Alterations in hippocampal serotonergic and INSR function in streptozotocin induced diabetic rats exposed to stress: neuroprotective role of pyridoxine and <Emphasis Type="Italic">Aegle marmelose</Emphasis>

Diabetes and stress stimulate hippocampal 5-HT synthesis, metabolism and release. The present study was carried out to find the effects of insulin, Aegle marmelose alone and in combination with pyridoxine on the hippocampal 5-HT, 5-HT_2A receptor subtype, gene expression studies on 5-HT_2A, 5-HTT, INSR, immunohistochemical studies and elevated plus maze in streptozotocin induced diabetic rats. 5-HT content showed a significant decrease (p < 0.001) and a significant increase (p < 0.001) in 5-HIAA in hippocampus of diabetic rats compared to control. 5-HT receptor binding parameters B_max and K_d showed a significant decrease (p < 0.001) whereas 5-HT_2A receptor binding parameters B_max showed a significant decrease (p < 0.001) with a significant increase (p < 0.05) in K_d in hippocampus of diabetic rats compared to control. Gene expression studies of 5-HT_2A, 5-HTT and INSR in hippocampus showed a significant down regulation (p < 0.001) in diabetic rats compared to control. Pyridoxine treated in combination with insulin and A. marmelose to diabetic rats reversed the 5-HT content, B_max , K_d of 5-HT, 5-HT_2A and gene expression of 5-HT_2A, 5-HTT and INSR in hippocampus to near control. The gene expression of 5-HT_2A and 5-HTT were confirmed by immunohistochemical studies. Behavioural studies using elevated plus maze showed that serotonin through its transporter significantly increased (p < 0.001) anxiety-related traits in diabetic rats which were corrected by combination therapy. Our results suggest that pyridoxine treated in combination with insulin and A. marmelose has a role in the regulation of insulin synthesis and release, normalising diabetic related stress and anxiety through hippocampal serotonergic function. This has clinical significance in the management of diabetes.     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background  

Paradoxical sleep deprivation (PSD) associated with cocaine has been shown to enhance genital reflexes (penile erection-PE and ejaculation-EJ) in Wistar rats. Since hypertension predisposes males to erectile dysfunction, the aim of the present study was to investigate the effects of PSD on genital reflexes in the spontaneously hypertensive rat (SHR) compared to the Wistar strain. We also extended our study to examine how PSD affect steroid hormone concentrations involved in genital events in both experimental models.     

Patterns of cervical metastasis from carcinoma of the oral tongue

Background  

Cancer of the oral tongue is the second most common cancer among males in various parts of India. Despite advances in diagnosis and treatment the failure rates in cancer of the oral tongue are high and survival poor. Majority of these failures occur in untreated neck.     

Ants climb plants because they cannot swim: ant presence on flowers during the flood season reduces the frequency of floral visitors

1. Seasonal changes in environments may not only affect habitat connectivity but may also affect its use by species and their interactions. Thus, during the flood season, ants are forced to develop survival strategies such as vertical plant migration.
2. According to this, it has been hypothesized that the presence of ants may directly affect plant-pollinator interactions.
3. Thus, we asked the following questions: (i) Are floral visitors of Hyptis brevipes expelled due to ant presence on inflorescences during the flood period? (ii) Is the ant effect mediated by the abundance of ants foraging on inflorescences? And, (iii) Does flower abundance predict the abundance of floral visits and ants?
4. We experimentally sampled 59 H. brevipes plants with and without ants during the flooded season, and observed no differences in flower abundance between ant treatments.
5. The probability of detaining floral visitors on H. brevipes increased with ant abundance and exceeded 50% possible repellency, but the probability of visitor deterrence was not related to flower abundance. Furthermore, the abundance of flowers did not predict the number of ants on H. brevipes individuals or the frequency of floral visits.
6. Consequently, ant repelling effects are pronounced when there are more ants foraging on plants. However, the ant repelling effect can be mitigated when plants flourish all year-round and exhibit higher concentrations of flowers in the dry months. Additionally, the different sexual functions of plants may present specific responses due to the explosive pollination mechanism associated with ant effects.

     

The MECP2-TRD domain interacts with the DNMT3A-ADD domain at the H3-tail binding site

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214–228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.     

Microtubule assembly and disassembly at alkaline pH

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of “treadmilling.” The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.     

