Twenty dyes which previously have been claimed to be excreted in pancreatic juice were reinvestigated to determine to what extent they could be eliminated through the pancreas. Exogenous secretin or cholecysto-kinin-pancreozymin(CCK-PZ) stimuli were used in dogs which had been given intravenous dye solutions at the rate of 1mg/min. In this experiment among the twenty dyes, only six were found to be eliminated through the pancreas. The intensity of dye color in pancreatic juice was estimated photometrically or macroscopically. The dye color intensity decreased as follows; basic fuchsin, acridine red, new fuchsin, rhodamin B, phenol red and rhodamin 6G. Basic fuchsin consistently appeared in CCK-PZ stimulated juice. However, it was seen in only a scant amount or not at all in juice stimulated by purified Vitrum (Sweden) secretin. Similar findings were observed in cats and conscious pigs. The content of basic fuchsin in pancreatic juice was more related to changes in the enzyme concentration than to other components. The chloride content of the juice was related to the amylase or basic fuchsin secretion. However, the chloride content was inversely related to the secreted volume. Vagal stimulation or the administration of parasympathomimetics produced a juice rich in enzyme content, but the dye response to vagal stimulation was weak. Usually the volume of secreted pancreatic juice following stimulation by Boots (England) secretin is greater than stimulated by purified Vitrum preparation. Basic fuchsin was slightly reduced during its elimination from pancreas or when present in alkaline pancreatic juice. Adding acid and formaldehyde revived the color. The acridine red and other pyronine dyes caused the juice to fluorescence. This effect lasted over 24 hours.
Animal ; Dogs ; Dyes/metabolism* ; Pancreas/metabolism* ; Pancreatic Juice/secretion* ; Rosaniline Dyes/metabolism*

A preliminary study was performed to investigate the staining characteristics of trabecula. endothelial cells with low density lipoprotein (LDL) and acetylated low density lipoprotein (Ac-LDL) labeled with a fluorescent probe, 1, 1`- dioctadecyl-3,3,3`, 3`- tetramethyl-indocarbocyanine perchlorate (Dil). Trabecular endothelial cells revealed a strong fluorescence with Dil-LDL, which was contradictory to the previous results obtained from other types of endothelial cells. These cells also showed a moderate fluorescence with Dil-Ac-LDL. Scleral fibroblasts and keratocytes showed a moderate to strong fluorescence with Dil-LDL and a weak fluorescence with Dil-Ac-LDL. Corneal endothelial cells revealed a very weak background fluorescence with Dil-LDL and a moderate fluorescence with Dil-Ar-LDL. Therefore, these four kinds of cells could not be definitely differentiated depending only on the staining characteristics with Dil-LDL and Dil-Ac-LDt.
Animals ; Cattle ; Endothelium/cytology ; Fluorescent Dyes/*diagnostic use ; Lipoproteins, LDL/*metabolism ; Trabecular Meshwork/*metabolism

OBJECTIVESTo evaluate the application of Coomassie brilliant blue (CBB) G250 staining for the detection of human sperm deformity rate, rate of intact acrosome and acrosome reaction.

METHODSThe smear of spermatozoa before and after capacitation and induced acrosome reaction with progesterone (P) were stained with 0.05% CBB G250 and Wright-Giemisa solution respectively, and visualized with light microscopy. The deformity rate of spermatozoa, rate of intact acrosome and acrosome reaction were calculated.

RESULTSThere was no any difference in detection of deformity rate of spermatozoa and rate of intact acrosome with CBB G250 and Wright-Giemisa staining(P < 0.05). The sperm population of acrosome reaction with induced P was divided by CBB staining into two types: positive staining with dark violet blue on acrosome cap and pale or negative staining on the same area. The rate of the latter was increasing with increasing inductive time, maybe representative of the rate of acrosome reaction. The mean rate was(75.1 +/- 3.8)% after induced for 1 h.

CONCLUSIONSCBB G250 staining is a reliable method for assessment of the human sperm morphology and acrosome reaction.

Acrosome ; metabolism ; Acrosome Reaction ; Humans ; Male ; Rosaniline Dyes ; chemistry ; metabolism ; Spermatozoa ; cytology ; enzymology ; Staining and Labeling

