Clinical value of rapid urine trypsinogen-2 test strip, urinary trypsinogen activation peptide, and serum and urinary activation peptide of carboxypeptidase B in acute pancreatitis

AIM: To assess the usefulness of urinary trypsinogen-2 test strip, urinary trypsinogen activation peptide (TAP),and serum and urine concentrations of the activation peptide of carboxypeptidase B (CAPAP) in the diagnosisof acute pancreatitis.METHODS: Patients with acute abdominal pain and hospitalized within 24 h after the onset of symptoms were prospectively studied. Urinary trypsinogen-2 was considered positive when a clear blue line was observed (detection limit 50 μg/L). Urinary TAP was measured using a quantitative solid-phase ELISA, and serum and urinary CAPAP by a radioimmunoassay method.RESULTS: Acute abdominal pain was due to acute pancreatitis in 50 patients and turned out to be extrapancreatic in origin in 22 patients. Patients with acute pancreatitis showed significantly higher median levels of serum and urinary CAPAP levels, as well as amylase and lipase than extrapancreatic controls. Median TAP levels were similar in both groups. The urinary trypsinogen-2 test strip was positive in 68% of patients with acute pancreatitis and 13.6% in extrapancreatic controls (P<0.01). Urinary CAPAP was the most reliable test for the diagnosis of acute pancreatitis (sensitivity 66.7%, specificity 95.5%, positive and negative predictive values 96.6% and 56.7%, respectively), with a 14.6 positive likelihood ratio for a cut-off value of 2.32 nmol/L.CONCLUSION: In patients with acute abdominal pain,hospitalized within 24 h of symptom onset, CAPAP in serum and urine was a reliable diagnostic marker of acute pancreatitis. Urinary trypsinogen-2 test strip showed a clinical value similar to amylase and lipase.Urinary TAP was not a useful screening test for the diagnosis of acute pancreatitis.

Carboxypeptidase A6 Promotes the Proliferation and Migration of Hepatocellular Carcinoma by Up-regulating AKT Signaling Pathway

Summary:Hepatocellular carcinoma(HCC)has a poor treatment prognosis and high mortality worldwide.Understanding the molecular mechanism underlying HCC development would benefit the identification of diagnostic biomarkers and the improvement of the treatment strategies.The expression of carboxypeptidase A6(CPA6)has been reported in epilepsy and febrile seizures rather than in any type of cancers.However,the function of CPA6 expression in HCC is not yet understood.In this study,we aimed to investigate the clinicopathological significance of the expression of CPA6 in HCC and the underlying mechanisms.We observed that the expression of the CPA6 protein was increased significantly in HCC tissues than in paracancerous tissues.To explore its function in HCC,both gain-and loss-of-function studies demonstrated that CPA6 played a vital role in promoting HCC growth and metastasis.When knocking down CPA6 with shRNA,HCC cell proliferation and migration could be suppressed.Meanwhile,CPA6 overexpression could promote proliferation and migration of HLF cells.Moreover,CPA6 could activate AKT serine/threonine kinase(AKT)signaling pathway as confirmed by Western blotting.In conclusion,our study revealed that CPA6 could promote HCC cell proliferation and migration via AKT-mediated signaling pathway.These findings suggest that CPA6 is a promising diagnostic biomarker and therapeutic target to improve the prognosis of HCC.

Optical 2-benzyl-5-hydroxy-4-oxopentanoic acids against carboxypeptidase A:Synthesis,kinetic evaluation and X-ray crystallographic study

2-Benzyl-5-hydroxy-4-oxopentanoic acid 1 and its enantiomers were designed,synthesized and assayed for inhibitory activity against carboxypeptidase A(CPA,EC 3.4.17.1).To verify the role of the terminal hydroxyl group in 1 binding to CPA,2-benzyl-5- benzyloxy-4-oxopentanoic acid 2 was also synthesized and evaluated.The inhibition constants show that both L-1 and D-1 were shown to have strong binding affinity with L-1 being more potent than its enantiomer by 165-fold.On the other hand,the inhibition constant ...

