Phytochemical mediated modulation of COX-3 and NFκB for the management and treatment of arthritis.

In this study, we investigated whether zerumbone (ZBN), ellagic acid (ELA) and quercetin (QCT), the plant-derived components, can modulate the role of COX-3 or cytokines liable in arthritic disorder. Initially, the effect of ZBN, ELA, and QCT on inflammatory process was investigated using in-vitro models. In-silico docking and molecular dynamics study of these molecules with respective targets also corroborate with in-vitro studies. Further, the in-vivo anti-arthritic potential of these molecules in Complete Freund's adjuvant (CFA)-induced arthritic rats was confirmed. CFA increases in TNF-α and IL-1ß levels in the arthritic control animals were significantly (***p < 0.001) attenuated in the ZBN- and ELA-treated animals. CFA-induced attenuation in IL-10 levels recovered under treatment. Moreover, ELA attenuated CFA-induced upregulation of COX-3 and ZBN downregulated CFA-triggered NFκB expression in arthritic animals. The bonding patterns of zerumbone in the catalytic sites of targets provide a useful hint in designing and developing suitable derivatives that can be used as a potential drug. To our best knowledge, the first time we are reporting the role of COX-3 in the treatment of arthritic disorders which could provide a novel therapeutic approach for the treatment of inflammatory disorders.

Influence of photoperiod variations on the mRNA expression pattern of melatonin bio-synthesizing enzyme genes in the pineal organ and retina: A study in relation to the serum melatonin profile in the tropical carp Catla catla.

Surface-dwelling C. catla were exposed to different photoperiods (8L:16D, 12L:12D, 12D:12L and 16L:8D) and the mRNA level profile of enzymes involved in melatonin synthesis was evaluated in the pineal gland and retina. Furthermore, a comparative analysis of the serum melatonin profile with the mRNA level was also performed. The results indicated diurnal variations in the transcripts of tph1, aanat and hiomt in the pineal organ and retina, and these variations change with the change in lighting regime. The serum melatonin profile showed rhythmicity in the natural photoperiod, but the serum melatonin level increased proportionally with increasing daylength. In short photoperiods, the peak value (though lower than in long photoperiods) of melatonin maintains a longer duration in serum. Moreover, the comparative analysis revealed a similar profile of mRNA of pineal aanat1 and aanat2 with serum melatonin under the same lighting conditions. This indicates that serum melatonin is produced by the pineal gland. Our results specify the importance of day length and the timing of onset or offset of the dark for maintaining the oscillating levels of serum melatonin and mRNA levels of melatonin biosynthesizing enzyme genes in the pineal organ and retina as well. The findings in this study highlight the distinctive pattern of mRNA levels in the pineal organ and retina under different photoperiods. The pineal melatonin biosynthesizing enzyme genes showed a similar pattern with serum melatonin levels while the retinal genes changed dramatically with photoperiod. We also revealed a light-dependent transcriptional regulation of pineal aanat genes in C. catla. Moreover, our results suggest that ALAN and skyglow can influence the levels of serum melatonin and its biosynthesis, resulting in desynchronization of the entire biological clock as well as the overall physiology of the animal.

Artificial Light at Night (ALAN): A Potential Anthropogenic Component for the COVID-19 and HCoVs Outbreak.

The origin of the coronavirus disease 2019 (COVID-19) pandemic is zoonotic. The circadian day-night is the rhythmic clue to organisms for their synchronized body functions. The "development for mankind" escalated the use of artificial light at night (ALAN). In this article, we tried to focus on the possible influence of this anthropogenic factor in human coronavirus (HCoV) outbreak. The relationship between the occurrences of coronavirus and the ascending curve of the night-light has also been delivered. The ALAN influences the physiology and behavior of bat, a known nocturnal natural reservoir of many Coronaviridae. The "threatened" and "endangered" status of the majority of bat species is mainly because of the destruction of their proper habit and habitat predominantly through artificial illumination. The stress exerted by ALAN leads to the impaired body functions, especially endocrine, immune, genomic integration, and overall rhythm features of different physiological variables and behaviors in nocturnal animals. Night-light disturbs "virus-host" synchronization and may lead to mutation in the genomic part of the virus and excessive virus shedding. We also proposed some future strategies to mitigate the repercussions of ALAN and for the protection of the living system in the earth as well.

