OBJECTIVE: To explore the role of reactive oxygen species (ROS) and lactate dehydrogenase isoenzyme X (LDH-X) in the changes of sperm mitochondrial membrane potential (MMP) in infertility patients with varicocele (VC). METHODS: This study included 38 infertility patients with VC (VCinf), 35 non-VC infertile males (NVCinf), and 30 normal fertile men as controls. We obtained the routine semen parameters using the sperm quality analysis system, examined the contents of LDH-X in the seminal plasma and sperm with the automatic biochemical analyzer, measured the level of malondialdehyde (MDA) in seminal plasma by thiobarbituric acid (TBA) colorimetry, and determined the expressions of mitochondrial membrane potential (MMP) and LDH-X mRNA in the sperm using JC-1 fluorescence probe and RT-PCR. RESULTS: No statistically significant differences were observed among the three groups of subjects in age, semen pH value, semen volume and sperm concentration (P > 0.05). Compared with the normal fertile controls, the patients in the VCinf and NVCinf groups showed significantly decreased sperm motility (［52.36 ± 12.48］% vs ［34.74 ± 15.23］% vs ［25.76 ± 13.73］%, P< 0.05), percentage of progressively motile sperm (PMS) (［42.54 ± 13.58］% vs ［29.10 ± 14.17］% vs ［20.95 ± 12.33］%, P< 0.05), sperm LDH-X (［16.46 ± 5.47］ vs ［13.63 ± 4.50］ vs ［10.18 ± 3.00］ mU/106, P< 0.05), sperm MMP (［48.04 ± 11.62］% vs ［40.86 ± 12.69］% vs ［34.41 ± 13.93］%, P< 0.05) and expression of sperm LDH-X mRNA (P< 0.05). but increased seminal plasma LDH-X (［935.36 ± 229.48］ vs ［1241.05 ± 337.07］ vs ［1425.08 ± 469.35］ U/L, P< 0.05), seminal plasma/whole sperm LDH-X (［1.06 ± 0.35］ vs ［1.40 ± 0.34］ vs ［1.63 ± 0.66］, P< 0.05), and content of seminal plasma MDA (［1.10 ± 0.19］ vs ［1.59 ± 0.27］ vs ［2.00 ± 0.22］ nmol/ml, P< 0.05). CONCLUSION Excessive ROS in the reproductive system of VCinf patients reduces the content of MMP and causes the overflow of LDH-X out of sperm cells. Therefore the decrease of sperm LDH-X may be accompanied by that of MMP.
Humans ; Male ; Infertility, Male/etiology* ; Varicocele/metabolism* ; Adult ; Reactive Oxygen Species/metabolism* ; Spermatozoa/metabolism* ; L-Lactate Dehydrogenase/metabolism* ; Membrane Potential, Mitochondrial ; Isoenzymes/metabolism* ; Case-Control Studies ; Young Adult ; Mitochondrial Membranes/metabolism*

Titin, the largest known protein in the body expressed in three isoforms (N2A, N2BA and N2B), is essential for muscle structure, force generation, conduction and regulation. Since the 1950s, muscle contraction mechanisms have been explained by the sliding filament theory involving thin and thick muscle filaments, while the contribution of cytoskeleton in force generation and conduction was ignored. With the discovery of insoluble protein residues and large molecular weight proteins in muscle fibers, the third myofilament, titin, has been identified and attracted a lot of interests. The development of single molecule mechanics and gene sequencing technology further contributed to the extensive studies on the arrangement, structure, elastic properties and components of titin in sarcomere. Therefore, this paper reviews the structure, isforms classification, elastic function and regulatory factors of titin, to provide better understanding of titin.
Connectin/genetics* ; Muscle Proteins/metabolism* ; Protein Isoforms/genetics* ; Sarcomeres/metabolism* ; Muscle Fibers, Skeletal/metabolism*

