Microcystin-leucine arginine (MC-LR), a potentially carcinogenic toxin, is produced by Cyanobacteria such as Microcystis and Ananabacteria during water bloom. Increasing evidence demonstrated that MC-LR induces male reproductive toxicity, mainly by inducing germ cell apoptosis, destroying cell cytoskeleton, interfering with DNA damage repair pathway, and damaging blood-testicular barrier (BTB), which eventually lead to male sterility. Testicular Sertoli cells are the somatic cells that directly contact with spermatogenic cells in seminiferous tubules. They not only regulate immune response to maintain testicular immune homeostasis by secreting a variety of cytokines and immunosuppressive factors, but also provide the protective effects of spermatogenic cells by forming BTB. MC-LR induces inflammation and apoptosis of Sertoli cells, and destroys the integrity of the BTB, and then causes spermatogenesis dysfunction.
Male ; Humans ; Sertoli Cells ; Leucine/pharmacology* ; Arginine/pharmacology* ; Microcystins/metabolism* ; Immunity

BACKGROUND: The objective of this study was to evaluate the role of proteases on the degradation of parathyroid hormone (PTH) in blood samples. METHODS: Protease inhibitors with specificity against serine proteases (aprotinin), cysteine proteases (E-64), serine and cysteine proteases (leupeptin), metalloproteases (EDTA), or a protease inhibitor cocktail with a broad spectrum of inhibitory activity were added to blood samples. After storage at room temperature (0-48 hr), PTH levels were measured. RESULTS: PTH levels in samples with the protease inhibitor cocktail did not change significantly after 48 hr of storage at room temperature, but the average PTH levels decreased by 40.7% and 20.1%, in samples stored at room temperature and stored at 4degrees C without protease inhibitors, respectively. PTH levels in samples with leupeptin were stable for up to 24 hr. After 48 hr, the mean PTH levels decreased by 17.1%, 16.0%, 26.2%, and 32.1%, with 500 KIU/mL aprotinin, 100 micro mol/L leupeptin, 10 micro mol/L E-64, and 10 micro mol/L EDTA, respectively, in the samples stored at room temperature. CONCLUSIONS: The decrease in PTH levels in blood samples seemed to be due to the degradation of PTH by proteases. Various proteases, including especially serine proteases, would act together to degrade PTH in blood specimen. The PTH degradation may be inhibited in blood specimen with protease inhibitor cocktail.
Aprotinin/pharmacology ; Blood Specimen Collection ; Edetic Acid/pharmacology ; Female ; Humans ; Leucine/analogs & derivatives/pharmacology ; Leupeptins/pharmacology ; Male ; Parathyroid Hormone/*blood/metabolism ; Protease Inhibitors/*pharmacology ; Time Factors

OBJECTIVETo investigate the effect of aminopeptidase N inhibitor ubenimex on differentiation induction of all-trans-retinoic acid (ATRA) in acute promyelocytic leukemia (APL) cells and its mechanism.

METHODSThe expression of CD11b was analyzed by flow cytometry and nitroblue-tetrazolium (NBT) reduction assay was performed to determine the cell differentiation of APL cells. The expressions of c-Myc, ERK1/2, p38MAPK protein and the phosphorylation of ERK1/2, p38MAPK protein in NB4 cells were detected by Western blot assay.

RESULTSUbenimex alone induced no significant changes in NBT reduction activity and CD11b expression but potentiated the differentiation induction activity of ATRA in APL cells. 100 microg/ml of ubenimex could enhance the NBT reduction activity induced by 10 nmol/L of ATRA, intensify the down-regulation of c-Myc protein expression and inhibit the phosphorylation of p38MAPK protein induced by 10 nmol/L of ATRA in NB4 cells.

CONCLUSIONSUbenimex could potentiate ATRA induced differentiation in APL cells, which may be correlated with the inhibition of p38 MAPK protein phosphorylation and regulation of c-Myc protein expression.

