Effect of amino acids on inhibition of lactate dehydrogenase-X by gossypol.
Gossypol acetic acid (GAA) has been shown to have male antifertility effects, but there are pronounced differences among animal species. In the search of endogenous effector molecules, which interfere with the functions of GAA, we have studied the in vitro effect of various amino acids on the inhibition of the purified LDH-X by GAA. Histidine, cysteine and glycine were shown to block the effect of GAA. The effects of these amino acids were concentration dependent. Histidine and glycine protection was found to be complex type in which both the Km and Vmax were decreased compared to control. Arginine, glutamic acid, phenylalanine and valine were found to be ineffective against the inhibitory action of GAA. (+info)
NAD- and NADP-dependent 11 beta-hydroxysteroid dehydrogenase isoforms in guinea pig kidney with gossypol inhibition.
AIM: To study the mechanism of gossypol-induced hypokalemia. METHODS: The 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) protein was prepared from guinea pig kidney. The activity of 11 beta-OHSD with NAD or NADP as the coenzyme was measured by HPLC in both control and gossypol treatment. RESULTS: The Vmax and K(m) were 0.64 mmol.h-1/g protein and 0.07 mumol (cortisol) for NAD-dependent 11 beta-OHSD, 1.75 mmol.h-1/g protein and 0.21 mumol (cortisol) for NADP-dependent 11 beta-OHSD, respectively when 80 micrograms of enzyme protein was used. The inhibitory effects of gossypol on these two 11 beta-OHSD isoforms were different. The IC50 (95% confidence limits) were 50.2 (48.3-52.0) mumol of gossypol for NAD-dependent 11 beta-OHSD and 1143 (1098-1188) mumol of gossypol for NADP-dependent 11 beta-OHSD. The Ki was gossypol 96 mmol.L-1 for the former and 340 mmol.L-1 for the latter. CONCLUSION: The NAD-dependent 11 beta-OHSD is a more critical physiologic mechanism than NADP-dependent 11 beta-OHSD for hypokalemia caused by gossypol. (+info)
Inhibitory effect of gossypol on basal and luteinization factor-stimulated progesterone synthesis in porcine granulosa cells.
Gossypol, a polyphenolic aldehyde, inhibits steroidogenesis and the reproductive system in both sexes. The present study was undertaken to investigate whether gossypol may affect progesterone biosynthesis in cultured porcine granulosa cells isolated from small (1-2 mm) follicles (SGC). SGC were cultured with gossypol, NO donor S-nitroso-N-acetylpenicillamine (S-NAP) or the specific NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), in the presence or absence of follicular fluid isolated from large (5-8 mm) follicles (LFF) or conditioned media (CM) of granulosa cells isolated from large follicles (LGC). Gossypol enhanced the nitrite content in culture media of SGC and inhibited basal progesterone secretion by SGC. S-NAP (10(-3) M) inhibited progesterone secretion and enhanced the formation of cGMP by SGC. L-NAME had no effect on progesterone accumulation by SGC. The stimulatory effect of LFF or CM media on progesterone production by SGC in culture was also inhibited by S-NAP (10(-3)) and gossypol (10(-4) M). Moreover, gossypol inhibited forskolin-stimulated progesterone secretion, as well as substrate-enhanced conversion of 22-OH-cholesterol and pregnenolone to progesterone. These results suggest that the inhibitory effect of gossypol on progesterone secretion in culture of SGC may be mediated via NO generation. (+info)
Gossypol induced apoptosis in the human promyelocytic leukemia cell line HL 60.
In human promyelocytic leukemia cell line HL60, apoptosis was induced by treatment with gossypol that is an inhibitor of protein kinase C. Gossypol acetic acid was added to HL 60 cells at 50, 100, 150 and 200 microM concentrations for six hours. Morphological features of apoptosis as well as internucleosomal DNA fragmentation were evaluated by light microscope, agarose gel electrophoresis and spectrofluorometric quantitation. Our results indicated that with the effective concentrations of gossypol (50 and 100 microM), apoptosis was induced in HL 60 cells. (+info)
Effects of diets containing gossypol on reproductive capacity of rainbow trout (Oncorhynchus mykiss).
