Depletion of the Central Metabolite NAD Leads to Oncosis-mediated Cell Death

Depletion of the central metabolite NAD in cells results in broad metabolic defects leading to cell death and is a proposed novel therapeutic strategy in oncology. There is, however, a limited understanding of the underlying mechanisms that connect disruption of this central metabolite with cell death. Here we utilize GNE-617, a small molecule inhibitor of NAMPT, a rate-limiting enzyme required for NAD generation, to probe the pathways leading to cell death following NAD depletion. In all cell lines examined, NAD was rapidly depleted (average t_½ of 8.1 h) following NAMPT inhibition. Concurrent with NAD depletion, there was a decrease in both cell proliferation and motility, which we attribute to reduced activity of NAD-dependent deacetylases because cells fail to deacetylate α-tubulin-K40 and histone H3-K9. Following depletion of NAD by >95%, cells lose the ability to regenerate ATP. Cell lines with a slower rate of ATP depletion (average t_½ of 45 h) activate caspase-3 and show evidence of apoptosis and autophagy, whereas cell lines with rapid depletion ATP (average t_½ of 32 h) do not activate caspase-3 or show signs of apoptosis or autophagy. However, the predominant form of cell death in all lines is oncosis, which is driven by the loss of plasma membrane homeostasis once ATP levels are depleted by >20-fold. Thus, our work illustrates the sequence of events that occurs in cells following depletion of a key metabolite and reveals that cell death caused by a loss of NAD is primarily driven by the inability of cells to regenerate ATP.     

Mutation of Npr2 Leads to Blurred Tonotopic Organization of Central Auditory Circuits in Mice

Tonotopy is a fundamental organizational feature of the auditory system. Sounds are encoded by the spatial and temporal patterns of electrical activity in spiral ganglion neurons (SGNs) and are transmitted via tonotopically ordered processes from the cochlea through the eighth nerve to the cochlear nuclei. Upon reaching the brainstem, SGN axons bifurcate in a stereotyped pattern, innervating target neurons in the anteroventral cochlear nucleus (aVCN) with one branch and in the posteroventral and dorsal cochlear nuclei (pVCN and DCN) with the other. Each branch is tonotopically organized, thereby distributing acoustic information systematically along multiple parallel pathways for processing in the brainstem. In mice with a mutation in the receptor guanylyl cyclase Npr2, this spatial organization is disrupted. Peripheral SGN processes appear normal, but central SGN processes fail to bifurcate and are disorganized as they exit the auditory nerve. Within the cochlear nuclei, the tonotopic organization of the SGN terminal arbors is blurred and the aVCN is underinnervated with a reduced convergence of SGN inputs onto target neurons. The tonotopy of circuitry within the cochlear nuclei is also degraded, as revealed by changes in the topographic mapping of tuberculoventral cell projections from DCN to VCN. Nonetheless, Npr2 mutant SGN axons are able to transmit acoustic information with normal sensitivity and timing, as revealed by auditory brainstem responses and electrophysiological recordings from VCN neurons. Although most features of signal transmission are normal, intermittent failures were observed in responses to trains of shocks, likely due to a failure in action potential conduction at branch points in Npr2 mutant afferent fibers. Our results show that Npr2 is necessary for the precise spatial organization typical of central auditory circuits, but that signals are still transmitted with normal timing, and that mutant mice can hear even with these deficits.     

Environmental and metaboliccontrol of LHCII protein phosphorylation: revealing the mechanismsfor dual regulation of the LHCII kinase