Procyanidin A2, an anti-diabetic condensed tannin extracted from Wendlandia glabrata, reduces elevated G-6-Pase and mRNA levels in diabetic mice and increases glucose uptake in CC1 hepatocytes and C1C12 myoblast cells.

To reduce the global burden of diabetes in an affordable way great attention has been paid to the search for functional foods and herbal remedies. One of the most popularly used functional foods in the North Eastern region of India is tender shoots of Wendlandia glabrata DC. In the current study identification of active anti-diabetic constituent of the tender shoots of W. glabrata was guided through α-glucosidase inhibition and procyanidin A2 was identified with IC50 0.27 ± 0.01 µg mL-1 making it potential source for postprandial management of DM type 2. The study has also demonstrated procyanidin A2 as a potent anti-diabetic agent that exhibits significant glucose-6-phosphatase inhibitory activities and downregulated mRNA level in diabetic mice as well as increases glucose uptake in hepatocytes and myoblast cells. This study revealed that easily available tender shoots of W. glabrata could be used to make specific dietary recommendations for consumption for affordable management of diabetes.

Artificial Light at Night (ALAN), an alarm to ovarian physiology: A study of possible chronodisruption on zebrafish (Danio rerio).

The ALAN is drawing the attention of researchers and environmentalists for its ever-increasing evidence on its capacity of "desynchronization" of organismal physiology. Photoperiod and circadian cycles are critical parameters to influence the biology of reproduction in several animals, including fish. The present study is the first proof of the development of an ovarian tumour with the effect of light in zebrafish (Danio rerio), an excellent model for circadian-related studies. Results of three experimental conditions, continuous light for one week, LLW, one month, LLM, and for one year, LLY revealed a clear desynchronization of clock associated genes (Clock1a, Bmal1a, Per2, and Cry2a). Interestingly, loss of rhythmicity and low concentration of melatonin found in these conditions in whole brain, retina, ovary, and serum through ELISA. RNA-Seq data of ovarian samples revealed the upregulation of Mid2, Tfg, Irak1, Pim2, Tradd, Tmem101, Nfkbib genes and ultimately increase the expression of NF-κB, a cellular transformer for tumourigenesis, confirmed by the western blot. The appearance of TNFα, inflammatory cytokines and activator of NF-κB also increased. Histology approved the formation of thecoma and granulosa cell tumour in the one year exposed ovarian sample. The whole transcriptome data analysis revealed 1791 significantly upregulated genes in an ovarian tumour. Among these genes, DAVID functional annotation tool identified 438 genes, directly linked to other physiological disorders. This study evidenced of an ovarian tumour induced by ALAN in zebrafish.

Interaction of melatonin and gonadotropin-inhibitory hormone on the zebrafish brain-pituitary-reproductive axis.

Circadian cycles and photoperiod are known to influence reproductive physiology in several animals. Neuropeptides, such as gonadotropin-inhibitory hormone (GNIH) and gonadotropin-releasing hormone (GNRH), are influenced by melatonin in birds and mammals. The present study demonstrates the role of melatonin in oocyte maturation in the zebrafish (Danio rerio), via the brain-pituitary-reproductive axis, under different photic conditions. Melatonin was significantly higher both in the whole brain and ovary under continuous dark (DD) compared to continuous light (LL) conditions. Transcription of gnih in the brain was high in LL, but low in DD; similarly, melatonin exogenous treatment reduced gnih in cultured brain in a dose-dependent manner. Expression of gnrh3, however, was high in both continuous photic conditions (DD and LL), whereas fshb and lhb were high only during DD. kiss2, another neuropeptide, was high in LL, but kiss1 remain unchanged among the conditions. At the gonad level, expression of fshr, lhcgr, mtnr1aa, and mtnr1ab tracked with the expression of their respective ligand in DD and LL. The expression of mprb is high in DD ovary, although intra-ovarian growth factors (tgfb1a and bmp15) were low. The measured increased percentages of germinal vesicle breakdown, expression of Cyclin B1, and reduced Cdc2p34 phosphorylation are consistent with increased maturation in the dark. Our study thus links melatonin to the inhibition of gnih in the brain-pituitary-reproductive axis of zebrafish in response to photic conditions.

