Proteolytic cleavage is one of the post-translational modifications and plays important roles in many biological processes, such as apoptosis and tumor cell metastasis. The identification of the cleavage events can improve our understanding of their biological functions in these processes. Although proteomic approaches using N-terminal labeling have resulted in the discovery of many proteolytic cleavages, this strategy has its own inherent drawbacks. Labeling of protein C-termini is an alternative approach. Here, we optimized the labeling procedure in the profiling protein C-termini by enzymatic labeling (ProC-TEL) and improved the labeling efficiency for the positive isolation of protein C-terminal peptides and mass spectrometric identification. We applied this approach to a complex protein mixture from Escherichia coli and identified many C-terminal peptides and internal cleaved peptides from more than 120 proteins. From the identified cleavages, we found several previously known internal proteolytic cleavage sites and many novel ones which may play roles in regulating normal biological processes. This work provides a potential new way, complementary to the N-terminomics, for the identification of proteolytic cleavages in complex biological systems.
Cathepsin A ; chemistry ; Protein C ; chemistry ; Protein Processing, Post-Translational ; Proteolysis ; Proteomics

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.
Animals ; Cattle ; Dogs ; Hemagglutinins, Viral ; chemistry ; metabolism ; Influenzavirus C ; physiology ; Orthomyxoviridae Infections ; metabolism ; virology ; Protein Conformation ; Protein Folding ; Protein Processing, Post-Translational ; Viral Fusion Proteins ; chemistry ; metabolism

We developed a high-sensitivity C-reactive protein quantifiable chemiluminescent immunoassay (hs-CRP CLIA). The high-purity native CRP was purified from hepatic cirrhosis patient ascetic fluid by affinity and ion exchange chromatography and used as an immunogen to develop the monoclonal antibodies (mAbs) against CRP. Twenty-two mAbs were identified reactive with CRP in ELISA and 13 of them were reactive in the phosphorycholine ligand capture ELISA. The mAbs 10C5 and 10C11 were selected to develop the hs-CRP CLIA. The linearity and performance of the hs-CRP CLIA was characterized. It was showed not reactive when testing against other serum materials (IgG, hemoglobin and triglyceride). The reliable correlation (R2 > 0.993) was obtained between testing value (RLU/S) and the concentration of human serum CRP calibrator. The linearity fell in the range of 0.04-20.38 mg/L. The assay has good accuracy and reproducibility, the mean recovery was 99% and the precision of the intra- and inter assay was CVs (4.2%-5.8%) and (9.0%-11.5%), respectively. In testing of 90 human sera, this assay performed well and correlated comparably with a commercial hs-CRP ELISA kit. Thus, hs-CRP CLIA is an accurate, reliable, quantifiable assay for detection of high-sensitive C-reactive protein in serum, it may be useful to improve the risk assessment of cardiovascular disease and the prognosis of inflammatory bowel disease.
C-Reactive Protein ; analysis ; chemistry ; Humans ; Immunoassay ; methods ; Luminescent Measurements ; methods ; Sensitivity and Specificity

OBJECTIVETo evaluate the relationship between periodontitis and the traditional risk factors of coronary heart disease (CHD), as well as the role in the mechanisms responsible for high-sensitivity C-reactive protein (hsCRP) in the relationship of peridontitis and CHD.

METHODSA periodontal examination was conducted on a total of 356 subjects, and community periodontal index of treatment needs (CPITN) was obtained from each subject. Periodontal status was categorized into TN < or =2, TN=3, TN=4 three groups according to the CPITN indexes. Fasting venous blood samples were collected from all the three group subjects, the serum hsCRP concentration and serological changes used in diagnosing CHD routinely were determined, and software of SPSS 16.0 were used to analyzed the relationship of periodontal, hsCRP concentration and routinely CHD serological indexes.

RESULTSIn the groups of TN < or =2, TN=3 and TN=4, the hsCRP level was (1.10 +/- 1.16), (1.86 +/- 2.34), (2.25 +/- 2.75) mg x L(-1), respectively. Compared with Group TN < or =2, the concentration of hsCRP in Group TN=3 and TN=4 were higher (OR = 1.24, OR = 1.31, respectively). Compared with group hsCRP < 3.0 mg x L(-1), more calculus and deep periodontal pockets were found in the Group hsCRP > or = 3.0 mg x L(-1) (P < 0.05).