Aminopeptidases ; antagonists & inhibitors ; Cell Differentiation ; drug effects ; Drug Synergism ; Flow Cytometry ; Humans ; In Vitro Techniques ; Leucine ; analogs & derivatives ; pharmacology ; Leukemia, Promyelocytic, Acute ; pathology ; Tretinoin ; pharmacology

Antiviral Agents/pharmacology* ; COVID-19 ; Coronavirus 3C Proteases ; Humans ; Lactams ; Leucine ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Nitriles ; Proline ; Protease Inhibitors/pharmacology* ; SARS-CoV-2

The C-terminus ends of the second putative transmembrane domains of both M-1 and M-2 Muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in M-1 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-LYT) in the sequence Of M-2 receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of M-1 receptors to the corresponding M-2 receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT M-1 receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant M-1 receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant M-1 receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in M-1 muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of M-1 receptors to the stimulation of phospholipase C.
Animals ; Artifacts ; Carbachol ; CHO Cells ; Cricetinae ; Cyclic AMP ; GTP-Binding Proteins ; Humans ; Hydrolysis ; Leucine ; Pharmacology ; Phospholipases* ; Receptors, Muscarinic* ; Threonine ; Trinucleotide Repeats* ; Triplets* ; Type C Phospholipases* ; Tyrosine

This study was purposed to investigate whether aminopeptidase inhibitor, bestatin, can potentiate all-trans retinoic acid (ATRA)-inducing differentiation in NB4 cells, and to explore its mechanism. The NB4 cells were exposed to either bestatin and ATRA alone or in combination, the morphological changes of NB4 cells were observed by optical microscopy, the CD11b expression was measured by flow cytometry, the function of defferentiation cells was analyzed by nitroblue-tetrazolium (NBT) reduction assay, the mRNA expressions of c-myc and c-EBPepsilon in NB4 cells were detected by RT-PCR, the c-Myc protein expression was determined by Western blot. The results showed that treatment with bestatin alone induced no significant changes in morphology, NBT reduction activity and CD11b expression in NB4 cells. NB4 cells incubated with 10 nmol/L ATRA plus 100 microg/ml bestatin showed more morphologic feature of metamyelocyte and band neutrophil than ATRA alone treated cells. 100 microg/ml bestatin enhanced the NBT reduction activity in NB4 cells induced by various concentrations of ATRA (10, 20, 40 nmol/L). The effects of various concentrations of ATRA in combination with 100 microg/ml bestatin were statistically different from the effect of ATRA alone (P < 0.01). From 48 to 96 hours, 100 microg/ml bestatin time-dependently increased NBT reduction in NB4 cells induced by 10 nmol/L ATRA (P < 0.01). 10 nmol/L ATRA plus 100 microg/ml bestatin for 72 hours prominently elevated CD11b expression in NB4 cells as compared with ATRA alone treated NB4 cells (P < 0.01). There was a substantial decrease in c-myc mRNA levels when 100 microg/ml bestatin was added to 10 nmol/L ATRA (P < 0.05). Various concentrations (50, 75, 100 microg/ml) of bestatin combined with 10 nmol/L ATRA down-regulated the expression of c-Myc protein, which was negatively correlated with the NBT reduction activity of NB4 cells induced by 10 nmol/L ATRA alone or plus bestatin at various concentrations (r = -0.940, P = 0.017). However, 100 microg/ml bestatin plus 10 nmol/L ATRA could not induce any significant changes in the levels of c-EBPepsilon mRNA as compared with ATRA alone treated NB4 cells. It is concluded that an aminopeptidase inhibitor bestatin can potentiate ATRA-inducing differentiation of NB4 cells, possibly by down-regulating c-myc expression in synergy with ATRA.
Aminopeptidases ; antagonists & inhibitors ; Antibiotics, Antineoplastic ; pharmacology ; Cell Transformation, Neoplastic ; drug effects ; Down-Regulation ; Drug Synergism ; Humans ; Leucine ; analogs & derivatives ; pharmacology ; Leukemia, Promyelocytic, Acute ; pathology ; Proto-Oncogene Proteins c-myc ; drug effects ; metabolism ; Tretinoin ; pharmacology ; Tumor Cells, Cultured

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.
Taenia solium/*enzymology ; Serum Albumin, Bovine/metabolism ; Leucine/analogs & derivatives/pharmacology ; Iodoacetic Acid/pharmacology ; Immunoglobulin G/metabolism ; Humans ; Cysteine Proteinase Inhibitors/pharmacology ; Cysteine Endopeptidases/chemistry/*isolation & purification/metabolism ; Collagen/metabolism ; Chromatography, Ion Exchange ; Chromatography, Gel ; Animals