Melatonin biosynthesizing enzyme genes and clock genes in ovary and whole brain of zebrafish (Danio rerio): Differential expression and a possible interplay.

The present study on zebrafish (Danio rerio) is the first attempt to demonstrate the circadian mRNA expression of melatonin biosynthesizing enzyme genes (Tph1a, Aanat1, Aanat2 and Hiomt) and clock associated genes (Bmal1a, Clock1a, Per1b, Per2 and Cry2a) in the ovary with a comparison to whole brain in normal (LD=12h L:12h D) and altered photic conditions (continuous dark, DD; continuous light, LL). Moreover, the present study also confirmed the ability of zebrafish ovary to biosynthesize melatonin both in vivo and in vitro with a significant difference at day and night. qRT-PCR analysis of genes revealed a dark acrophase of Aanat2 in both organs while Tph1 is in whole brain in LD condition. On the contrary, Bmal1a and Clock1a giving their peak in light, thereby showing a negative correlation with Tph1a and Aanat2. In LD-ovary, the acrophase of Tph1a, Bmal1a and Clock1a is in light and thus display a positive correlation. This trend of relationship in respect to Tph1a is not changing in altered photic conditions in both organs (except in DD-ovary). On the other hand this association for Aanat2 is varying in ovary under altered photic conditions but only in DD-whole brain. Both in LD and LL the expression of Aanat2 in brain presenting an opposite acrophase with both Bmal1a and Clock1a of ovary and consequently displaying a strong negative correlation among them. Interestingly, all ovarian clock associated genes become totally arrhythmic in DD, representing a loss of correlation between the melatonin synthesizing genes in brain and clock associated genes in ovary. The result is also indicating the formation of two heterodimers namely Clock1a:Bmal1a and Per2:Cry2a in the functioning of clock genes in both organs, irrespective of photic conditions, as they are exhibiting a strong significant positive correlation. Collectively, our data suggest that ovary of zebrafish is working as peripheral oscillator having its own melatonin biosynthesizing machinery and signifying a possible correlation with central oscillating system in various photic conditions.

Rhinovirus Delays Cell Repolarization in a Model of Injured/Regenerating Human Airway Epithelium.

Rhinovirus (RV), which causes exacerbation in patients with chronic airway diseases, readily infects injured airway epithelium and has been reported to delay wound closure. In this study, we examined the effects of RV on cell repolarization and differentiation in a model of injured/regenerating airway epithelium (polarized, undifferentiated cells). RV causes only a transient barrier disruption in a model of normal (mucociliary-differentiated) airway epithelium. However, in the injury/regeneration model, RV prolongs barrier dysfunction and alters the differentiation of cells. The prolonged barrier dysfunction caused by RV was not a result of excessive cell death but was instead associated with epithelial-to-mesenchymal transition (EMT)-like features, such as reduced expression of the apicolateral junction and polarity complex proteins, E-cadherin, occludin, ZO-1, claudins 1 and 4, and Crumbs3 and increased expression of vimentin, a mesenchymal cell marker. The expression of Snail, a transcriptional repressor of tight and adherence junctions, was also up-regulated in RV-infected injured/regenerating airway epithelium, and inhibition of Snail reversed RV-induced EMT-like features. In addition, compared with sham-infected cells, the RV-infected injured/regenerating airway epithelium showed more goblet cells and fewer ciliated cells. Inhibition of epithelial growth factor receptor promoted repolarization of cells by inhibiting Snail and enhancing expression of E-cadherin, occludin, and Crumbs3 proteins, reduced the number of goblet cells, and increased the number of ciliated cells. Together, these results suggest that RV not only disrupts barrier function, but also interferes with normal renewal of injured/regenerating airway epithelium by inducing EMT-like features and subsequent goblet cell hyperplasia.