Comparison of Substrate Specificity of <em>Escherichia Coli</em>p-Aminobenzoyl-Glutamate Hy-drolase with <em>Pseudomonas</em>Carboxypeptidase G

Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/μg). Relative to its activity with 10 μM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in treatment of chemotherapy overdose.

Cloning and Expression of the Serine Carboxypeptidase Gene in Zea mays and Its Antifungal Activity against Rhizoctonia solani

The authors cloned and identified a new maize serine carboxypeptidase gene named ZmSCP from R15 inbred lines seedlings which were induced by Rhizoctonia solani AGI-IA. ZmSCP encodes a 332 amino acid protein with a predicted molecular mass of 36.5 kDa and pI of 4.75. Phylogenetic analysis revealed that ZmSCP showed closer kinship with Oryza sativa and sorghum, which belong to the same evolutionary branch. Amino acid sequence analysis revealed that there are four types of amino acids in ZmSCP, the percentages of them are 43.1%, 26.9%, 13.9% and 13.1%. The authors subsequently purified the recombinant protein which expressed in Escherichia coli BL21 and analyzed its antimicrobial activities in vitro. Results showed that the recombinant protein inhibited hyphal growth of Rhizoctonia solani. The study suggests that the expression of ZmSCP is closely related to maize sheath blight resistance caused by Rhizoctonia solani. Further, the antifungal activity showed that ZmSCP may play at role in the disease resistance response.

Crystal Structure of Carboxypeptidase a Complexed with an Inactivator in a New Crystalline Form

A new crystalline form of carboxypeptidase A (CPA) complexes withan inactivator was obtained by the method of hanging drop vapor diffusion. The inacti-’vator, 2-benzyl-3-iodo-propanoic acid (BIPA ), binds covalently to an active siteresidue Glu-27O of CPA. The complex was crystallized in space group P212121 with a= 48. 8 A, b=66. 9 A, c= 96. 0 A. The complex structure was determined by molecu-lar replacement using the native CPA crystal structure as the search model. The finalcrystallographic residual is 0. 152. Except for the modification of Glu-270, the inactiva-tor exhibits normal binding mode compared with other ligand complexes of CPA. Inthe final different electron density maps (2Fo-Fc, Fo-Fc), the density of the iodo ioncould not be found while the conserved molecule remains coordinated to Zn2+ as in thenative CPA. Comparisons of complex of CPA-BIPA with the native CPA and theCPA/D-Phe complex are presented. The mechanism of inactivation of CPA was dis-cussed.

Carboxypeptidase E (NF-α1):a new trophic factor in neuroprotection

Carboxypeptidase E （CPE） is a prohormone-processing enzyme and sorting receptor that functions intracellularly. However, recent studies have demonstrated that CPE acts as atrophic factor extracellularly to up-regulate the expression of a pro-survival gene. This mini-review summarizes the roles of CPE in neuroprotection and the implications for neurodegenerative diseases.

Abrogation of immune complex glomerulonephritis by native carboxypeptidase and pharmacological antagonism of the C5a receptor

Activation of complement generates C5a which leads to signaling through C5aR 1. This is tightly controlled, including by the plasma proteins factor H （FH） and carboxypeptidase N. Here we studied a chronic serum sickness （CSS） model of glomerulonephritis （GN） in which there is an active humoral immune response, formation of glomerular immune complexes （ICs）, and resulting glomerular inflammation. The antibody response, glomerular IC deposition, the degree of GN, and consequent renal functional insufficiency in CSS were all worse in FH-/- mice compared to wild-type FH＋/＋ animals. This was ameliorated in the former by giving a C5aR1 antagonist for the final 3 weeks of the 5-week protocol. In contrast, blocking CP-mediated inactivation of C5a increased these disease measures. Thus, complement regulation by both plasma FH and CP to limit the quantity of active C5a is important in conditions where the humoral immune response is directed to a continuously present foreign antigen. Signaling through C5aR1 enhances the humoral immune response as well as the inflammatory response to ICs that have formed in glomeruli. Both effects are relevant even after disease has begun. Thus, pharmacological targeting of C5a in IC-mediated GN has potential clinical relevance.