Isoenzymes

Objective: To explore the value of paraquat (PQ) intake, urine protein and myocardial enzyme indexes in judging the prognosis of patients with acute PQ poisoning. Methods: From September to December 2021, all 201 patients with acute PQ poisoning admitted to Guangzhou Twelfth People's Hospital from January 2010 to December 2019 were selected as the research objects. Based on follow-up results 60 days after poisoning, the research objects were divided into survival group (n=78) and death group (n=123) . The differences in information about poisoning, treatment plan, PQ intake, urine protein, creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase between the two groups of patients were compared and analyzed. Logistic regression and Cox regression were used to analyze the correlation between poisoning outcome and PQ intake, urine protein and myocardial enzymes. ROC curve and principal component analysis were used to explore high-efficiency indicators for predicting the outcome of acute PQ poisoning. Results: The PQ intake[50 (20, 100) ml], urine protein (total rank 15570.50) , creatine kinase[ (336.36±261.96) U/L], creatine kinase isoenzyme[ (43.91±43.74) U/L], lactate dehydrogenase [ (346.01±196.50) U/L], α-hydroxybutyrate dehydrogenase content[ (271.23±11.92) U/L] of patients in the death group were all higher than the survival group[15 (10, 20) ml, 4730.50, (187.78±178.06) U/L, (18.88±15.50) U/L, (190.92±60.50) U/L, (152.60±48.34) U/L, respectively] (P<0.05) . The outcome of acute PQ poisoning was positively correlated with PQ intake, urine protein, creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase (P<0.05) . Multivariate logistic regression and multivariate Cox regression analysis showed that creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase and α-hydroxybutyrate dehydrogenase was positively correlated with the prognosis of patients with acute PQ poisoning (P<0.05) . ROC curve analysis and principal component analysis showed that the combined indexes of PQ intake, urine protein and myocardial enzymes had the highest efficacy and weight in judging the prognosis of patients (AUC=0.91, weight coefficient=0.19, sensitivity=0.76, specificity=0.89) . When the combined score was ≥4, the probability of accurately predicting the death of patients was as high as 91% (positive predictive value=0.91) . Conclusion: PQ intake, urine protein combined with creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase has high value in predicting the prognosis of patients with acute PQ poisoning.
Humans ; Creatine ; Creatine Kinase ; Isoenzymes ; Lactate Dehydrogenases ; Paraquat/poisoning* ; Prognosis ; Retrospective Studies ; Myocardium/enzymology* ; Urine/chemistry*

Wnt5a is a secreted Wnt ligand that plays a critical role in cellular pathways and inflammatory diseases. The WNT5A gene encodes two protein isoforms, Wnt5a-long and Wnt5a-short, which differ based on different promoter methylation and have distinct functions. However, the mechanisms of the promoter methylation are unclear. Depending on the extent of promoter methylation, Wnt5a exerts both anti-inflammatory and pro-inflammatory effects in inflammatory diseases, which may be involved in different Wnt5a isoforms. Therefore, the Wnt5a isoforms may be potential diagnostic markers for inflammatory diseases and the mechanisms of the WNT5A gene promoter methylation need to be further investigated.
DNA Methylation ; Wnt-5a Protein ; Promoter Regions, Genetic ; Protein Isoforms/genetics*

Vascular endothelial growth factor-A (VEGF-A) is a critical angiogenic factor which is mainly secreted from podocytes and epithelial cells in kidney and plays an important role in renal pathophysiology. In recent years, functions of different isoforms of VEGF-A and the new secretion approach via extracellular vesicles (EVs) have been identified. Thus, further understanding are needed for the role of VEGF-A and its isoforms in renal injury and repair. In this review, we summarized the expression, secretion and regulation of VEGF-A, its biological function, and the role of different isoforms of VEGF-A in the development of different renal diseases. Meanwhile, the research progress of VEGF-A as diagnostic marker and therapeutic target for renal diseases were discussed.
Humans ; Kidney/metabolism* ; Kidney Diseases ; Protein Isoforms/metabolism* ; Vascular Endothelial Growth Factor A/physiology*

This study intends to obtain recombinant proteins of ALT1 and ALT2 isozymes by using genetic recombination technology. Monoclonal antibodies ALT1 and ALT2 with high specificity and high activity were prepared and screened (ALT1 monoclonal antibody has been successfully prepared and published). The localization, distribution and expression of ALT1 and ALT2 isozymes in human tissues were discussed. The ALT2 genes were amplified from human liver cancer cell (HepG2) by RT-PCR method. The mature ALT2 gene was subcloned into the pET32a-ALT2 prokaryotic expression vector. Its ligation product was transformed into BL21(DE3) competent cells, and transformed into competent cells to express ALT2 proteins induced by IPTG. The recombinant proteins of ALT2 were purified by nickel column (Ni⁺) affinity chromatography. Balb/c mice were immunized with recombinant proteins of ALT2. Positive serum mouse spleen cells and myeloma cells SP2/0 were selected for cell fusion. The positive cell lines were selected by indirect ELISA and subcloned by limited dilution method. Affinity chromatography was used to purify ALT2 antibodies. The expression and distribution of ALT2 in human normal tissues were detected by RT-PCR and Western blotting. Results show that the expression of ALT isoenzyme in tissues was almost the same at gene mRNA level and protein level. ALT1 is highly expressed in liver, kidney and skeletal muscle, and moderately expressed in gastrointestinal smooth muscle. ALT2 is highly expressed in fat, skeletal muscle and myocardium, and is poorly expressed in gastrointestinal smooth muscle. Immunohistochemical studies show that ALT1 is highly expressed in hepatocytes, renal medullary tubules and muscle fibers, ALT2 is highly expressed in adipocytes and myocardial cells, and ALT1 and ALT2 in gastrointestinal tissues are mainly expressed in mucosa of upper intestinal wall region. The results showed that the isoenzymes ALT1 and ALT2 were mainly expressed in the mucosa of the upper part of the intestinal wall. It is widely distributed in the tissues, providing theoretical basis for understanding the mechanism of ALT activity increase under different pathological conditions.
Alanine Transaminase ; Animals ; Cloning, Molecular ; Humans ; Isoenzymes/genetics* ; Liver ; Mice ; Recombinant Proteins