Light‐harvesting complex II (LHCII) protein phosphorylation inplant chloroplasts is under complex regulation. Combination of the invivo monitoring of LHCII protein phosphorylation (by immunoblotting)with the in vitro[γ‐³²P]ATPphosphorylation assays revealed that the basic activation/deactivationmodel of the LHCII kinase, regulated by reversible occupation/releaseof plastoquinol at the plastoquinol oxidation (Q_o) siteof the cytochrome b₆f (cyt b₆f) complex, isconsistent with, but not sufficient to explain the data obtainedwith isolated chloroplasts, leaf discs or intact leaves. Not onlythe light conditions but also the metabolic state of the entireplant, particularly the sugar metabolism, exerted a control overLHCII protein phosphorylation. Feeding of leaves with glucose (alsowith glutathione) activated the LHCII kinase in darkness. On the otherhand, independently of the basic activation/deactivationmechanism of the kinase, a strong inhibition of LHCII protein phosphorylationoccurred in vivo at increasing irradiances and even at lowlight conditions, depending on the metabolic state of the plant.Both the experiments with intact chloroplasts and the reconstitutionexperiments with isolated thylakoids to mimic LHCII kinase inhibition,disclosed that the kinase in its activated state (plastoquinol at theQ_o site of cyt b₆f complex) is protected againstinhibition by thiol reductants. However, directly upon deactivationof the kinase (release of plastoquinol from the Q_o site) itbecomes a target for inhibition by thiol reductants. Thus the twointerdependent regulatory systems of the LHCII kinase, the constantlyoccurring activation and deactivation on the one hand and the inhibitionby thiol reductants on the other, are strongly dependent on theconcentration of reducing equivalents in the chloroplast stroma.A scheme demonstrating the interconversion of activated, deactivated andinhibited states of the LHCII kinase in the chloroplast environmentof intact leaves is presented.     

Family Aggregation and Risk Factors in Phobic Disorders over Three-Generations in a Nation-Wide Study

Objective

This nation-wide register-based study investigated how often phobic disorders (PHO) and co-morbid disorders occur in affected families compared to control families. Furthermore, the study addressed the impact of sex, year of birth, and degree of urbanization in terms of risk factors.

Method

A total of N = 746 child and adolescent psychiatric participants born between 1969 and 1986 and registered in the Danish Psychiatric Central Research Register (DPCRR) with a diagnosis of a mental disorder before the age of 18, and developed PHO at some point during their life-time until a maximum age of 40 years were included. In addition, N = 2229 controls without any diagnosis of mental disorders before age 18 and that were matched for age, sex, and residential region were included. Diagnoses of mental disorders were also obtained from the first- degree relatives as a part of the Danish Three Generation Study (3GS). A family load component was obtained by using various mixed regression models.

Results

PHO occurred significantly more often in case than in control families, in particular, in mothers and siblings. Substance use disorders (SUD), Depressive disorders (DEP), anxiety disorders (ANX) and personality disorders (PERS) in the family were significantly associated with specific phobia in the case-probands. After controlling for various mental disorders comorbid to PHO it was found that some of the family transmission could be caused by various other mental disorders in family members rather than the PHO itself. Female sex and more recent year of birth were further risk factors while region of residence was not related to the manifestation of PHO. Case-relatives did not develop PHO earlier than control relatives. After adjusting for various additional explanatory variables, the family load explained only 0.0013% of the variance in the manifestation of PHO in the case-probands

Discussion

These findings, based on a very large and representative dataset, provide evidence for the family aggregation and further risk factors in PHO. In contrast to anxiety disorders and other major mental disorders the family load of PHO in this nation-wide study was rather low.     

Aggregation Properties of the Acetylcholinesterase from the Central Nervous System of Manduca sexta

Abstract: The CNS of the tobacco hornworm, Manduca sexta , provides a rich source of true acetylcholinesterase (AChE, acetylcholine hydrolase, EC 3.1.1.7.). Optimal extraction of the enzyme was obtained with a nonionic detergent at high ionic strength (1% Triton X-100, 0.5 M NaCl). Velocity sedimentation of the Triton + salt-extracted enzyme demonstrated a single peak whose sedimentation coefficient was dependent upon the enzyme concentration layered on top of the gradient. When more than 20 units were applied to the gradient, a sedimentation coefficient of 8.6 S (205,000) was obtained, and extrapolation to zero units yielded a 5.7 S (110,500) species. Sedimentation in the absence of detergents (1.0 M NaCl or 10 mM phosphate buffer, pH 7.4) yielded pelleted enzyme and species with mean values of 18.6 S (650,000) and 17.5 S (600,000), respectively. The detergent-extracted enzyme also demonstrated a concentration-dependent size in gel filtration experiments. When less than 300 units were applied to the column, a single species was recovered, with a molecular radius of 40.15 ± 2.08 Å, (108,000) or 43.4 ± 2.38 Å, (117,000) calculated by different methods. If the sample contained 300 to 1300 units, two species were observed, with molecular radii of 40.15 ± 2.08 Å or 43.4 ± 2.38 Å and 78.4 ± 3.94 Å, (319,000) or 80.25 ± 3.01 Å (326,000). Velocity sedimentation and gel filtration of AChE have demonstrated that the enzyme has a minimum molecular weight of approximately 110,000 and also exists as higher-molecular-weight aggregates of this value.     

