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The Bioenergetics of Mammalian Sperm Motility
Published in Claude Gagnon, Controls of Sperm Motility, 2020
Bovine cauda epididymal spermatozoa from organs which had been stored at 4°C for periods up to 24 h were vigorously motile when freshly resuspended, exhibited high fructolytic rates, and contained 10.5 μmol ATP/108 cells. After 1 h, incubation motility and fructolysis declined markedly and the concentration of ATP had increased to 20 μmol/108 cells. The presence of caffeine prevented or restored the decline in motility and maintained a high fructolytic rate. The concentration of ATP declined to about 5 μmol/108 cells. The changes in ATP concentration were accompanied by opposite changes in the concentration of ADP and AMP. It was concluded that glycolysis is mediated by the concentrations of adenine nucleotides acting to modify the activity of glyceraldehyde 3-phosphate dehydrogenase.118 Boar spermatozoa have a comparatively low rate of glycolysis and this was not enhanced by caffeine despite a significant decline in ATP concentration.119
Vitamin C and Cancer
Published in Qi Chen, Margreet C.M. Vissers, Cancer and Vitamin C, 2020
Channing Paller, Tami Tamashiro, Thomas Luechtefeld, Amy Gravell, Mark Levine
While pharmacologic ascorbate appears to have cytotoxic effects on many cancer cells through hydrogen-peroxide-mediated pro-oxidant damage, in a subset of cancer cells, additional related mechanisms have been described. Cytotoxicity may be due to oxidation of ascorbate into an unstable metabolite and reversible oxidized form of ascorbate, dehydroascorbic acid [70]. Tumor cells internally reduce dehydroascorbic acid to ascorbate-triggering glutathione scavenging, inducing oxidative stress, inactivating glyceraldehyde 3-phosphate dehydrogenase, inhibiting glycolytic flux, and ultimately leading to an energy crisis leading to cell death [71,72]. For example, cultured human colorectal cancer cells with KRAS or BRAF mutations were selectively killed by pharmacologic ascorbate by depletion of intracellular glutathione. This is followed by inactivation of glyceraldehyde 3-phosphate dehydrogenase, leading to inhibition of glycolysis and death in cancer cells highly dependent on glycolysis [71]. Pharmacologic ascorbate can also induce metabolic stress by depletion of NAD in several cancer cell lines [73,74].
Oxidative Stress and the Effects of Dietary Supplements on Glycemic Control in Type 2 Diabetes
Published in Emmanuel C. Opara, Sam Dagogo-Jack, Nutrition and Diabetes, 2019
It is clear that oxidative stress is strongly associated with type 2 diabetes, as shown by various studies using different approaches, as outlined in the preceding section. The crucial question from these studies showing association between oxidative stress and type 2 diabetes is the role, if any, that oxidative stress may play in the pathogenesis of type 2 diabetes. To address this question, it is pertinent to review the metabolic pathways of glucose disposal. The primary pathway of glucose metabolism is glycolysis, through which pyruvate is generated and enters the second pathway, known as the Krebs cycle, for complete oxidation [47]. The complete oxidation of glucose to yield ATP is achieved by oxidative phosphorylation that is coupled to an electron transport chain. In the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase enzyme catalyzes the degradation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. This enzyme is a heme-containing protein that can be inhibited when oxidized severely by a burden of oxidants [48,49]. Also, the cytochrome enzymes of the electron transport chain contain the transition metal copper (Cu++), which can also be inhibited when oxidized by the abundance of ROS. The consequence of these blockages to glucose metabolism by oxidative stress would contribute to an elevation of blood glucose or hyperglycemia.
Feasibility of olfactomedin 4 as a molecular biomarker for early diagnosis of gastric neoplasia after intestinal metaplasia
Published in Scandinavian Journal of Gastroenterology, 2023
Lixing Pang, Xin Yan, Dongxing Su, Xianbin Wu, Haixing Jiang
A quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the mRNA expression level of OLFM4. Briefly, the total RNA was extracted from the tissues of GS, GC, and GCP using a TRIzol™ reagent. The RNA concentration and purity were detected by a spectrophotometer. The PCR amplification was conducted on cDNA synthesized from the total RNA using a reverse transcription kit. The OLFM4 primers were synthesized by Sangon Biotech (Shanghai) Co. Ltd. and sequenced as forwarding 5′-TAGGCAGCGGAGGTTCTGTGTC-3′ and reverse 5′-AATTCCAAGCGTTCCACTC TGTCC- 3′. The PCR was performed using a two-step procedure (step 1: 95 °C for 30 s [pre-denaturation]; step 2: 95 °C for 5 s and 60 °C for 34 s, 40 cycles [amplification]). Glyceraldehyde-3-phosphate dehydrogenase served as the internal control. The relative mRNA expression level of OLFM4 was calculated using the 2−ΔΔCt method.
Genistein affects gonadotrophin-releasing hormone secretion in GT1-7 cells via modulating kisspeptin receptor and key regulators
Published in Systems Biology in Reproductive Medicine, 2022
Jingyuan Xiong, Ye Tian, Aru Ling, Zhenmi Liu, Li Zhao, Guo Cheng
Total RNA was extracted from GT1-7 cells using cell total RNA isolation kit. For cDNA generation, 1 μg of total RNA was reverse transcribed using Prime Script first strand cDNA synthesis kit, according to the instruction from the manufacturer. Primer pairs used for each gene are summarized in Table 1. qRT-PCR was performed using Premix Ex taq kit following the manufacturer’s description. Precision data, including intra-assay and inter-assay CV of the kits, were below 5% according to the manufacturer. PCR cycle numbers at which the fluorescence reached threshold (CT) were recorded using CFX96 touch real-time PCR detection system (Bio-Rad, Hercules, CA). Relative changes in the transcription of target genes were analyzed based on the techniques described in a recent report (Kuwano et al. 2021). Transcriptions of glyceraldehyde-3-phosphate dehydrogenase (GADPH) were determined. Mean CT values of GAPDH across different treatment groups (0, 5, 10, 20 μM genistein) were 16.5 ± 0.4, 16.7 ± 0.8, 16.8 ± 0.5 and 16.6 ± 0.6, respectively, justifying the use of GADPH as the internal control. The experiments were performed in triplicates.
Histological and Biochemical Changes in Adult Male Rat Liver after Spinal Cord Injury with Evaluation of the Role of Granulocyte-Colony Stimulating Factor
Published in Ultrastructural Pathology, 2020
Dalia A. Mohamed, Noura Mostafa Mohamed, Shaimaa Abdelrahaman
PCR was conducted in a final volume of 25 μl consisting of 1 μl cDNA, 1 μl of 10 pM of each primer (forward and reverse), and 12.5 μl PCR master mix (Promega Corporation, Madison, WI, USA). The volume was brought up to 25 μl using sterilized deionized water. PCR was carried out using Bio-Rad T100™ Thermal Cycle machine with a cycle sequence of 94℃ for 5 min, followed by variable cycles, each of which consists of denaturation at 94℃ for 1 min, annealing at the specific temperature corresponding to each primer of IL1B, IL 10 and TNF-α and extension at 72℃ for 1 min with an additional final extension at 72℃ for 7 min. The expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was examined as a reference. The expression of IL1B, IL10 and TNF-α was reported as the D cycle threshold (DCt) value (Table 1).