The Mechanisms Underlying α-Amanitin Resistance in Drosophila melanogaster: A Microarray Analysis

The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study; however, very little is known about how Drosophila species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in Drosophila melanogaster. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in D. melanogaster and perhaps in mycophagous Drosophila species has evolved as cross-resistance to pesticides, other xenobiotic substances, or environmental stress factors.     

Transport of poly-β-hydroxybutyrate in human plasma

Poly-β-hydroxybutyrate (PHB) is an amphiphilic lipid that has been found to be a ubiquitous component of the cellular membranes of bacteria, plants and animals. The distribution of PHB in human plasma was investigated using chemical and immunological methods. PHB concentrations proved highly variable; in a random group of 24 blood donors, total plasma PHB ranged from 0.60 to 18.2 mg/l, with a mean of 3.5 mg/l. In plasma separated by density gradient ultracentrifugation, lipoproteins carried 20–30% of total plasma PHB; 6–14% in the very low density lipoproteins (VLDL), 8–16% in the low density lipoproteins (LDL), and < 3% in the high density lipoproteins (HDL). The majority of plasma PHB (70–80%) was found in protein fractions of density > 1.22 g/ml. Western blot analysis of the high density fractions with anti-PHB F(ab')₂ identified albumin as the major PHB-binding protein. The affinity of albumin for PHB was confirmed by in vitro studies which demonstrated transfer of ¹⁴C-PHB from chloroform into aqueous solutions of human and bovine serum albumins. PHB was less tightly bound to LDL than to other plasma components; the polymer could be isolated from LDL by extraction with chloroform, or by digestion with alkaline hypochlorite, but it could not similarly be recovered from VLDL or albumin. PHB in the LDL correlated positively with total plasma cholesterol and LDL cholesterol, and negatively with HDL cholesterol. The wide concentration range of PHB in plasma, its presence in VLDL and LDL and absence in HDL, coupled with its physical properties, suggest it may have important physiological effects.     

Enhancing Water Solubility and Stability of Natamycin by Molecular Encapsulation in Methyl-β-Cyclodextrin and its Mechanisms by Molecular Dynamics Simulations

Food Biophysics - In this study, the antifungal compound natamycin was encapsulated in methyl-β-cyclodextrin (heptakis(2,6-di-O-methyl)-β-cyclodextrin, Me-β-CD) to improve its...     

Cellular Mechanisms Underlying the Formation of HCO_3-Rich Uterine Fluid and Possible Implications in Fertility

Cellular Mechanisms Underlying the Formation of HCO_3-Rich Uterine Fluid and Possible Implications in Fertility     

Mechanisms Underlying Domoic Acid–Induced Dopamine Release from Striatum: An in Vivo Microdialysis Study

The brain microdialysis technique has been used to examine the in vivo effects of the neurotoxin domoic acid (an ionotropic glutamate receptor agonist) on dopamine (DA) release in the striatum of conscious and freely moving rats. Local application of domoic acid (500M) through the microdialysis probe produced an increase in striatal DA content (597±96% with respect to basal levels). The release of DA induced by domoic acid was not attenuated in a Ca⁺²-free medium (469±59%) or after pretreatment with 10mg/kg reserpine (533±79%). Intrastriatal infusion of 1M tetrodotoxin (TTX) partially reduced the domoic acid–evoked DA release (278±34%). Moreover, domoic acid perfusion had no effect on K⁺-evoked DA release. The results suggest that domoic acid increases the striatal DA release according to a reserpine-independent, calcium-independent and partially TTX-insensitive mechanism, suggesting that these effects probably involve a nonexocytotic process. On the other hand, the inhibitor of DA uptake nomifensine (10M) reduced the domoic acid–evoked DA release (356±59%), suggesting that a carrier-dependent mechanism could be involved in the effect of domoic acid on the striatal DA levels.     

β-Hydroxybutyrate in the Brain: One Molecule,Multiple Mechanisms

β-Hydroxybutyrate (βOHB), a ketone body, is oxidised as a brain fuel. Although its contribution to energy metabolism in the healthy brain is minimal, it is an interesting metabolite which is not only oxidised but also has other direct and collateral effects which make it a molecule of interest for therapeutic purposes. In brain βOHB can be produced in astrocytes from oxidation of fatty acids or catabolism of amino acids and is metabolised in the mitochondria of all brain cell types although uptake across the blood brain barrier is a metabolic control point. βOHB possesses an intrinsic high heat of combustion, making it an efficient mitochondrial fuel, where it can alter the NAD⁺/NADH and Q/QH₂ couples and reduce production of mitochondrial reactive oxygen species. It can directly interact as a signalling molecule influencing opening of K⁺ channels and regulation of Ca²⁺ channels. βOHB is an inhibitor of histone deacetylases resulting in upregulation of genes involved in protection against oxidative stress and regulation of metabolism. It interacts with an inflammasome in immune cells to reduce production of inflammatory cytokines and reduce inflammation. Use of βOHB as an efficient neurotherapeutic relies on increasing blood βOHB levels so as to encourage entry of βOHB to the brain. While use of βOHB as a sole therapeutic is currently limited, with employment of a ketogenic diet a more widely used approach, recent development and testing of esterified forms of βOHB have shown great promise, with the approach elevating plasma βOHB while allowing consumption of normal diet. An improved understanding of the mechanisms by which βOHB acts will allow better design of both diet and supplemental interventions.     