CONCLUSIONThe serum hsCRP level is correlated with the severity of periodontal disease.

C-Reactive Protein ; chemistry ; Coronary Disease ; Humans ; Periodontal Index ; Periodontitis ; blood ; diagnosis ; Risk Factors

OBJECTIVETo investigate the content and activity of M-phase promoting factor (MPF) in pleomorphic adenoma, mucoepidermoid carcinoma, buccal carcinoma and normal tissue, in order to evaluate the role of MPF in the development of tumor and the relationship between MPF and malignant degree.

METHODSThe content and activity of MPF were assessed by immunobloting and Gollicano method.

RESULTSThe cdc2 and cyclinB (two subunits of MPF) were found both in normal and tumor tissues, and their content in tumor was higher than normal tissues. Buccal carcinoma was 64% higher than normal tissues. The activity of MPF in carcinoma was higher than normal tissue and had positive relation with the malignant extent.

CONCLUSIONSThe content and activity of MPF in tumor are higher than normal tissue. PKC can activate MPF. These results show PKC may promote tumor proliferation by activating MPF and also, the activity of MPF has some relation with malignant extent.

CDC2 Protein Kinase ; analysis ; Cyclin B ; analysis ; Humans ; Immunoblotting ; Maturation-Promoting Factor ; analysis ; Mouth ; chemistry ; Mouth Neoplasms ; chemistry ; Protein Kinase C ; physiology

The present study was aimed to investigate the role of cerebrospinal fluid-contacting nucleus (CSF-CN) neurons in modulation of inflammatory pain and underlying mechanism. The inflammatory pain model was made by subcutaneous injection of the complete Freund's adjuvant (CFA) into the left hind paw of rats. The phosphorylation level of PKC (p-PKC) was examined by Western blot. Thermal withdrawal latency (TWL) of the rats was measured to assess inflammatory pain. The results showed that, compared with the sham controls, the inflammatory pain model rats showed shortened TWL on day 1, 3, and 7 after CFA injection, as well as increased level of p-PKC in CSF-CN neurons at 24 h after CFA injection. The administration of GF109203X, a PKC inhibitor, into lateral ventricle decreased the level of p-PKC protein expression and increased TWL in the model rats. These results suggest that blocking the PKC pathway in CSF-CN neurons may be an effective way to reduce or eliminate the inflammatory pain.
Animals ; Freund's Adjuvant ; Inflammation ; enzymology ; Neurons ; enzymology ; Pain ; enzymology ; Phosphorylation ; Protein Kinase C ; cerebrospinal fluid ; chemistry ; Rats ; Rats, Sprague-Dawley

PURPOSE: Cytokines found at a inflammatory site may be a good indicator of clinical severity of an infectious inflammatory disease. We assayed interleukin 6 (IL-6) concentrations in cerebrospinal fluid (CSF) from patients affected with viral meningitis, and verified the relationship between IL-6 and inflammatory parameters and whether IL-6 can be used as a diagnostic marker in the diagnosis of viral meningitis. METHODS: We measured CSF IL-6 concentration in viral meningitis (30 cases) and healthy children (3 cases) by using ELISA, and also measured serum and cerebral spinal fluid (CSF) chemistry and inflammatory markers, including C-reactive protein (CRP). We compared the data and analyzed the relationship between the results. RESULTS: The CSF IL-6 levels of viral meningitis (520.1+/-384.3pg/dL) were significantly higher than that of normal control (2.3+/-4.0pg/dL) (P<0.05). The relationship between CSF IL-6 level and serum CRP was significant (P=0.0041). The relationship between CSF protein level and CSF IL-6 level was significant (P=0.001). There was no significant correlation between CSF IL-6 level and CSF WBC count. CONCLUSION: According to these results, we concluded that viral meningitis is highly associated with CSF IL-6, and we predict CSF IL-6 is useful in the diagnosis and prediction of treatment for viral meningitis.
C-Reactive Protein ; Cerebrospinal Fluid* ; Chemistry ; Child ; Cytokines ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-6* ; Meningitis, Viral*