Viral and cellular determinants involved in hepadnaviral entry

Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus （HBV） constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses （e.g. HBV） and avihepadnaviruses （e.g. duck hepatitis B virus [DHBV]）. Two important discoveries have been made recently regarding HBV： （1） primary hepatoo/tes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and （2） a human hepatoma cell line （HepaRG） was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D （CPD） has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes （PDH）, investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to： （1） functionally dissect the hepadnaviral entry pathways, （2） perform reverse genetics （e.g. test the fitness of escape mutants）, （3） titrate and map neutralizing antibodies, （4） improve current vaccines to combat acute and chronic infections of hepatitis B, and （5） develop entry inhibitors for future clinical applications.

Early dynamic transcriptomic changes during preoperative radiotherapy in patients with rectal cancer: A feasibility study

AIM: To develop novel biomarkers of rectal radiotherapy, we measured gene expression profiles on biopsies taken before and during preoperative radiotherapy. METHODS: Six patients presenting with a locally advanced rectal cancer (T>T2, N0/Nx, M0) eligible for preoperative radiotherapy (45 Gy in 25 fractions) were selected in a pilot study. Six tumor and 3 normal tissues biopsies were taken before and during radiotherapy,after a dose of 7.2 Gy at a median time of 1 h following irradiation (0:27-2:12). Tumor or normal tissue purity was assessed by a pathologist prior to RNA extraction. Mean RNA content was 23 μg/biopsy (14-37) before radiotherapy and 22.7 μg/biopsy (12-35) during radiotherapy. After RNA amplification, biopsies were analysed with 54K HG-U133A Plus 2.0 Affymetrix expression micro-arrays. Data were normalized according to MAS5 algorithm. A gene expression ratio was calculated as: (gene expression during radiotherapy-gene expression before radiotherapy)/gene expression before radiotherapy. Were selected genes that showed a ratio higher than ± 0.5 in all 6 patients. RESULTS: Microarray analysis showed that preoperative radiotherapy significantly up-regulated 31 genes and down-regulated 6 genes. According to the Gene Ontology project classification, these genes are involved in protein metabolism (ADAMDEC1 ; AKAP7 ; CAPN5 ; CLIC5 ; CPE ; CREB3L1 ; NEDD4L ; RAB27A), ion transport (AKAP7 ; ATP2A3 ; CCL28 ; CLIC5 ; F2RL2 ; NEDD4L ; SLC6A8), transcription (AKAP7 ; CREB3L1 ; ISX ; PAB-PC1L ; TXNIP), signal transduction (CAPN5 ; F2RL2 ; RA- B27A ; TNFRSF11A), cell adhesion (ADAMDEC1 ; PXDN ; SPON1 ; S100A2), immune response (CCL28 ; PXDN ; TNFRSF11A) and apoptosis (ITM2C ; PDCD4 ; PVT1). Up-regulation of 3 genes (CCL28 ; CLIC5 ; PDCD4) was detected by 2 different probes and up-regulation of 2 genes (RAB27A ; TXNIP) by 3 probes. CONCLUSION: Micro-arrays can efficiently assess early transcriptomic changes during preoperative radiotherapy for rectal cancer, and may help better understand tumor radioresistance.

Solid-Phase Enzymatic Peptide Synthesis to Produce an Antioxidant Dipeptide

Peptide bond synthesis is favorable to the production of bioactive small peptides. However, the abuse of toxic reagents remains an issue for chemical synthesis method, whereas the low product yield and purity limit the widespread use of enzymatic method. In this study, a new solid-phase enzymatic peptide synthesis(SPEPS) strategy was developed to produce an antioxidant tyrosine-alanine dipeptide(Tyr-Ala) by using recombinant carboxypeptidase Y(CPY) as the catalyst. The general SPEPS procedure involves three steps. First, the N-protected acyl donor was covalently attached to solid resin. Second,the peptide bond was condensed between the acyl donor and the nucleophile under the catalysis of CPY. Finally, one-step cleavage was performed to remove the protecting group and cleave the peptides from solid resin. Upon the optimization of reaction conditions, 77.92%(±2.723%) yield of Tyr-Ala with high product purity of 90.971%(±2.695%) was obtained.In addition, the antioxidant activity of Tyr-Ala was determined by ABTS method, indicating that the synthesized Tyr-Ala obtained by SPEPS showed a superior antioxidant capability compared with commercial glutathione.