Tonic GABAergic inhibition of taste-responsive neurons in the nucleus of the solitary tract

The effects of gamma-aminobutyric acid (GABA) and the GABAA receptor antagonist bicuculline methiodide (BICM) on the activity of taste- responsive neurons in the nucleus of the solitary tract (NST) were examined electrophysiologically in urethane-anesthetized hamsters. Single neurons in the NST were recorded extracellularly and drugs (21 nl) were microinjected into the vicinity of the cell via a multibarrel pipette. The response of each cell was recorded to lingual stimulation with 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid and 0.032 M quinine hydrochloride (QHCl). Forty-six neurons were tested for the effects of GABA; the activity of 29 cells (63%) was inhibited by 5 mM GABA. Whether activity was elicited in these cells by repetitive anodal current stimulation (25 microA, 0.5 s, 0.1 Hz) of the tongue (n = 13 cells) or the cells were spontaneously active (n = 13 cells), GABA produced a dose-dependent (1, 2 and 5 mM) decrement in activity. Forty- seven NST neurons were tested for the effects of BICM on their responses to chemical stimulation of the tongue; the responses of 28 cells (60%) were enhanced by 10 mM BICM. The gustatory responses of 26 of these cells were tested with three concentrations (0.2, 2 and 10 mM) of BICM, which produced a dose-dependent increase in both spontaneous activity and taste-evoked responses. Nine of these neurons were sucrose- best, seven were NaCl-best, eight were acid-best and two responded best to QHCl. The responses to all four tastants were enhanced, with no difference among neuron types. For 18 cells that were tested with two or more gustatory stimuli, BICM increased their breadth of responsiveness to their two most effective stimuli. These data show that approximately 60% of the taste-responsive neurons in the rostral NST are inhibited by GABA and/or subject to a tonic inhibitory influence, which is mediated by GABAA receptors. The modulation of these cells by GABA provides a mechanism by which the breadth of tuning of the cell can be sharpened. Modulation of gustatory activity following a number of physiological changes could be mediated by such a GABAergic circuit.      

Induction of eosinophil apoptosis by hydrogen peroxide promotes the resolution of allergic inflammation

Eosinophils are effector cells that have an important role in the pathogenesis of allergic disease. Defective removal of these cells likely leads to chronic inflammatory diseases such as asthma. Thus, there is great interest in understanding the mechanisms responsible for the elimination of eosinophils from inflammatory sites. Previous studies have demonstrated a role for certain mediators and molecular pathways responsible for the survival and death of leukocytes at sites of inflammation. Reactive oxygen species have been described as proinflammatory mediators but their role in the resolution phase of inflammation is poorly understood. The aim of this study was to investigate the effect of reactive oxygen species in the resolution of allergic inflammatory responses. An eosinophilic cell line (Eol-1) was treated with hydrogen peroxide and apoptosis was measured. Allergic inflammation was induced in ovalbumin sensitized and challenged mouse models and reactive oxygen species were administered at the peak of inflammatory cell infiltrate. Inflammatory cell numbers, cytokine and chemokine levels, mucus production, inflammatory cell apoptosis and peribronchiolar matrix deposition was quantified in the lungs. Resistance and elastance were measured at baseline and after aerosolized methacholine. Hydrogen peroxide accelerates resolution of airway inflammation by induction of caspase-dependent apoptosis of eosinophils and decrease remodeling, mucus deposition, inflammatory cytokine production and airway hyperreactivity. Moreover, the inhibition of reactive oxygen species production by apocynin or in gp91^phox−/− mice prolonged the inflammatory response. Hydrogen peroxide induces Eol-1 apoptosis in vitro and enhances the resolution of inflammation and improves lung function in vivo by inducing caspase-dependent apoptosis of eosinophils.Eosinophils express numerous receptors and secrete a wide variety of inflammatory mediators that influence many innate and adaptive immune responses. These multifunctional cells are important in the defense against helminth infection and are involved in the pathogenesis of many eosinophilic dominant allergic diseases.¹ High levels of eosinophil granule proteins (such as major basic protein (MBP)) have been found in bronchoalveolar lavage fluid from patients with asthma and evidence indicates that high-concentration granule products have contributed to the development of airway hyperreactivity (AHR), a cardinal feature of asthma.² Asthma is an inflammatory disease of the airways with participation of many cell types including leukocytes especially eosinophils and lymphocytes.^{3, 4} Activation of these cells (mainly lymphocytes) leads to the release of proinflammatory mediators and cytokines such as leukotriene B₄, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-9 (IL-9), interleukin-13 (IL-13) and colony-stimulating factor granulocyte-macrophage (GM-CSF).^{3, 5, 6, 7} Investigations using preclinical animal models of asthma and clinical studies in patients with asthma have demonstrated that the presence of eosinophils in the lungs are associated with epithelial damage, goblet cell hyperplasia, smooth muscle hypertrophy and airway hyperresponsiveness resulting in airflow limitation which can be fatal.^{3, 8, 9, 10} Recently, anti-IL-5 treatment has been shown to ameliorate lung function in patients with eosinophilic asthma.¹¹Apoptosis of leukocytes is regarded as an important process for the successful resolution of inflammatory responses. Reduced eosinophil apoptosis in bronchoalveolar lavage (BAL) fluid has been shown to correlate positively with severity of asthma.^{3, 12, 13, 14} Indeed, defective leukocyte apoptosis and subsequent removal of apoptotic cells by phagocytes is thought to be important for the initiation and propagation of chronic inflammatory diseases such as asthma.¹⁵ Therefore, a balance in the tissue microenvironment between pro- and antiapoptotic signals is likely to greatly influence the load of eosinophils in the asthmatic lung.¹⁶ Thus, there is a great interest in understanding the mechanisms responsible for the elimination of eosinophils and other leukocytes and inactivation of proinflammatory mediators in inflammatory sites.¹⁷Several molecular pathways have been shown to modulate the survival and death of leukocytes at sites of inflammation, including reactive oxygen species (ROS).¹⁸ ROS are a family of molecules containing oxygen and includes hydrogen peroxide (H₂O₂), superoxide O₂⁻, hydroxyl radical (OH) and nitric oxide (NO).¹⁹ In inflammatory conditions, ROS are increased as they help in neutralizing invading organisms during infection either directly or indirectly by formation of extracellular traps (ETs).²⁰ ROS have traditionally been regarded as quintessentially proinflammatory. However, evidence for ROS-mediated anti-inflammatory actions has been described.²¹ The importance for ROS production in the context of infection can be exemplified in patients with chronic granulomatous disease (CGD) where defective production in ROS results in multiple infections and often early death.^{22, 23} Furthermore, studies in mouse models have shown that NADPH oxidase is key for regulating lung inflammation and injury as well as NF-κB activation and downstream cytokine production in response to LPS.²⁴ More recently, our group has demonstrated that NADPH oxidase-derived H₂O₂ is directly linked to induction of apoptosis of neutrophils and resolution of inflammation in a model of antigen-induced arthritis.¹⁸ However, the role of ROS in the context of the resolution of allergic inflammation is still unknown.Here, we evaluated whether H₂O₂ drives apoptosis of eosinophils and thereby influences the resolution of established eosinophilic inflammation and reduction of airflow obstruction. Our study provides evidence that H₂O₂ is released during allergic inflammation in a gp91^phox−/−-dependent manner and induces a caspase-dependent proapoptotic effect in eosinophils, thus having a crucial role in the resolution of allergic inflammation.     

