Decreased GABA receptor in the cerebral cortex of epileptic rats: effect of Bacopa monnieri and Bacoside-A

Abstact

Background

Gamma amino butyric acid (GABA), the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tones that counter balances neuronal excitation. When this balance is perturbed, seizures may ensue.

Methods

In the present study, alterations of the general GABA, GABA_A and GABA_B receptors in the cerebral cortex of the epileptic rat and the therapeutic application of Bacopa monnieri were investigated.

Results

Scatchard analysis of [³H]GABA, [³H]bicuculline and [³H]baclofen in the cerebral cortex of the epileptic rat showed significant decrease in B_max (P < 0.001) compared to control. Real Time PCR amplification of GABA receptor subunits such as GABA_Aά1, GABA_Aγ, GABA_Aδ, GABA_B and GAD where down regulated (P < 0.001) in epileptic rats. GABA_Aά5 subunit and Cyclic AMP responsible element binding protein were up regulated. Confocal imaging study confirmed the decreased GABA receptors in epileptic rats. Epileptic rats have deficit in radial arm and Y maze performance.

Conclusions

Bacopa monnieri and Bacoside-A treatment reverses epilepsy associated changes to near control suggesting that decreased GABA receptors in the cerebral cortex have an important role in epileptic occurrence; Bacopa monnieri and Bacoside-A have therapeutic application in epilepsy management.     

The murine double-stranded RNA-dependent protein kinase PKR is required for resistance to vesicular stomatitis virus

Interferon (IFN)-induced antiviral responses are mediated through a variety of proteins, including the double-stranded RNA-dependent protein kinase PKR. Here we show that fibroblasts derived from PKR(-/-) mice are more permissive for vesicular stomatitis virus (VSV) infection than are wild-type fibroblasts and demonstrate a deficiency in alpha/beta-IFN-mediated protection. We further show that mice lacking PKR are extremely susceptible to intranasal VSV infection, succumbing within days after instillation with as few as 50 infectious viral particles. Again, alpha/beta-IFN was unable to rescue PKR(-/-) mice from VSV infection. Surprisingly, intranasally infected PKR(-/-) mice died not from pathology of the central nervous system but rather from acute infection of the respiratory tract, demonstrating high virus titers in the lungs compared to similarly infected wild-type animals. These results confirm the role of PKR as the major component of IFN-mediated resistance to VSV infection. Since previous reports have shown PKR to be nonessential for survival in animals challenged with encephalomyocarditis virus, influenza virus, and vaccinia virus (N. Abraham et al., J. Biol. Chem. 274:5953-5962, 1999; Y. Yang et al., EMBO J. 14:6095-6106, 1995), our findings serve to highlight the premise that host dependence on the various mediators of IFN-induced antiviral defenses is pathogen specific.     

Environmental Challenges and Physiological Solutions: Comparative Energetic Daily Rhythms of Field Mice Populations from Different Ecosystems

Daily and seasonal variations in physiological characteristics of mammals can be considered adaptations to temporal habitat variables. Across different ecosystems, physiological adjustments are expected to be sensitive to different environmental signals such as changes in photoperiod, temperature or water and food availability; the relative importance of a particular signal being dependent on the ecosystem in question. Energy intake, oxygen consumption (VO₂) and body temperature (T_b) daily rhythms were compared between two populations of the broad-toothed field mouse Apodemus mystacinus, one from a Mediterranean and another from a sub-Alpine ecosystem. Mice were acclimated to short-day (SD) ‘winter’ and long-day (LD) ‘summer’ photoperiods under different levels of salinity simulating osmotic challenges. Mediterranean mice had higher VO₂ values than sub-Alpine mice. In addition, mice exposed to short days had higher VO₂ values when given water with a high salinity compared with mice exposed to long days. By comparison, across both populations, increasing salinity resulted in a decreased T_b in SD- but not in LD-mice. Thus, SD-mice may conserve energy by decreasing T_b during (‘winter’) conditions which are expected to be cool, whereas LD-mice might do the opposite and maintain a higher T_b during (‘summer’) conditions which are expected to be warm. LD-mice behaved to reduce energy expenditure, which might be considered a useful trait during ‘summer’ conditions. Overall, increasing salinity was a clear signal for Mediterranean-mice with resultant effects on VO₂ and T_b daily rhythms but had less of an effect on sub-Alpine mice, which were more responsive to changes in photoperiod. Results provide an insight into how different populations respond physiologically to various environmental challenges.     

Experimental selection of long‐term intracellular mycobacteria

Some intracellular bacteria are known to cause long‐term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long‐term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re‐infection and also with the specific expression of stress‐ and survival‐related genes. Our findings identify bacterial traits implicated in the establishment of long‐term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.     

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α(2) β(2) (Pro100Leu) )

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O(2) affinity compared with normal cells (P(50) = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O(2) affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO(2) , decreasing the rate approximately twofold. These kinetic data help explain the high O(2) affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure.     