STUDY DESIGN: A retrospective study. PURPOSE: We evaluated the results of the use of anterior debridement and interbody fusion followed by posterior spinal instrumentation. OVERVIEW OF LITERATURE: An early diagnosis of pyogenic spondylitis is difficult to obtain. The disease can be treated with various surgical methods (such as anterior debridement and bone graft, anterior instrumentation, and posterior instrumentation). METHODS: This study included 20 patients who received anterior debridement and interbody fusion with strut bone graft followed by posterior spinal fusion for pyogenic spondylitis between 1996 and 2005. We analyzed the culture studies, the correction of the kyphotic angle, blood chemistry, the bony union period, and the amount of symptom relief. RESULTS: In terms of clinical symptoms relief, eight patients were grouped as "excellent", eleven patients as "good", and one patient as "fair". The vertebral body cultures were positive in 14 patients showing coagulase (-) streptococcus and S. aureus. The average times for normalization of the erythrocyte sedimentation rate and C-reactive protein level were 3.3 and 1.9 months, respectively. Four months was required for bony union. For complications, meralgia paresthetica was found in two cases. CONCLUSIONS: Due to early ambulation and the correction of the kyphotic angle, anterior interbody fusion with strut bone graft and posterior instrumentation could be another favorable method for the treatment of pyogenic spondyulitis.
Blood Sedimentation ; C-Reactive Protein ; Chemistry ; Coagulase ; Debridement* ; Early Ambulation ; Early Diagnosis ; Humans ; Retrospective Studies ; Spinal Fusion ; Spondylitis* ; Streptococcus ; Transplants*

Objective: To explore the application value of T lymphocyte subsets combined with procalcitonin (PCT), C-reactive protein (CRP), neutrophil to lymphocyte ratio (NLR) and white blood cell count (WBC) in the auxiliary diagnosis and prognosis evaluation of sepsis. Methods: In a retrospective study, seventy-two patients with sepsis diagnosed and treated in Tianjin First Central Hospital from June 2018 to April 2021 were selected as the research objects, and included in the sepsis group were 46 males and 26 females, aged 68 (57.3, 80.3) years. In addition, 111 patients with local infection admitted to hospital during the same period were included in the local infection group, including 62 males and 49 females, aged 68 (51, 77) years. Sepsis patients were divided into survival group (43 cases) and death group (29 cases) according to the 28-day outcome. CD3⁺, CD4⁺, CD8⁺, CD4⁺/CD8⁺ ratio were detected by flow cytometry within 24 h after admission, PCT was detected by ELISA, CRP was detected by immunoturbidimetry, blood routine examination, blood lactic acid (Lac) and oxygen partial pressure (PO₂) were detected by instrumental method. Multivariate Logistic regression analysis was used to evaluate the correlation between each indicator and sepsis, and receiver operating characteristic curve (ROC) was drawn to evaluate the diagnostic value of each indicator for sepsis. Multivariate Logistic regression analysis and Kaplan Meier survival analysis were used to evaluate the prognostic value of each index for patients with sepsis. Results: Peripheral blood CD3⁺, CD4⁺, CD8⁺, CD4⁺/CD8⁺ ratio and PLT in sepsis group were significantly lower than those in local infection group(Z=-8.184,P<0.001;Z=-7.210,P<0.001;Z=-5.936,P<0.001;Z=-2.700,P=0.007;Z=-6.381,P<0.001); PCT, CRP, NLR and Lac levels were significantly higher than those in local infection group(Z=-8.262,P<0.001;Z=-3.094,P=0.002;Z=-9.004,P<0.001;Z=-4.770,P<0.001). Multivariate Logistic regression model showed that PCT, NLR, CD3⁺, CD8⁺, CD4⁺/CD8⁺ were independent risk factors for sepsis. According to ROC curve analysis, AUC of sepsis patients diagnosed by each indicator were 0.862, 0.894, 0.858, 0.760 and 0.618, respectively. The cut-off values were 3.075 ng/ml, 10.715, 44.935×10⁹/L, 27.463×10⁹/L and 0.750, respectively. The NLR sensitivity was 80.6%, and the CD3⁺ specificity was 94.6%. The AUC of combined detection of PCT and NLR was 0.947, sensitivity was 87.5% and specificity was 91.9%. The combined detection AUC of PCT, NLR, CD3⁺, CD4⁺/CD8⁺ was 0.958, the sensitivity and specificity were 90.3% and 91.0% respectively(P<0.001). PCT and Lac in death group were significantly higher than those in survival group(Z=-2.302,P=0.021;Z=-3.095,P=0.002);Peripheral blood CD4⁺/CD8⁺ levels were significantly lower than those in survival group(Z=-3.691,P<0.001),Multivariate Logistic regression model showed that CD4⁺/CD8⁺ ratio was an independent risk factor for 28 d mortality in patients with sepsis (P<0.001). The ROC curve showed that the AUC was 0.758, and the Youden index reached the maximum when the cut-off value was 1.27, the sensitivity and specificity were 79.3% and 60.5%, respectively. Compared with patients with CD4⁺/CD8⁺ ≥1.27, 28-day mortality was significantly increased in patients with CD4⁺/CD8⁺<1.27 (P=0.032). Conclusion: The combined detection of PCT, NLR, CD3⁺ and CD4⁺/CD8⁺ can improve the auxiliary diagnostic efficiency of sepsis, and the ratio of CD4⁺/CD8⁺ in peripheral blood may have certain predictive value for the prognosis of sepsis.
Aged ; C-Reactive Protein/analysis* ; Female ; Humans ; Male ; Middle Aged ; Procalcitonin ; Retrospective Studies ; Sepsis/diagnosis* ; T-Lymphocyte Subsets/chemistry*