Aldose reductase deficiency in mice protects from ragweed pollen extract (RWE)-induced allergic asthma

Background

Childhood hospitalization related to asthma remains at historically high levels, and its incidence is on the rise world-wide. Previously, we have demonstrated that aldose reductase (AR), a regulatory enzyme of polyol pathway, is a major mediator of allergen-induced asthma pathogenesis in mouse models. Here, using AR null (AR^-/-) mice we have investigated the effect of AR deficiency on the pathogenesis of ragweed pollen extract (RWE)-induced allergic asthma in mice and also examined the efficacy of enteral administration of highly specific AR inhibitor, fidarestat.

Methods

The wild type (WT) and AR^-/- mice were sensitized and challenged with RWE to induce allergic asthma. AR inhibitor, fidarestat was administered orally. Airway hyper-responsiveness was measured in unrestrained animals using whole body plethysmography. Mucin levels and Th2 cytokine in broncho-alveolar lavage (BAL) were determined using mouse anti-Muc5A/C ELISA kit and multiplex cytokine array, respectively. Eosinophils infiltration and goblet cells were assessed by H&E and periodic acid Schiff (PAS)-staining of formalin-fixed, paraffin-embedded lung sections. T regulatory cells were assessed in spleen derived CD4⁺CD25⁺ T cells population.

Results

Deficiency of AR in mice led to significantly decreased PENH, a marker of airway hyper-responsiveness, metaplasia of airway epithelial cells and mucus hyper-secretion following RWE-challenge. This was accompanied by a dramatic decrease in infiltration of eosinophils into sub-epithelium of lung as well as in BAL and release of Th2 cytokines in response to RWE-challenge of AR^-/- mice. Further, enteral administration of fidarestat significantly prevented eosinophils infiltration, airway hyper-responsiveness and also markedly increased population of T regulatory (CD4⁺CD25⁺FoxP3⁺) cells as compared to RWE-sensitized and challenged mice not treated with fidarestat.

Conclusion

Our results using AR^-/- mice strongly suggest the role of AR in allergic asthma pathogenesis and effectiveness of oral administration of AR inhibitor in RWE-induced asthma in mice supports the use of AR inhibitors in the treatment of allergic asthma.