The new effector AbSCP1 of foliar nematode(Aphelenchoides besseyi) is required for parasitism rice

Plant parasitic nematodes secrete effector proteins to parasitize hosts successfully. Of these proteins, serine carboxypeptidases have critical roles in pathogenicity. This study investigated the role of new effector AbSCP1 in Aphelenchoides besseyi pathogenicity. In situ hybridization and qRT-PCR analyses indicated that AbSCP1 was exclusively expressed in the esophageal glands and upregulated in juveniles. Subcellular localization assays indicated that the protein was expressed in the nucleus. The ability to hydrolyze C-terminal amino acid residues was proven for AbSCP1. Moreover, RNAi significantly reduced the expression of AbSCP1 and RNAi-treated nematodes’ reproductive potential. Pathogenicity assays on rice showed that RNAi-treated nematodes were less pathogenic than the untreated control groups. These results suggest the important role of AbSCP1 in the A. besseyi infection process.

A New Competitive Inhibitory Monoclonal Antibody to Carboxypcptidase A(CPA)

The monoclonal antibody ID11D7 to carboxypeptidase A (CPA) was prepared. Theinhibitory effect of McAb ID11D7 on the peptidase activity of CPA was measured. The result reflectsthat the McAb ID11D7 can competitively inhibit the peptidase activity of CPA with an apparentinhibitory constant of 6.3 × 10-9M. Based on this work. the McAb ID11D7 can be used as idiotypicantigen to prepare anti-idiotypic catalytic antibody with the peptidase activity similar to CPA.

A rice tubulin tyrosine ligase-like 12 protein affects the dynamic and orientation of microtubules

The detyrosination/retyrosination cycle is the most common post-translational modification of α-tubulin.Removal of the conserved C-terminal tyrosine of α-tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase(TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase-like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL-like 12 protein(Os TTLL12)andgeneratedoverexpression Os TTLL12-RFP lines in both rice and tobacco BY-2 cells. We found, unexpectedly, that overexpression of this Os TTLL12-RFP increased the relative abundance of detyrosinated α-tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of Os TTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth.

Pancreatic biomarkers:role in diabetes mellitus

Diabetes mellitus refers to a group of diseases that cause high blood sugar levels.The most common type is type 2 diabetes,which is caused by insulin resistance and inadequate insulin production.However,diabetes can also result from conditions affecting the exocrine pancreas.Both type 1 and type 2 diabetes patients may experience changes in their pancreatic exocrine function,leading to reduced levels of fecal elastase-1 in many cases.This review article focuses on the role of specific pancreatic biomarkers in diabetes mellitus,including cholecystokinin,trypsin,chymotrypsin,carboxypeptidase,amylase,lipase,secretin,elastase-1,and retinol-binding protein 4 about recent advances and discoveries,significant gaps in the literature,current debates,and potential directions for future research related to these biomarkers about diabetes mellitus.This review article discusses various biomarkers related to pancreatic exocrine and endocrine function and their implications in diabetes.It suggests that gut cholecystokinin may play a role in lowering glucose synthesis through a neural network and resistance to it could contribute to hyperglycemia in diabetic patients.It also discusses the use of various markers such as serum trypsin concentration,amylase and lipase levels,pancreatic elastase levels,and fasting secretin levels to assess pancreatic exocrine function.Additionally,the article explores the role of carboxypeptidase E in the endocrine and neurological systems and its association with disorders.Moreover,it also highlights the involvement of retinol-binding protein 4 in the development of type 2 diabetes and insulin resistance.