StartReact Effects Support Different Pathophysiological Mechanisms Underlying Freezing of Gait and Postural Instability in Parkinson’s Disease

Introduction

The pathophysiology underlying postural instability in Parkinson’s disease is poorly understood. The frequent co-existence with freezing of gait raises the possibility of shared pathophysiology. There is evidence that dysfunction of brainstem structures contribute to freezing of gait. Here, we evaluated whether dysfunction of these structures contributes to postural instability as well. Brainstem function was assessed by studying the StartReact effect (acceleration of latencies by a startling acoustic stimulus (SAS)).

Methods

We included 25 patients, divided in two different ways: 1) those with postural instability (HY = 3, n = 11) versus those without (HY<3, n = 14); and 2) those with freezing (n = 11) versus those without freezing (n = 14). We also tested 15 matched healthy controls. We tested postural responses by translating a balance platform in the forward direction, resulting in backward balance perturbations. In 25% of trials, the start of the balance perturbation was accompanied by a SAS.

Results

The amplitude of automatic postural responses and length of the first balance correcting step were smaller in patients with postural instability compared to patients without postural instability, but did not differ between freezers and non-freezers. In contrast, the StartReact effect was intact in patients with postural instability but was attenuated in freezers.

Discussion

We suggest that the mechanisms underlying freezing of gait and postural instability in Parkinson’s disease are at least partly different. Underscaling of automatic postural responses and balance-correcting steps both contribute to postural instability. The attenuated StartReact effect was seen only in freezers and likely reflects inadequate representation of motor programs at upper brainstem level.     

Methyl-β-cyclodextrin alters adipokine gene expression and glucose metabolism in swine adipose tissue

This study was designed to determine whether methyl-β-cyclodextrin (MCD) can substitute for albumin in incubation medium for neonatal swine adipose tissue explants, or whether MCD affects metabolism and cytokine expression. Subcutaneous adipose tissue explants (100 ± 10 mg) were prepared from 21-day-old pigs. Explants were incubated in medium 199 supplemented with 25 mM HEPES, 1.0 nM insulin at 37°C. The medium also contained bovine serum albumin (BSA) or MCD at 0%, 0.05%, 0.1%, 0.2% or 0.3%. Tissue explants were treated with these media for 1 h and then switched to the same basal incubation medium containing 0.05% BSA. Explants were removed from basal medium at 2 or 8 h of incubation, and real-time PCR was performed to assess expression of tumor necrosis α (TNF) and interleukin 6 (IL6), acetyl CoA carboxylase (ACAC) and fatty acid synthase (FASN). Alternatively, rates of ¹⁴C-glucose oxidation and lipogenesis were monitored ± insulin (100 nM), following MCD treatment. Incubation with BSA had minimal effects on gene expression or adipose tissue metabolism, only producing a doubling in TNF mRNA abundance (P < 0.01). Treatment with MCD increased TNF mRNA abundance by eightfold (P < 0.009), whereas IL6 gene expression increased a 100-fold (P < 0.001) with a suppression in ACAC and FASN expression (P < 0.01). This was paralleled by MCD inhibition of insulin-stimulated glucose oxidation and lipogenesis (P < 0.001). Addition of a TNF antibody to the incubation medium alleviated this inhibition of insulin-stimulated glucose metabolism by ∼30% (P < 0.05).     

Endogenous Interleukin-1β in Neuropathic Rats Enhances Glutamate Release from the Primary Afferents in the Spinal Dorsal Horn through Coupling with Presynaptic N-Methyl-d-aspartic Acid Receptors

Excessive activation of glutamate receptors and overproduction of proinflammatory cytokines, including interleukin-1β (IL-1β) in the spinal dorsal horn, are key mechanisms underlying the development and maintenance of neuropathic pain. In this study, we investigated the mechanisms by which endogenous IL-1β alters glutamatergic synaptic transmission in the spinal dorsal horn in rats with neuropathic pain induced by ligation of the L5 spinal nerve. We demonstrated that endogenous IL-1β in neuropathic rats enhances glutamate release from the primary afferent terminals and non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Myeloid differentiation primary response protein 88 (MyD88) is a mediator used by IL-1β to enhance non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Presynaptic NMDA receptors are effector receptors used by the endogenous IL-1β to enhance glutamate release from the primary afferents in neuropathic rats. This is further supported by the fact that NMDA currents recorded from small neurons in the dorsal root ganglion of normal rats are potentiated by exogenous IL-1β. Furthermore, we provided evidence that functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is mediated by the neutral sphingomyelinase/ceramide signaling pathway. Hence, functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is a crucial mechanism leading to enhanced glutamate release and activation of non-NMDA receptors in the spinal dorsal horn neurons in neuropathic pain conditions. Interruption of such functional coupling could be an effective approach for the treatment of neuropathic pain.     