Daily and Seasonal Expression Profile of Serum Melatonin and Its Biosynthesizing Enzyme Genes (tph1, aanat1, aanat2, and hiomt) in Pineal Organ and Retina: A Study under Natural Environmental Conditions in a Tropical Carp, Catla catla.

The tropical carp Catla catla is gaining importance for the studies of the impact of environmental changes on aquatic animals due to its surface dwelling habitat. To date, no information is available on the transcriptional profile of melatonin biosynthesizing enzyme genes in any tropical carp under either natural or artificial photothermal conditions in pineal and retina. The present study is an attempt to demonstrate the temporal pattern of expression of melatonin biosynthesizing enzyme genes, tryptophan hydroxylase 1 (tph1), arylalkylamine N-acetyltransferase (aanat1 and aanat2), and hydroxyindole-O-methyltransferase (hiomt) collectively and simultaneously in pineal organ and retina in tropical fish, C. catla, on a daily and seasonal basis under natural environmental conditions along with the serum melatonin levels. Depending upon the changes of the natural photothermal conditions, in four phases of an annual cycle, the variation and/or shifting of the rhythm parameters of different melatonin biosynthesizing enzyme genes in these two organs are different. Moreover, relative expression of these genes varies based on tissue and season. The serum melatonin levels correspond to the expression pattern of pineal aanat2 and hiomt. This finding indicates a possible organization of melatonin biosynthesizing enzyme genes with reproductive phases differently in these two photoreceptive organs for maintaining its physiological functions.

Melatonin: a potent candidate in the regulation of fish oocyte growth and maturation.

Recent studies on several fish species, especially carp, implicated pineal hormone melatonin (N-acetyl-5-methoxytryptamine) as a potent candidate in the regulatory mechanism of seasonal reproduction. Under natural conditions, the temporal pattern of serum melatonin varied with daily light-dark cycle and the reproductive status of the fish as well. Carefully controlled study revealed that exogenous administration of melatonin may result in stimulation or inhibition or no influences at all on the gonadal functions depending on the reproductive status of fish. Cross-talk between the melatonin and ovarian steroid has been evident from in vitro study, in which melatonin accelerated the action of 17α,20ß-dihydroxy-4-pregnen-3-one or maturation inducing hormone (MIH) on meiotic cell cycle resumption in carp oocytes by formation of maturation promoting factor (MPF) - a complex of two proteins, cyclin B and cyclin dependant kinase Cdk1. While several lines of evidence suggest melatonin effects on hypothalamo-hypophyseal-gonadal axis, localization and dynamics of a 37-kDa melatonin receptor protein in carp oocytes argued in favor of extra-hypothalamic direct action of melatonin on fish reproduction. A recent study in carp indicated that influences of an identical regimen of photoperiods in different parts of annual cycle on ovarian functions vary in relation to the profiles of serum melatonin, but not to any rhythm parameters of MT1 or MT2 receptors on the gonad or brain. The purpose of this short review is to bring together the current knowledge on the biological effects of melatonin on fish reproduction mainly focusing the recent findings on carp.

Rhinovirus-induced barrier dysfunction in polarized airway epithelial cells is mediated by NADPH oxidase 1.