As a classic fluorescent detect technique, fluorescence resonance energy transfer (FRET) has been widely used in biological researches. Researchers have developed a series of fluorescence detect probes which were based on FRET. Caspase family plays an important role in apoptosis pathway, especially Caspase-8 which located, at the initial of death receptor mediated apoptosis pathway, whose its activation can trigger subsequent precaspases' activation and lead to apoptosis. So it is of great significance to detect the activation of Caspase-8 in apoptosis assay. In this study, a fluorescent probe based on FRET has been designed which can detect the activity change of Caspase-8 in cells. To identify the effectiveness and specificity of the probe, we measure the Caspase-8 activity under the Caspase-8 specifically activated apoptosis inducer RGD-TRAIL with the flow cytometry FRET detection platform. The results show that the probe can respond to the activity change of Caspase-8 in apoptotic cells, and the change can be quantified rapidly by flow cytometry. The study provides a more efficient and convenient detection method of Caspase-8 activity in living cells.
Apoptosis ; Caspase 8 ; metabolism ; Flow Cytometry ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes ; Humans

OBJECTIVETo observe the influence of electroacupuncture (EA) on indocyanine green (ICG) metabolism in the liver and the action mechanism of acupuncture for hepatic regulation.

METHODSForty Kunming mice were randomly divided into an EA group and a control group, 20 cases in each one. Combined with classical hepatic metabolism test of ICG, after tail vein injection of ICG, twenty mice were treated with EA at "Zusanli" (ST 36) for 20 min. The ICG fluorescence intensity and distribution in the liver were observed with animal in vivo fluorescence imaging apparatus during 20 min of EA and 60 min of needle withdrawal. Twenty mice, taken as control group, were treated with ICG injection and no EA. The differences of fluorescence intensity and distribution in both groups were compared.

RESULTS(1) There was blocking and gathered fluorescent sign in both groups when ICG was injected immediately. With the extension of observation time, the fluorescence brightness and area were increased until 30 min of injection. This was in accordance with known metabolism activities of ICG in the liver. (2) At 10 min and 20 min of EA and after needle withdrawal, compared with the control group, the fluorescence intensity in the liver of the EA group was weaker and the distribution area was smaller. (3) According to quantitative statistics of fluorescence intensity, at each time point of treatment, the value in the control group was higher than that in the EA group, which appeared regularly all along. As for the total mean difference of ICG fluorescence intensity at all time points, there was statistical difference between the two groups (P < 0.05).

CONCLUSIONEA could reduce fluorescence intensity and lasting time of ICG, indicating that EA accelerates metabolic process of ICG in the liver. Also it is feasible to apply animal in vivo fluorescence imaging technology to the principle research of acupuncture effect.

Animals ; Electroacupuncture ; Female ; Fluorescent Dyes ; chemistry ; metabolism ; Indocyanine Green ; chemistry ; metabolism ; Liver ; chemistry ; metabolism ; Male ; Mice ; Whole Body Imaging

Redox signal transduction, especially the oxidative modification of proein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection of protein thiols and their oxidative modification in living cells. In this article, we review the current chemical strategies for the detection and quantification of protein thiols and related cysteine oxidation. We also look into the future of the development of fluorescent probes for protein thiols and their potential application in the research of reactive cysteine proteomes and early detection of redox-related diseases.
Animals ; Cysteine ; metabolism ; Fluorescent Dyes ; Humans ; Nitrosation ; Oxidation-Reduction ; Proteins ; chemistry ; metabolism ; Reactive Nitrogen Species ; metabolism ; Reactive Oxygen Species ; metabolism ; Sulfenic Acids ; analysis ; Sulfhydryl Compounds ; analysis ; chemistry ; metabolism

OBJECTIVE: To investigate the relationship between postmortem interval (PMI) and the metabolic law of the amount of DNA in cells. METHODS: After different PMI from the heart, liver, spleen and kidney were taken into pieces respectively, then centrifuged and digested to get suspending cells fluid. The amount of DNA of rats'viscera were detected by flow cytometry after stained by fluorescence, and also inspect the amount of DNA in different periods to find out the law of its variation. RESULTS: It showed a descendent trend of the amount of DNA in cells after different PMI, especially in spleen. CONCLUSION The amount of DNA of all the viscera grows downwards after death, this might be applyed in forensic estimation of PMI.
Animals ; Cell Nucleus/metabolism* ; DNA/metabolism* ; Flow Cytometry ; Kidney/metabolism* ; Liver/metabolism* ; Postmortem Changes ; Rats ; Rats, Sprague-Dawley ; Rosaniline Dyes ; Spleen/metabolism* ; Time Factors

OBJECTIVE: To study the relationship between degradation of marrow DNA and late postmortem interval (PMI). METHODS: Marrow were left on natural condition for 0,1,3,5,7 day after death respectively, Marrow DNA were detected by using Feulgen staining and computerized image analysis. RESULTS: The content of marrow DNA could be detected till 7 days after death yet. CONCLUSION The degradation of marrow DNA may be used on estimation the late PMI.
Bone Marrow/metabolism* ; Cell Nucleus/metabolism* ; DNA/metabolism* ; Humans ; Image Processing, Computer-Assisted ; In Vitro Techniques ; Postmortem Changes ; Rosaniline Dyes ; Sternum/metabolism* ; Time Factors