Microstructural White Matter Changes Underlying Cognitive and Behavioural Impairment in ALS – An In Vivo Study Using DTI

Background

A relevant fraction of patients with amyotrophic lateral sclerosis (ALS) exhibit a fronto-temporal pattern of cognitive and behavioural disturbances with pronounced deficits in executive functioning and cognitive control of behaviour. Structural imaging shows a decline in fronto-temporal brain areas, but most brain imaging studies did not evaluate cognitive status. We investigated microstructural white matter changes underlying cognitive impairment using diffusion tensor imaging (DTI) in a large cohort of ALS patients.

Methods

We assessed 72 non-demented ALS patients and 65 matched healthy control subjects using a comprehensive neuropsychological test battery and DTI. We compared DTI measures of fiber tract integrity using tract-based spatial statistics among ALS patients with and without cognitive impairment and healthy controls. Neuropsychological performance and behavioural measures were correlated with DTI measures.

Results

Patients without cognitive impairment demonstrated white matter changes predominantly in motor tracts, including the corticospinal tract and the body of corpus callosum. Those with impairments (ca. 30%) additionally presented significant white matter alterations in extra-motor regions, particularly the frontal lobe. Executive and memory performance and behavioural measures were correlated with fiber tract integrity in large association tracts.

Conclusion

In non-demented cognitively impaired ALS patients, white matter changes measured by DTI are related to disturbances of executive and memory functions, including prefrontal and temporal regions. In a group comparison, DTI is able to observe differences between cognitively unimpaired and impaired ALS patients.     

Cx43/β-Gal Inhibits Cx43 Transport in the Golgi Apparatus

A connexin construct consisting of bacterial β-galactosidase fused to the C-terminus of connexin43 (Cx43/β-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/β-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/β-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/β-gal were assembled into a sub-hexameric complex, and that Cx43/β-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/β-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.     

Dopamine-β-hydroxylase-Induced Changes in the Electrical Conductance of Bimolecular Lipid Membranes

Dopamine-β-hydroxylase (DBH), an enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE) in adrenal medullary chromaffin granules, increases the electrical conductance of bimolecular lipid membranes. The conductance increase requires both DA and Ca²⁺ and occurs in discrete steps. The conductance, which increases as the square of the DBH concentration, is nonselective for cations over anions and requires the native conformation of DBH. NE cannot replace DA.     

Distinct Mechanisms Underlying α1-Adrenoceptor and P2x Purinoceptor Operated ATP Release and Contraction in the Guinea-Pig Vas Deferens

The temperature-dependence of ATP release and contraction response evoked by different agonists were investigated in superfused guinea-pig vas deferens. -Adrenoceptor agonists, i.e. noradrenaline (300 M), and -methyl-noradrenaline (300 M), increased the basal ATP outflow, measured by the luciferin-luciferase assay, and induced biphasic contractile response. Cooling the bath temperature to 12°C almost completely inhibited ATP release and twitch contraction evoked by -adrenoceptor agonists, whereas the phasic contraction remained unaffected. In contrast, twitch contraction and subsequent ATP release induced by ,-methylene-ATP, a selective P2 receptor agonist (100 M), was not reduced by low temperature. The ectoATPase activity, measured by HPLC technique was not significantly different at 37°C and 12°C. Nifedipine (1 M), the voltage sensitive Ca²⁺ channel blocker eliminated ,-methylene-ATP evoked twitch contraction but not ATP release. In conclusion, -adrenoceptor and P2 receptor agonists utilize distinct mechanisms to elicit ATP release and contraction: -adrenoceptor-mediated ATP release and contraction is temperature-dependent, indicating the involvement of a carrier-mediated process in it, whereas P2x purinoceptor evoked ATP release and twitch is mediated by a different mechanism.     

The Odyssey of a Young Gene: Structure–Function Studies in Human Glutamate Dehydrogenases Reveal Evolutionary-Acquired Complex Allosteric Regulation Mechanisms

Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35–40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or l-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed.     

Methyl-β-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK

The drug 2-hydroxypropyl-β-cyclodextrin (HPβCD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease, type C (NPC) and has been advanced to human clinical trials. However, its mechanism of action for reducing cholesterol accumulation in NPC cells is uncertain and its molecular target is unknown. We found that methyl-β-cyclodextrin (MβCD), a potent analog of HPβCD, restored impaired macroautophagy/autophagy flux in Niemann-Pick disease, type C1 (NPC1) cells. This effect was mediated by a direct activation of AMP-activated protein kinase (AMPK), an upstream kinase in the autophagy pathway, through MβCD binding to its β-subunits. Knockdown of PRKAB1 or PRKAB2 (encoding the AMPK β1 or β2 subunit) expression and an AMPK inhibitor abolished MβCD-mediated reduction of cholesterol storage in NPC1 cells. The results demonstrate that AMPK is the molecular target of MβCD and its activation enhances autophagy flux, thereby mitigating cholesterol accumulation in NPC1 cells. The results identify AMPK as an attractive target for drug development to treat NPC.