Previously, we showed that rhinovirus (RV), which is responsible for the majority of common colds, disrupts airway epithelial barrier function, as evidenced by reduced transepithelial resistance (R(T)), dissociation of zona occludins 1 (ZO-1) from the tight junction complex, and bacterial transmigration across polarized cells. We also showed that RV replication is required for barrier function disruption. However, the underlying biochemical mechanisms are not known. In the present study, we found that a double-stranded RNA (dsRNA) mimetic, poly(I:C), induced tight junction breakdown and facilitated bacterial transmigration across polarized airway epithelial cells, similar to the case with RV. We also found that RV and poly(I:C) each stimulated Rac1 activation, reactive oxygen species (ROS) generation, and Rac1-dependent NADPH oxidase 1 (NOX1) activity. Inhibitors of Rac1 (NSC23766), NOX (diphenylene iodonium), and NOX1 (small interfering RNA [siRNA]) each blocked the disruptive effects of RV and poly(I:C) on R(T), as well as the dissociation of ZO-1 and occludin from the tight junction complex. Finally, we found that Toll-like receptor 3 (TLR3) is not required for either poly(I:C)- or RV-induced reductions in R(T). Based on these results, we concluded that Rac1-dependent NOX1 activity is required for RV- or poly(I:C)-induced ROS generation, which in turn disrupts the barrier function of polarized airway epithelia. Furthermore, these data suggest that dsRNA generated during RV replication is sufficient to disrupt barrier function.

Quercetin prevents progression of disease in elastase/LPS-exposed mice by negatively regulating MMP expression.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis, emphysema and irreversible airflow limitation. These changes are thought to be due to oxidative stress and an imbalance of proteases and antiproteases. Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. We hypothesized that quercetin reduces lung inflammation and improves lung function in elastase/lipopolysaccharide (LPS)-exposed mice which show typical features of COPD, including airways inflammation, goblet cell metaplasia, and emphysema. METHODS: Mice treated with elastase and LPS once a week for 4 weeks were subsequently administered 0.5 mg of quercetin dihydrate or 50% propylene glycol (vehicle) by gavage for 10 days. Lungs were examined for elastance, oxidative stress, inflammation, and matrix metalloproteinase (MMP) activity. Effects of quercetin on MMP transcription and activity were examined in LPS-exposed murine macrophages. RESULTS: Quercetin-treated, elastase/LPS-exposed mice showed improved elastic recoil and decreased alveolar chord length compared to vehicle-treated controls. Quercetin-treated mice showed decreased levels of thiobarbituric acid reactive substances, a measure of lipid peroxidation caused by oxidative stress. Quercetin also reduced lung inflammation, goblet cell metaplasia, and mRNA expression of pro-inflammatory cytokines and muc5AC. Quercetin treatment decreased the expression and activity of MMP9 and MMP12 in vivo and in vitro, while increasing expression of the histone deacetylase Sirt-1 and suppressing MMP promoter H4 acetylation. Finally, co-treatment with the Sirt-1 inhibitor sirtinol blocked the effects of quercetin on the lung phenotype. CONCLUSIONS: Quercetin prevents progression of emphysema in elastase/LPS-treated mice by reducing oxidative stress, lung inflammation and expression of MMP9 and MMP12.

Melatonin formation in mammals: in vivo perspectives.

Melatonin is a hormone secreted from the pineal gland specifically at night and contributes to a wide array of physiological functions in mammals. Melatonin is one of the most well understood output of the circadian clock located in the suprachiasmatic nucleus. Melatonin synthesis is controlled distally via the circadian clock located in the suprachiasmatic nucleus and proximally regulated by norepinephrine released in response to the circadian clock signals. To understand melatonin synthesis in vivo, we have performed microdialysis analysis of the pineal gland, which monitors melatonin as well as the precursor (serotonin) and intermediate (N-acetylserotonin) of melatonin synthesis in freely moving animals in realtime at high resolution. Our data revealed a number of novel features of melatonin production undetected using conventional techniques, which include (1) large inter-individual variations of melatonin onset timing; (2) circadian regulation of serotonin synthesis and secretion in the pineal gland; and (3) a revised view on the rate-limiting step of melatonin formation in vivo. This article will summarize the main findings from our laboratory regarding melatonin formation in mammals.