We evaluated five practical diets in which 0%, 25%, 50%, 75%, and 100% (dietary treatments 1-5) of fish meal protein was replaced by solvent-extracted cottonseed meal protein. Adult rainbow trout (initial average weight 247 +/- 8 g) were fed the diets over a period of 131 days during which a general 2-fold body weight increase occurred. The total diet gossypol concentration (free and protein-bound) showed a gradual increase with increased cottonseed meal substitution. Blood samples were collected on Days 0, 64, 112, and 131 for hematological and steroid hormone determination in plasma of males and females. Hemoglobin content was significantly reduced in fish from treatment 5 (7.9 +/- 0.3 g/dl) in comparison to treatments 1-3 (10.3-10.9 g/dl). After 112 and 131 days of feeding, testis weights, concentrations of testosterone, and 11-ketotestosterone were elevated in fish from dietary treatments 2 and 3 in comparison to control and diets 4 and 5. On Day 71, sperm were collected from 6 fish per dietary treatment to assess sperm quality. No significant differences in sperm concentrations (7.2-9.8 x 10(9)/ml), motility (78-89%), and standardized (300 x 10(5) sperm/egg) fertilizing ability (18.9-22.6% hatched embryos) were found. Total gossypol concentrations in blood plasma differed significantly among treatments, and the levels were among the highest ever recorded in animals fed cottonseed-supplemented diets (2.9 +/- 0.2, 11.7 +/- 4.1, 21.7 +/- 1.4, and 29.9 +/- 3.9 microg/ml, for treatments 2-5, respectively). The major portion of gossypol in blood plasma was protein-bound (81-93%). This was in contrast to minute amounts of gossypol present in seminal plasma, mostly in free form (0.02-0.18 microg/ml), which indicates the presence of a barrier between general circulation and the testis with respect to gossypol distribution in lower vertebrates. Thus, the reproductive parameters of male rainbow trout examined in this study were not significantly affected by feeding cottonseed meal for 131 days. (+info)
Novel effects of gossypol, a chemical contraceptive in man: mobilization of internal Ca(2+) and activation of external Ca(2+) entry in intact cells.
The effect of gossypol on Ca(2+) signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Gossypol evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 2 and 20 microM. The response was decreased by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with gossypol nearly abolished the [Ca(2+)](i) increase induced by carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, and thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with CCCP and thapsigargin only partly inhibited gossypol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 5 microM gossypol in Ca(2+)-free medium. This Ca(2+) entry was decreased by 25 microM econazole, 50 microM SKF96365 and 40 microM aristolochic acid (a phospholipase A(2) inhibitor). Pretreatment with aristolochic acid inhibited 5 microM gossypol-induced internal Ca(2+) release by 55%, but suppression of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) had no effect. Gossypol (5 microM) also increased [Ca(2+)](i) in human bladder cancer cells and neutrophils. Collectively, we have found that gossypol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca(2+) influx from external space. (+info)
Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy.
The accumulation of insoluble protein aggregates in intra and perinuclear inclusions is a hallmark of Huntington's disease (HD) and related glutamine-repeat disorders. A central question is whether protein aggregation plays a direct role in the pathogenesis of these neurodegenerative diseases. Here we show by using a filter retardation assay that the mAb 1C2, which specifically recognizes the elongated polyglutamine (polyQ) stretch in huntingtin, and the chemical compounds Congo red, thioflavine S, chrysamine G, and Direct fast yellow inhibit HD exon 1 protein aggregation in a dose-dependent manner. On the other hand, potential inhibitors of amyloid-beta formation such as thioflavine T, gossypol, melatonin, and rifampicin had little or no inhibitory effect on huntingtin aggregation in vitro. The results obtained by the filtration assay were confirmed by electron microscopy, SDS/PAGE, and MS. Furthermore, cell culture studies revealed that the Congo red dye at micromolar concentrations reduced the extent of HD exon 1 aggregation in transiently transfected COS cells. Together, these findings contribute to a better understanding of the mechanism of huntingtin fibrillogenesis in vitro and provide the basis for the development of new huntingtin aggregation inhibitors that may be effective in treating HD. (+info)
Effect of level of whole cottonseed on intake, digestibility, and performance of growing male goats fed hay-based diets.
Twenty-four purebred Boer (Capra hircus hircus) and 12 male kids of 1/2 Boer breeding (initial BW 21 +/- .5 kg) were used in a randomized complete block design to study the effects of increasing level of whole cottonseed (WCS) on ADG, serum urea N, plasma gossypol, live grades, and intake of DM, CP, NDF, ADF, hemicellulose, cellulose, lignin, ether extract, and free gossypol. A subgroup of 16 purebred goats was used to determine digestibility using a 5-d total fecal collection. Goats were blocked by BW and then assigned at random to one of four diets containing 0, 8, 16, or 24% WCS. All diets contained 71% chopped orchardgrass (Dactylis glomerata L.) hay and 29% concentrate; WCS replaced corn and soybean meal to maintain calculated TDN and CP concentrations at 68 and 15% of DM, respectively. Concentrations of NDF in diet DM were 52.4, 55.9, 59.3, and 62.1% with increasing WCS, and the Ca:P ratio was maintained at 2:0. Goats were given ad libitum access to feed and water. Over the 90-d performance phase, DMI (P < .05), CP intake (P < .02), and ADG (P < .01) decreased linearly with increasing WCS in the diet, whereas ether extract (EE) intake increased in a cubic fashion (P < .01). Gain:feed decreased linearly (P < .02) with increasing level of WCS. Addition of WCS resulted in linear decreases in apparent digestibility coefficients of DM (P < .02) and NDF (P < .05), a linear increase in total plasma gossypol (P < .01), and a quadratic increase in serum urea N (P < .04). Apparent digestibility of CP was not affected by WCS level. At the 16 and 24% WCS levels, EE constituted 4.2 and 4.8% of total DMI, respectively. Adding WCS to diets for growing goats had detrimental effects on animal performance, and, based on the possible negative effects of dietary EE and NDF rather than gossypol, economics should dictate whether to use WCS in feeding programs. (+info)