Background: Dipeptidyl-peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis. Results: DPP5, which is specific for Ala and hydrophobic residues, is expressed in the periplasmic space of P. gingivalis. Conclusion: DPP5 was discovered in prokaryotes for the first time. Significance: The discovery of DPP5 expands understanding of amino acid and energy metabolism in prokaryotes.
Porphyromonas gingivalis, an asaccharolytic gram-negative rod-shaped bacterium, expresses the novel Asp/Glu-specific dipeptidyl-peptidase (DPP) 11 (Ohara-Nemoto, Y. et al., (2011) J. Biol. Chem. 286, 38115-38127), which has been categorized as a member of the S46/DPP7 family that is preferential for hydrophobic residues at the P1 position. From that finding, 129 gene products constituting five clusters from the phylum Bacteroidetes have been newly annotated to either DPP7 or DPP11, whereas the remaining 135 members, mainly from the largest phylum Proteobacteria, have yet to be assigned. In this study, the substrate specificities of the five clusters and an unassigned group were determined with recombinant DPPs from typical species, i.e., P. gingivalis, Capnocytophaga gingivalis, Flavobacterium psychrophilum, Bacteroides fragilis, Bacteroides vulgatus, and Shewanella putrefaciens.Consequently, clusters 1, 3, and 5 were found to be DPP7 with rather broad substrate specificity, and clusters 2 and 4 were DPP11. An unassigned S. putrefaciens DPP carrying Ser 673 exhibited Asp/Glu-specificity more preferable to Glu, in contrast to the Asp preference of DPP11 with Arg 673 from Bacteroidetes species. Mutagenesis experiments revealed that Arg 673 /Ser 673 were indispensable for the Asp/Glu-specificity of DPP11, and that the broad specificity of DPP7 was mediated by Gly 673 . Taken together with the distribution of the two genes, all 264 members of the S46 family could be attributed to either DPP7 or DPP11 by an amino acid at position 673. A more compelling phylogenic tree based on the conserved C-terminal region suggested two gene duplication events in the phylum Bacteroidetes, one causing the development of DPP7 and DPP11 with altered substrate specificities, and the other producing an additional DPP7 in the genus Bacteroides.
Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with kcat/Km of 10.62 ± 2.51 μM−1 s−1, while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2- as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1- and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing kcat and lowering Km values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2- and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria.
Background:The optimal effective and safe dose of alloxan for inducing stable diabetes in rats has been long arguable. Lower doses can result in auto-reversion from diabetogenic state to normal state. On the other hand, higher doses cause toxicity and loss of experimental animals since it completely destroys the insulin-producing pancreatic β cells. Therefore, determination of effective as well as safe dose of alloxan to induce stable diabetes in experimental Long Evan rats is crucial for investigating on diabetes. Objective: To determine effective and safe dose of alloxan for inducing a stable diabetes mellitus in Long Evans Rats to facilitate availability of diabetic rats for studying on diabetes. Methods: Healthy adult Long Evans rats (80-156 g) were divided into eight groups and each group contains six rats. One group treated as a nondiabetic control (C) while the other seven groups (Group-2 to Group-8) were used as experimental diabetic groups. Different doses of alloxan (80, 100, 120, 130, 140, 150 and 160 mg/kg body weight) were injected through intraperitoneal routes in overnight fasting group-2 to group-8 rats respectively. Blood glucose levels were monitored before and after administering alloaxan dose (for 7 consecutive weeks) by using a blood glucose meter and test strips. Results: In the case of non-diabetic rats, the normal blood glucose levels were found between 7.75 to 10.8 mmol/L. On the other hand, a slow gradual increment of blood glucose levels were measured among the rats (group-2, group-3 and group-4) treated with low doses of alloxan (80, 100 and 120 mg/kg BW) until 5 weeks. However, the increased blood glucose levels were reverted back to its normal by the following two weeks. Group-5 and Group-6 rats treated with alloxan doses of 130 mg/kg and 140 mg/kg respectively stably increased blood glucose levels during the experimental periods. While rats in group-7 and group-8 (treated with 150 and 160 mg/kg BW) were expired by the experimental period might be due to severe alloxan toxicity. Group-6 rats treated with 140 mg/kg alloxan were more preferable because the diabetic level is much more stable and significant than group-5. Conclusion: The optimum effective dose of alloxan was found to be 140 mg/kg BW for induction of stable diabetes in Long Evan rats.
The modern trend of consuming mustard oil in raw, fried and cooked forms is increasing day by day in the general people irrespective of economic status in Bangladesh. But unfortunately they are not aware of the adverse effects of this oil. Because, mustard oils have been evidenced to have high level of erucic acid and erucic acid has been evidenced to be a causative agent for cardiovascular diseases. It is therefore very important to know the erucic acid level both in traditional and commercially available mustard oils in Bangladesh. The aim of the study was to investigate the fatty acid profile of the commercial mustard oil (Industrially manufactured) and ghani (traditional method of oil processing) mustard oil. After preparation of the purified fatty acid methyl esters of the above oils, Gas-Liquid Chromatographic (GLC) analysis was carried out. Results showed that the percentage of erucic acid (22:1) in the commercial and ghani mustard oils were 41.80% and 51.98% respectively. In context to percentage of erucic acid, the commercial mustard oil seemed better compared to ghani. The reasons behind this discrepancy still remains unclear but the possibility of mixed oil effects in the commercial mustard oil could be investigated in the near future. The author also established a method to partially eliminate erucic acid from mustard oil. After partially eliminate erucic acid from commercial mustard oil erucic acid reduces from 41.80% to 20.14%.
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