Posttranscriptional regulation of pineal melatonin synthesis in Octodon degus.

Small laboratory animals have provided significant information about melatonin regulation, yet most of these organisms are nocturnal and regulate melatonin synthesis by mechanisms that diverge from those of humans. For example, in all rodents examined, melatonin secretion occurs with a time lag of several hours after the onset of darkness; in addition, arylalkylamine N-acetyltransferase (AANAT), the key enzyme in melatonin synthesis, displays dynamic transcriptional activation specifically at night in all rodents studied to date. In ungulates and primates including humans, on the other hand, melatonin secretion occurs immediately during the early night and is controlled by circadian posttranscriptional regulation of AANAT. We hypothesize that the diurnal Octodon degus (an Hystricognath rodent) could serve as an improved experimental model for studies of human melatonin regulation. To test this, we monitored melatonin production in degus using pineal microdialysis and characterized the regulation of melatonin synthesis by analyzing degu Aanat. Degu pineal melatonin rises with little latency at night, as in ungulates and primates. In addition, degu Aanat mRNA expression displays no detectable diurnal variation, suggesting that, like ungulates and primates, melatonin in this species is regulated by a posttranscriptional mechanism. Compared with AANAT from all rodents examined to date, the predicted amino acid sequence of degu AANAT is phylogenetically more closely related to ungulate and primate AANAT. These data suggest that Octodon degus may provide an ideal model system for laboratory investigation of mechanisms of melatonin synthesis and secretion in diurnal mammals.

Localization and dynamics of Mel(1a) melatonin receptor in the ovary of carp Catla catla in relation to serum melatonin levels.

We studied the localization, sub-cellular distribution and daily rhythms of a 37 kDa melatonin receptor (Mel(1a)R) in the ovary to assess its temporal relationship with the serum melatonin levels in four different reproductive phases in carp Catla catla. Our immunocytochemical study accompanied by Western blot analysis of Mel(1a)R in the ovary revealed that the expression of this 37-kDa protein was greater in the membrane fraction than in the cytosol. Ovarian Mel(1a)R protein peaked at midnight and fell at midday in each reproductive phase. Conversely, serum melatonin levels in the same fish demonstrated a minimum diurnal value at midday in all seasons, but a peak at midnight (during pre-spawning, spawning, and post-spawning phases) or at late dark phase (during preparatory phase). In an annual cycle, band intensity of Mel(1a)R protein showed a maximum at night in the spawning phase and a minimum in the post-spawning phase, demonstrating an inverse relationship with the levels of serum melatonin. Our data provide first evidence of the presence of Mel(1a) melatonin receptor in carp ovary and offer interesting perspectives especially for the study of the mechanisms of the control of its rhythmicity and its response to external factors.

Posttranslational regulation of TPH1 is responsible for the nightly surge of 5-HT output in the rat pineal gland.

Serotonin (5-hydroxytryptamine, 5-HT), a precursor for melatonin production, is produced abundantly in the pineal gland of all vertebrate animals. The synthesis of 5-HT in the pineal gland is rate limited by tryptophan hydroxylase 1 (TPH1) whose activity displays a twofold increase at night. Earlier studies from our laboratory demonstrate that pineal 5-HT secretion exhibits dynamic circadian rhythms with elevated levels during the early night, and that the increase is controlled by adrenergic signaling at night. In this study, we report that (a) 5-HT total output from the pineal gland and TPH1 protein levels both display diurnal rhythms with a twofold increase at night; (b) stimulation of cAMP signaling elevates 5-HT output in vivo; (c) 5-HT total output and TPH1 protein content in rat pineal gland are both acutely inhibited by light exposure at night. Consistent with these findings, molecular analysis of TPH1 protein revealed that (a) TPH1 is phosphorylated at the serine 58 in vitro and in the night pineal gland; and (b) phosphorylation of TPH1 at this residue is required for cAMP-enhanced TPH1 protein stability. These data support the model that increased nocturnal 5-HT synthesis in the pineal gland is mediated by the phosphorylation of TPH1 at the serine 58, which elevates the TPH1 protein content and activity at night.

