. The regression analyses (simple linear regression and stepwise multiple regression) have shown that, with respect to basic geographic, orographic, and 19 bioclimatic characteristics of each population, bioclimatic factor temperature of habitat represented the dominant abiogenetic factor, which, in chemical sense, led to differentiation of populations in the studied region. Also, the regression analysis have shown that some constituents of essential oils are independent of any single bioclimatic factors. However, some constituents display statistically significant correlations with some abiotic factors.     

Body mass index, waist circumference and waist-to-hip ratio: which anthropometric indicator is better predictor for the hypertension development in women population of the island Cres

The aim of this research was to investigate the prevalence of obesity and high blood pressure and to prove which of three anthropometric indicators of obesity - waist circumference, body mass index (BMI) waist-to-hip ratio - is better predictor for the development of hypertension in women population of the island of Cres. We approached separately groups of women with measured high blood pressure and with previously diagnosed. The research was preformed within the research project "Genetic and biomedical characteristics of the population of the island of Cres". This was the cross sectional study and data were obtained on the sample of 247 females over 18 years old that voluntarily participated in this study. In our study group the prevalence of overweight was 39.0%, obesity 27.5%, increased waist circumference was present in 69.4% while increased blood pressure was found in 53.0% examinees. Our results indicate that age, BMI, impaired glucose concentration and serum cholesterol could be considered as predictors for the development of arterial hypertension, whether measured or previously diagnosed.     

1,2,3,4-Tetrahydroisoquinoline Derivatives as a Novel Deoxyribonuclease I Inhibitors

Herein we report an assessment of 24 1,2,3,4-tetrahydroisoquinoline derivatives for potential DNase I (deoxyribonuclease I) inhibitory properties in vitro. Four of them inhibited DNase I with IC₅₀ values below 200 μM. The most potent was 1-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)propan-2-one ( 2 ) (IC₅₀=134.35±11.38 μM) exhibiting slightly better IC₅₀ value compared to three other active compounds, 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]-1-phenylethan-1-one ( 15 ) (IC₅₀=147.51±14.87 μM), 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one ( 18 ) (IC₅₀=149.07±2.98 μM) and 2-[6,7-dimethoxy-2-(p-tolyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one ( 22 ) (IC₅₀=148.31±2.96 μM). Cytotoxicity assessment of the active DNase I inhibitors revealed a lack of toxic effects on the healthy cell lines MRC-5. Molecular docking and molecular dynamics simulations suggest that interactions with Glu 39, His 134, Asn 170, Tyr 211, Asp 251 and His 252 are an important factor for inhibitors affinity toward the DNase I. Observed interactions would be beneficial for the discovery of new active 1,2,3,4-tetrahydroisoquinoline-based inhibitors of DNase I, but might also encourage researchers to further explore and utilize potential therapeutic application of DNase I inhibitors, based on a versatile role of DNase I during apoptotic cell death.     

The first structure of dipeptidyl-peptidase III provides insight into the catalytic mechanism and mode of substrate binding

Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-A crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.     

Effects of clonidine preemptive analgesia on acute postoperative pain in abdominal surgery

Preemptive analgesia refers to blockade of afferent nerve fibers before a painful stimulus, which prevents or reduces subsequent pain even beyond the effect of the block. The aim of the study was to compare the effect of clonidine used before and at the end of operation on pain control in abdominal surgery. A total of 77 patients admitted for colorectal surgery were randomly classified into three groups: epidural clonidine before operation, epidural clonidine at the end of operation, and control group. After the operation on patient demand, analgesia with boluses of epidural morphine was instituted. The parameters of postoperative pain level using VAS score (visual analog scale), sedation and analgesics consumption were determined as outcome measures at 1, 2, 6, and 24 h of the operation. Clonidine administered before operation provided lowest pain scores at 6 and 24 h (p < 0.05). Clonidine administered at the end of operation had low pain scores at 1 and 2 h, with a significant pain breakthrough thereafter (6.93 +/- 1.66 at 6 h and 4.04 +/- 2.39 at 24 h) compared with the group administered clonidine before operation (3.60 +/- 2.94 and 3.71 +/- 1.82). Clonidine administered before operation provided less sedation (p < 0.05) and a significantly lower use of analgesics (p < 0.05). Blockade of nociceptive stimulus using the centrally acting alpha2-adrenergic agonist clonidine before the onset of pain stimulus resulted in reduced pain levels, sedation and analgesic requirement.