LFA-1 on CD4+ T cells is required for optimal antigen-dependent activation in vivo

The leukocyte-specific integrin, LFA-1, plays a critical role in trafficking of T cells to both lymphoid and nonlymphoid tissues. However, the role of LFA-1 in T cell activation in vivo has been less well understood. Although there have been reports describing LFA-1-deficient T cell response defects in vivo, due to impaired migration to lymphoid structures and to sites of effector function in the absence of LFA-1, it has been difficult to assess whether T cells also have a specific activation defect in vivo. We examined the role of LFA-1 in CD4(+) T cell activation in vivo by using a system that allows for segregation of the migration and activation defects through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 Ag-specific TCR transgenic mice into wild-type BALB/c mice. We find that in addition to its role in trafficking to peripheral lymph nodes, LFA-1 is required for optimal CD4(+) T cell priming in vivo upon s.c. immunization. CD18(-/-) DO11.10 CD4(+) T cells primed in the lymph nodes demonstrate defects in IL-2 and IFN-gamma production. In addition, recipient mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate a defect in OVA-specific IgG2a production after s.c. immunization. The defect in priming of CD18(-/-) CD4(+) T cells persists even in the presence of proliferating CD18(+/-) CD4(+) T cells and in lymphoid structures to which there is no migration defect. Taken together, these results demonstrate that LFA-1 is required for optimal CD4(+) T cell priming in vivo.     

TLR4-initiated and cAMP-mediated abrogation of bacterial invasion of the bladder

The remarkable resistance of the urinary tract to infection has been attributed to its physical properties and the innate immune responses triggered by pattern recognition receptors lining the tract. We report a distinct TLR4 mediated mechanism in bladder epithelial cells (BECs) that abrogates bacterial invasion, a necessary step for successful infection. Compared to controls, uropathogenic type 1 fimbriated Escherichia coli and Klebsiella pneumoniae invaded BECs of TLR4 mutant mice in 10-fold or greater numbers. TLR4 mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Artificially increasing intracellular cAMP levels in BECs of TLR4 mutant mice restored resistance to type 1 fimbriated bacterial invasion. This finding reveals a novel function for TLR4 and another facet of bladder innate defense.     

Effect of Oxidation of αA- and αB-Crystallins on their Structure, Oligomerization and Chaperone Function

The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H₂O₂ on the structure, oligomerization, and chaperone function of αA- and αB-crystallins. Recombinant αA-and αB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H₂O₂ and 0.1 mM FeCl₃ at 37 ^∘C for 24 hrs and the reaction was stopped by adding catalase. Structural changes due to oxidation were ascertained by circular dichroism (CD) measurements and chaperone activity was assayed with alcohol dehydrogenase (ADH) and insulin as target proteins. The oligomeric nature of the oxidized proteins was assessed by molecular sieve HPLC. The secondary structure of the oxidized αA- and αB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in β-sheet or α-helix contents. The tertiary structure also showed significant changes indicative of different mode of folding of the secondary structural elements. Chaperone function was significantly compromised as supported by nearly 50% loss in chaperone activity. Oxidation also resulted in the formation of higher molecular weight (HMW) proteins as well as lower molecular weight (LMW) proteins. Thus, oxidation leads to disintegration of the oligomeric structure of αA- and αB-crystallins. Chaperone activity of the HMW fraction is normal whereas the LMW fraction lacks any chaperone activity. So, it appears that the formation of the LMW proteins is the primary cause of the chaperone activity loss due to oxidation.     

Risk and protective factors for mental disorders beyond genetics: an evidence‐based atlas

Decades of research have revealed numerous risk factors for mental disorders beyond genetics, but their consistency and magnitude remain uncer­tain. We conducted a “meta‐umbrella” systematic synthesis of umbrella reviews, which are systematic reviews of meta‐analyses of individual studies, by searching international databases from inception to January 1, 2021. We included umbrella reviews on non‐purely genetic risk or protective factors for any ICD/DSM mental disorders, applying an established classification of the credibility of the evidence: class I (convincing), class II (highly suggestive), class III (suggestive), class IV (weak). Sensitivity analyses were conducted on prospective studies to test for temporality (reverse causation), TRANSD criteria were applied to test transdiagnosticity of factors, and A Measurement Tool to Assess Systematic Reviews (AMSTAR) was employed to address the quality of meta‐analyses. Fourteen eligible umbrella reviews were retrieved, summarizing 390 meta‐analyses and 1,180 associations between putative risk or protective factors and mental disorders. We included 176 class I to III evidence associations, relating to 142 risk/protective factors. The most robust risk factors (class I or II, from prospective designs) were 21. For dementia, they included type 2 diabetes mellitus (risk ratio, RR from 1.54 to 2.28), depression (RR from 1.65 to 1.99) and low frequency of social contacts (RR=1.57). For opioid use disorders, the most robust risk factor was tobacco smoking (odds ratio, OR=3.07). For non‐organic psychotic disorders, the most robust risk factors were clinical high risk state for psychosis (OR=9.32), cannabis use (OR=3.90), and childhood adversities (OR=2.80). For depressive disorders, they were widowhood (RR=5.59), sexual dysfunction (OR=2.71), three (OR=1.99) or four‐five (OR=2.06) metabolic factors, childhood physical (OR=1.98) and sexual (OR=2.42) abuse, job strain (OR=1.77), obesity (OR=1.35), and sleep disturbances (RR=1.92). For autism spectrum disorder, the most robust risk factor was maternal overweight pre/during pregnancy (RR=1.28). For attention‐deficit/hyperactivity disorder (ADHD), they were maternal pre‐pregnancy obesity (OR=1.63), maternal smoking during pregnancy (OR=1.60), and maternal overweight pre/during pregnancy (OR=1.28). Only one robust protective factor was detected: high physical activity (hazard ratio, HR=0.62) for Alzheimer’s disease. In all, 32.9% of the associations were of high quality, 48.9% of medium quality, and 18.2% of low quality. Transdiagnostic class I‐III risk/protective factors were mostly involved in the early neurodevelopmental period. The evidence‐based atlas of key risk and protective factors identified in this study represents a benchmark for advancing clinical characterization and research, and for expanding early intervention and preventive strategies for mental disorders.