Influence of serotonin on the action of melatonin in MIH-induced meiotic resumption in the oocytes of carp Catla catla.

The influences of serotonin (5-hydroxytryptamine) on the action of melatonin (N-acetyl-5-methoxytryptamine) in MIH (maturation inducing hormone)-induced meiotic resumption were evaluated in the oocytes of carp Catla catla using an in vitro model. Oocytes from gravid female carp were isolated and incubated separately in Medium 199 containing either (a) only melatonin (MEL; 100 pg/mL), or (b) only serotonin (SER; 100 pg/mL), or (c) only MIH (1 microg/mL), or (d) MEL and MIH (e) or MEL (4 h before) and MIH, or (f) MEL and SER, (g) or SER and MIH, or (h) SER (4 h before) and MIH, or (i) luzindole (L-antagonist of MEL receptors; 10 microM) and MEL, or (j) MEL, L and MIH, or (k) MEL (4 h before), L and MIH, or (l) metoclopramide hydrochloride (M-antagonist of SER receptors; 10 microM) and SER, or (m) M, MEL, SER, or (n) M, SER and MIH, or (o) M, SER (4 h before) and MIH, or (p) M, MEL SER and MIH, or (q) MEL, L, SER and M, or (r) MEL, L, SER, M, and MIH, or (s) MEL, SER, L and MIH. Control oocytes were incubated in the medium alone. Oocytes were incubated for 4, or 8, or 12, or 16 h and effects were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). At the end of 16 h incubation, 93.24+/-1.57% oocytes underwent GVBD following incubation with only MIH, while incubation with only MEL or only SER resulted in 77.15+/-1.91% or 14.42+/-0.43% GVBD respectively. Interestingly, incubation with MEL 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD (92.58+/-1.10% at 12 h). In contrast, SER, irrespective of its time of application in relation to MIH, resulted in a maximum of 64.57+/-0.86% GVBD. While L was found to reduce the stimulatory actions of melatonin, M suppressed the inhibitory actions of serotonin. In each case, both electrophoretic and immunoblot studies revealed that the rate of GVBD was associated with the rate of formation of maturation promoting factor (a complex of two proteins: a regulatory component--cyclin B and the catalytic component--Cdk1 or cdc2). Collectively, the present study reports for the first time that SER not only inhibits the independent actions of MIH, but also the actions of MEL on the MIH-induced oocytes maturation in carp.

Melatonin in the regulation of annual testicular events in carp Catla catla: evidence from the studies on the effects of exogenous melatonin, continuous light, and continuous darkness.

The physiological significance of melatonin in the regulation of annual testicular events in a major carp Catla catla was evaluated through studies on the effects of graded dose (25, 50, or 100 microg/100 g body wt.) of melatonin exogenously administered for different durations (1, 15, or 30 days) and manipulation of the endogenous melatonin system by exposing the fish to constant darkness (DD) or constant light (LL) for 30 days. An identical experimental schedule was followed during the preparatory (February-March), pre-spawning (April-May), spawning (July-August), and post-spawning (September-October) phases of the annual cycle. Irrespective of the reproductive status of the carp, LL suppressed while DD increased the mid-day and mid-night values of melatonin compared to respective controls. Influences of exogenous melatonin varied in relation to the dose and duration of treatment and the reproductive status of the carp. However, testicular response to exogenous melatonin (at 100 microg, for 30 days) and DD in each reproductive phase was almost identical. Notably, precocious testicular maturation occurred in both DD and melatonin-injected fish during the preparatory phase and in LL carps during the pre-spawning phase. In contrast, testicular functions in both the melatonin-treated and DD fish were inhibited during the pre-spawning and spawning phases, while the testes did not respond to any treatment during the post-spawning phase. In conclusion, this study provided the first experimental evidence that melatonin plays a significant role in the regulation of annual testicular events in a sub-tropical surface-dwelling carp Catla catla, but the influence of this pineal hormone on the seasonal activity of testis varies in relation to the reproductive status of the concerned fish.