OBJECTIVETo explore the pathogenesis of protein C deficiency in two pedigrees through mutation detection and model analysis.

METHODSChromogenic substrate method and enzyme linked immunosorbent assay (ELISA) were used to determine the plasma protein C activity (PC: A) and protein C antigen (PC: Ag) in the two probands and their family members. All of the 9 exons and intron-exon boundaries of the PROC gene were amplified by PCR and analyzed with Sanger sequencing after purification. Corresponding mutate sites of the family members were also amplified and sequenced. The PolyPhen-2 software was used to analyze the perniciousness of the mutations and Clustal X was to analyze the conservatism. The protein model and amino acids interaction of the mutations were analyzed by Swiss-PdbViewer software.

RESULTSThe PC: A and PC: Ag of proband 1 was 30% and 35%, while PC:A of his father, mother and aunt were all slightly under the reference range. Two heterozygous missense mutations were found in exons 7 and 5 of the PROC gene, namely c.565 C>T (p.Arg147Trp) and c.383 G>A (p.Gly86Asp). His father and aunt were carriers for c.565 C>T, while his mother had carried c.383 G>A. The PC: A of proband 2 and his son were 50% and 64%, respectively. And they were both positive for p.Arg147Trp. Analysis of PolyPhen-2 indicated that p.Arg147Trp was benign, while p.Gly86Asp was damaging. Clustal X analysis indicated that the p.Arg147Trp was non-conservative, while the p.Gly86Asp was highly conservative. Modeling for the mutant proteins revealed that the simple aromatic ring of Trp147 in p.Arg147Trp destroyed the two hydrogen bonds between Arg147-Lys146 and Arg147-Lys151, and steric hindranted with Arg178. The side chain of Asp86 extended and generated steric clash with Gln90 with the occurrence of p.Gly86Asp. The change of hydrogen bonds and steric effects has altered the spatial configuration of amino acids, which led to unstable mutate proteins and interfered with the secretion.

CONCLUSIONBoth probands had hereditary protein C deficiencies, for which their parents were all carriers. The heterozygous mutations p.Arg147Trp and p.Gly86Asp were the main cause for PC: A activity decrease. Among these, p.Gly86Asp was discovered for the first time.

Base Sequence ; Child ; DNA Mutational Analysis ; methods ; Family Health ; Female ; Heterozygote ; Humans ; Hydrogen Bonding ; Male ; Middle Aged ; Models, Molecular ; Mutation ; Pedigree ; Phenotype ; Protein C ; chemistry ; genetics ; metabolism ; Protein C Deficiency ; blood ; genetics ; Protein Domains