This study was purposed to investigate the effect of aminopeptidase N/CD13 on bestatin enhancing all-trans-retinoic acid (ATRA)-inducing differentiation in NB4 cells. The nitroblue-tetrazolium (NBT) reduction assay was performed to determine the differentiation of NB4 cells, MR2 cells and primary APL blasts. The expression of P38 MAPK protein and the phosphorylation of P38 MAPK protein in NB4, MR2 and K562 cells were detected by Western blot. The results showed that pre-incubation with 5 µg/ml WM-15 blocked the enhancement effect of bestatin on differentiation of NB4 cells induced by ATRA. 5 µg/ml CD13 antibody WM-15 partly blocked the inhibition of bestatin on the phosphorylation of P38 MAPK in NB4 cells. 100 µg/ml bestatin inhibited the phosphorylation of P38 MAPK in NB4 cells and MR2 cells in a time-dependent manner. 100 µg/ml bestatin had no effect on the phosphorylation of P38 MAPK in K562 cells with low level of CD13. Bestatin could not restore the sensitivity to ATRA in ATRA-resistant primary APL blasts and MR2 cells. It is concluded that aminopeptidase N/CD13 inhibitor bestatin may enhance the differentiation-inducing activity of ATRA through inhibiting the phosphorylation of P38 MAPK in NB4 cells mediated by the cell surface APN/CD13.
Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents ; CD13 Antigens ; antagonists & inhibitors ; metabolism ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Cell Line, Tumor ; Humans ; Leucine ; analogs & derivatives ; pharmacology ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Phosphorylation ; Tretinoin ; p38 Mitogen-Activated Protein Kinases ; metabolism

In order to investigate the molecular mechanisms of SARI expression regulation in chronic myeloid leukemia (CML), 46 patients with CML and 40 healthy volunteers were recruited in this study. SARI expression in the peripheral blood mononuclear cells (PBMNC) of CML patients and healthy volunteers was assayed by using real-time quantitative PCR. K562 cells were in vitro incubated with the BCR-ABL inhibitor STI571 (imatinib) at 37°C and 5% CO2 for 24 hours, then SARI expression was detected by using real-time quantitative PCR. All experiments were repeated three times. The results showed that as compared with healthy volunteers, the expression of SARI mRNA in PBMNC of CML patients presented a lower level (p < 0.001). After exposure of K562 cells to STI571 (2.5 µmol/L) for 24 hours, the SARI expression was higher than that in K562 cells treated without STI571 (p < 0.001). It is concluded that the suppression of SARI expression is involved in CML pathogenesis, and BCR-ABL mediates the down-regulation of SARI mRNA expression in K562 cells. These findings suggest a new orientation for gene therapy in CML patients.
Basic-Leucine Zipper Transcription Factors ; genetics ; Benzamides ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Imatinib Mesylate ; K562 Cells ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology ; Tumor Suppressor Proteins ; genetics

Astragaloside Ⅳ(AS-Ⅳ) has protective effects against ischemia-reperfusion injury(IRI), but its mechanism of action has not yet been determined. This study aims to investigate the protective effects and mechanism of AS-Ⅳ on H9c2 cardiomyocyte injury induced by hypoxia-reoxygenation(H/R). The H/R model of myocardial cells was established by hypoxic culture for 12 hours and then reoxygenation culture for 8 hours. After AS-Ⅳ treatment, cell viability, the reactive oxygen species(ROS) levels, as well as the content or activity of superoxide dismutase(SOD), malondialdehyde(MDA), interleukin 6(IL-6), and tumor necrosis factor alpha(TNF-α), were measured to evaluate the effect of AS-Ⅳ treatment. The effect of AS-Ⅳ on HO-1 protein expression and nuclear Nrf2 and Bach1 protein expression was determined by Western blot. Finally, siRNA was used to knock down HO-1 gene expression to observe its reversal effect on AS-Ⅳ intervention. The results showed that as compared with the H/R model group, the cell viability was significantly increased(P<0.01), ROS level in the cells, MDA, hs-CRP and TNF-α in cell supernatant and nuclear protein Bach1 expression in the cells were significantly decreased(P<0.01), while SOD content, HO-1 protein expression in cells and expression of nuclear protein Nrf2 were significantly increased(P<0.01) in H/R+AS-Ⅳ group. However, pre-transfection of HO-1 siRNA into H9c2 cells by liposome could partly reverse the above effects of AS-Ⅳ after knocking down the expression of HO-1. This study suggests that AS-Ⅳ has significant protective effect on H/R injury of H9c2 cardiomyocytes, and Nrf2/Bach1/HO-1 signaling pathway may be a key signaling pathway for the effect.
Apoptosis ; Basic-Leucine Zipper Transcription Factors ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Heme Oxygenase-1 ; metabolism ; Humans ; Myocytes, Cardiac ; drug effects ; NF-E2-Related Factor 2 ; metabolism ; Saponins ; pharmacology ; Signal Transduction ; Triterpenes ; pharmacology