Arsenic-induced changes in optic tectal histoarchitecture and acetylcholinesterase-acetylcholine profile in Channa punctatus: amelioration by selenium.

Fish (Channa punctatus Bloch) were exposed in vivo for 14 days to non-lethal doses of As2O3 (10% LC50 and 5% LC50). Several endpoints related to histoarchitectural and acetylcholine-acetylcholinesterase (ACh-AChE) profile in the optic tectum were evaluated. Histological examination showed aggregated, disorganized and necrotic cells with irregular outlines in the different layers of optic tectum in the As-treated fish. The histopathological changes were more pronounced on day 7 than on other days and the damage was found to recover on day 14. ACh content and AChE activity demonstrated the usual inverse trend. Arsenic treatment was associated with a dose-dependent increase in AChE activity on day 1, a decrease on day 2 and reactivation on day 7, returning to the basal level on day 14. In vitro inhibition kinetics were set up to determine I50 (35 microM) concentration of As2O3. The ameliorative potential of selenium on arsenic-mediated inhibition of AChE revealed a positive role of Se, especially when Se preceded As2O3 treatment, either in vitro or in vivo.

Melatonin accelerates maturation inducing hormone (MIH): induced oocyte maturation in carps.

The present communication is an attempt to demonstrate the influence of melatonin on the action of maturation inducing hormone (MIH) on the maturation of oocytes in carps. The oocytes from gravid female major carp Labeo rohita were isolated and incubated separately in Medium 199 containing (a) only MIH (1 microg/ml), (b) only melatonin (at concentrations of 50, 100 or 500 pg/ml), and (c) both melatonin and MIH, but at different time intervals. In the latter group, melatonin was added to the incubating medium either (i) 4 h before addition of MIH, (ii) 2 h before addition of MIH, (iii) co-administered with MIH (0 h interval) or (iv) 2 h after addition of MIH. In each case, oocytes were further incubated for 4, 8, 12 or 16 h post- administration of MIH, and the effects of treatment on oocyte maturation were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). Incubation of oocytes in a medium containing only melatonin did not result in GVBD of any oocyte. Nearly all the oocytes underwent GVBD when incubated with MIH for 16 h. Administration of melatonin along with MIH (at 0 h interval) or 2 h after addition of MIH did not result in any significant change in the rate of GVBD compared to that in a medium containing only MIH. However, it was quite interesting to observe that incubation of oocytes with melatonin especially 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD in the oocytes. Experiments with the oocytes of another major carp Cyprinus carpio following an identical schedule depicted similar results except a difference in the optimum melatonin dose. In L. rohita, 50 pg/ml melatonin had maximum acceleratory effect on MIH-induced GVBD of oocytes, while it was 100 pg/ml in C. carpio. Further study revealed that pre-incubation with melatonin accelerates the action of MIH on the formation of a complex of two proteins (MPF), a regulatory component called cyclin B and the catalytic component protein kinase known as cyclin-dependent kinase, Cdk1. Densitometric analysis of the immunoblot data collected from the melatonin pre-treated MIH incubated oocytes showed that cyclin B level continued to increase even after 4 h of incubation, and reached the peak after 12 h. Moreover, determination of H1 kinase activity as an indicator of MPF activity in oocytes revealed that melatonin pre-incubation considerably increased MIH stimulation of histone H1 phosphorylation as compared to MIH alone. Thus, the present study demonstrates for the first time that prior incubation with melatonin accelerates the action of MIH on carp oocyte maturation.