OBJECTIVE: To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22). METHODS: Cultured HT22 cells were pretreated with 30 or 60 μmol/L berberine for 2 h before exposure to 0.5 μmol/L erastin for 8 h, and the cell proliferation, intracellular ferric iron level, changes in intracellular reactive oxygen species (ROS) and cell apoptosis were detected using CCK-8, Fe²⁺ fluorescent probe, fluorescent dye (DAPI) and fluorescent probe (H2DCFH-DA). RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Nrf2, HO-1 and GPX4 in the cells. We further tested the effects of treatments with 2 μmol/L ML385 (a Nrf2 inhibitor), 60 μmol/L berberine and erastin in the cells to explore the protective mechanism of berberine against erastin-induced ferroptosis in the neuronal cells. RESULTS: Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells (P < 0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level (P < 0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells (P < 0.05) and lowered the levels of intracellular ROS and ferric iron content (P < 0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells (P < 0.05), and treatment with ML385 significantly inhibited the Nrf2-HO-1/GPX4 pathway, increased intracellular ROS and ferric iron contents and mitigated the protective effect of berberine against erastin-induced ferroptosis (P < 0.05). CONCLUSION Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/ GPX4 pathway.
Animals ; Berberine/pharmacology* ; Ferroptosis ; Fluorescent Dyes ; Hippocampus/metabolism* ; Iron/metabolism* ; Mice ; NF-E2-Related Factor 2/metabolism* ; Piperazines ; Reactive Oxygen Species/metabolism*

Objective: To study the underlying mechanism of cadmium-induced apoptosis of mouse spermatocytes (GC-2 spd) . Methods: In March 2021, GC-2 spd cells were exposed to different concentrations of CdCl(2) for 24 hours, namely 5 μmol/L CdCl(2) (low-dose) group and 10 μmol/L CdCl(2) (high-dose) group, and unexposed GC-2 spd cells were used as control group. Mitochondrial morphology was observed in the cells stained with Mito-Track Red CMXRos fluorescent probes by confocal microscopy and the mitochrondrial membrane potential was measured by flow cytometry with JC-1 fluorescent probes. Mitochrondrial proteins, cytosolic proteins and total cellular proteins of GC-2 spd cells were extracted using cell mitochondria isolation kit and RIPA buffer, respectively. The expression of mitochondrial homeostasis regulatory proteins (FIS1 and OPA1), and apoptosis-related proteins (Cytochrome c and cleaved Caspase-3) were examined by Western blot. Results: Compared with the cells in the control group, the relative ratio of JC-1 red/green fluorescence signal in the cells of the low-dose and high-dose CdCl(2) groups decreased significantly (0.740±0.071, 0.570±0.028), with a statistically significant difference (P=0.017, 0.004) ; The morphology of mitochondria changed from long tube to point, and the proportion of cells containing point mitochondria increased significantly (45.1%±3.7% and 25.7%±4.9%), the difference was statistically significant (P=0.005, 0.001) ; The relative expression level of mitochondrial FIS1 in cells of low and high dose CdCl(2) groups was significantly higher (1.271±0.120, 1.693±0.155), the difference was statistically significant (P=0.046, 0.000) ; The relative expression level of OPA1 decreased significantly (0.838±0.050, 0.682±0.040), and the difference was statistically significant (P=0.049, 0.001). Compared with the control group, the relative expression level of cytochrome c protein in the cytoplasm of cells in the low dose group of CdCl(2) was not significantly increased (1.249±0.151), and the difference was not statistically significant (P=0.075). However, the relative expression level in the cytoplasm of cells in the high dose group of CdCl(2) was significantly increased (2.355±0.110), and the difference was statistically significant (P=0.000) ; The relative expression level of Cytochrome c in mitochondria of low and high dose CdCl(2) groups decreased significantly (0.681±0.043, 0.619±0.114), with a statistically significant difference (P=0.004, 0.001) ; Moreover, the level of cleaved Caspase-3 protein in cells gradually increased (5.486±0.544, 11.493±1.739), the difference was statistically significant (P=0.004, 0.000) . Conclusion: Cadmium induced cleaved Caspase-3 mediated apoptosis of GC-2 spd cells via promoting mitochrondrial fission and the release of Cytochrome c from the mitochrondria to the cytosol.
Male ; Mice ; Animals ; Mitochondrial Dynamics ; Caspase 3/metabolism* ; Cadmium/toxicity* ; Cytochromes c/metabolism* ; Fluorescent Dyes ; Apoptosis ; Apoptosis Regulatory Proteins/metabolism* ; Membrane Potential, Mitochondrial