OBJECTIVE: To explore the clinical value of serum isoform [-2] proprostate-specific antigen (p2PSA) and its derivatives %p2PSA and prostate health index (PHI) in predicting aggressive prostate cancer (PCa). METHODS: The pre-operation serum and basic clinical data of 322 patients with PCa (including 143 patients diagnosed with PCa by transrectal ultrasound-guided prostate biopsy and 179 patients undergoing radical prostatectomy) in Peking University First Hospital were collected from August 2015 to May 2018. Serum total prostate-specific antigen (tPSA), free prostate antigen (fPSA) and fPSA/tPSA (f/t) and the p2PSA level of all these patients were measured on automatic immune analyzers DxI800, and then %p2PSA and PHI were calculated. The prostate pathologic result was considered as the gold standard to evaluate the Gleason score of the patients with PCa. Receiver operator curves (ROC) were used to assess the ability of p2PSA, %p2PSA and PHI to predict aggressive PCa (pathologic Gleason score≥7) compared with those traditional markers tPSA, fPSA and f/t. RESULTS: Among these patients, the p2PSA, %p2PSA and PHI median levels were significantly higher in patients with pathologic Gleason score≥7 than those with Gleason score<7 (p2PSA: 30.22 ng/L vs. 18.33 ng/L; %p2PSA: 2.50 vs. 1.27; PHI: 91.81 vs. 35.44; all P<0.01). The area under curve (AUC) of %p2PSA and PHI (0.770, 0.760) in predicting Gleason score≥7 were higher than those of the traditional indicators tPSA, fPSA and f/t (AUC were 0.648, 0.536 and 0.693, respectively). Among those patients diagnosed with PCa by transrectal ultrasound-guided prostate biopsy, the AUC of %p2PSA and PHI (AUC were 0.808 and 0.801, respectively) in predicting Gleason score≥7 were higher than those of the traditional indicators tPSA, fPSA and f/t (AUC were 0.729, 0.655 and 0.665 respectively). Among those patients undergoing radical prostatectomy, PHI and %p2PSA also had the trend of higher predictive value than those of the traditional indicators. The AUC of %p2PSA and PHI were 0.798 and 0.744, respectively while the AUC of tPSA, fPSA and f/t were 0.625, 0.507 and 0.697, respectively. CONCLUSION Compared with traditional markers tPSA, fPSA and f/t, %p2PSA and PHI had much higher predictive value for aggressive PCa, which may help clinicians to evaluate the therapeutic regime and make more appropriate management plan for the patients.
Humans ; Male ; Neoplasm Grading ; Prostate-Specific Antigen ; Prostatectomy ; Prostatic Neoplasms ; Protein Isoforms ; ROC Curve

Galectin-4 (Gal-4) is a β-galactoside-binding protein mostly expressed in the gastrointestinal tract of animals. Although intensive functional studies have been done for other galectin isoforms, the immunoregulatory function of Gal-4 still remains ambiguous. Here, we demonstrated that Gal-4 could bind to CD14 on monocytes and induce their differentiation into macrophage-like cells through the MAPK signaling pathway. Gal-4 induced the phenotypic changes on monocytes by altering the expression of various surface molecules, and induced functional changes such as increased cytokine production and matrix metalloproteinase expression and reduced phagocytic capacity. Concomitant with these changes, Gal-4-treated monocytes became adherent and showed elongated morphology with higher expression of macrophage markers. Notably, we found that Gal-4 interacted with CD14 and activated the MAPK signaling cascade. Therefore, these findings suggest that Gal-4 may exert the immunoregulatory functions through the activation and differentiation of monocytes.
Animals ; Antigens, CD14 ; Cell Differentiation ; Galectin 4 ; Galectins ; Gastrointestinal Tract ; Macrophages ; Monocytes ; Protein Isoforms

Peroxiredoxins (Prxs) are antioxidant enzymes that protect cells from oxidative stress by reducing intracellular accumulation of reactive oxygen species (ROS). In mammalian cells, the six Prx isoforms are ubiquitously expressed in diverse intracellular locations. They are involved in the regulation of various physiological processes including cell growth, differentiation, apoptosis, immune response and metabolism as well as intracellular ROS homeostasis. Although there are increasing evidences that Prxs are involved in carcinogenesis of many cancers, their role in cancer is controversial. The ROS levels in cancer cells are increased compared to normal cells, thus promoting cancer development. Nevertheless, for various cancer types, an overexpression of Prxs has been found to be associated with poor patient prognosis, and an increasing number of studies have reported that tumorigenesis is either facilitated or inhibited by regulation of cancer-associated signaling pathways. This review summarizes Prx isoforms and their basic functions, the relationship between the expression level and the physiological role of Prxs in cancer cells, and their roles in regulating cancer-associated signaling pathways.
Apoptosis ; Carcinogenesis ; Homeostasis ; Humans ; Metabolism ; Oxidative Stress ; Peroxiredoxins ; Physiological Processes ; Prognosis ; Protein Isoforms ; Reactive Oxygen Species