Redistribution of stress due to a circular hole in a nonlinear anisotropic membrane

Many clinical procedures introduce holes into thin tissues that are typically under multiaxial stresses and finite strains. Such incisions change the stresses and strains from their homeostatic values, which may induce cells to alter their orientation and cytoskeletal organization as well as to migrate, proliferate, change their synthesis of matrix, or even to enter the cell death cycle. To correlate such changes in cellular activity with changes in the mechanics, we need solutions for the native and the perturbed boundary value problems. Such problems will often be complex and require a finite element solution; weak solutions should be evaluated independently, however, at least for special cases. Herein, we present a numerical solution of the governing nonlinear ordinary differential equation for the special case of stress redistribution due to the introduction of a circular hole into a finitely deformed, Fung-type, circular membrane that exhibits a cylindrical orthotropy. Among other results, we show that the anisotropy plays an increasingly greater role as the size of the hole becomes smaller, which is of course a goal of minimally invasive procedures.     

Free Radical Transients in Photobleaching of Xanthophylls and Carotenes

Carotenoicls in chloroform and carbon tetrachloriclc photobleach upon nanosecond laser flash photolysis in two steps: instantaneously and in a second-order reaction. The rate constant for second-order reaction (first-order in a solvent derived radical and first-order in (excess) ccirotenoid) is largest for carotenes (9.8·10⁸ M^-1 s^-1 for β-carotene), intermediate for hydroxylated carotenoids, and smallest for carbonyl containing carotenoids (1.0·10⁸ M^-1 s^-1 for astaxanthin) in chloroform at 20°C. Near infrared, ibsorbing transients are formed concomitant with pliotohleaching in chloroform (not detected in cxbon tetrachloride). A species formed instantaneously is tentatively identified as either a carotenoid/solvent adduct or an ion-pair. A second species is formed by decay of the instantaneously formed species and is identified as the carotenoid radical cation. This species is formed in a first-order reaction with a rate constant of approx. 5·10⁴ s^-1 and absorbing at longer wavelength than the precursor. The lifetime (second-order decay) of the interniediates appears to be longest for the carotenoids with the longest conjugated system. The results indicate that carotenes are better antioxidants than xantliophylls as the carotenes, at least in the present lipophilic solvents, react faster with free radicals.     

Changes in the functional composition of a Central European urban flora over three centuries

Documents on historical floras provide unique opportunities to analyze past changes and to show trends in biodiversity. We studied the historical and recent flora of the city of Halle in Central Germany. Our earliest records date back to the year 1687; the youngest were sampled in 2008. More than 20 other floras provide information for time in-between, covering ca. 320 years in total. We checked all historical plant occurrences for plausibility. The species turnover of 22% that took place in the study period should also yield changes in the functional composition of the flora. We identified native species and archaeophytes that went extinct since 1689 and 1856, respectively, and all neophytes that were introduced since 1689 or 1856. This ‘double’ calculation minimized the influence of so-called possible pseudo-absences. Contingency tables assisted to identify trait states which were associated with extinction or introduction. Time-series analysis identified temporal trends in trait state ratio development after testing for temporal autocorrelation. Within the study period, species of bogs, nitrogen-poor habitats or plants with helomorphic leaves got extinct more often than expected by chance. Species dispersed by humans, plants preferring nitrogen-rich or warm habitats, shrubs and trees, and species with mesomorphic leaves were, amongst others, over-represented among introduced neophytes. Land-use changes such as the transformation from agriculture to urban land use or the drainage of bogs are discussed as main drivers of these developments. Additionally, climatic changes, contamination of habitats and gardeners’ preferences for specific plants are presumed to having caused floristic changes. Our study shows the vast influence humans had and still have on biodiversity by intentionally or unintentionally selecting specific functional plant types and thus changing the composition of the flora.     

Antibodies to the photosystem I chlorophyll a + b antenna cross-react with polypeptides of CP29 and LHCII

A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.     

Phase Variation Leads to the Misidentification of a Neisseria Gonorrhoeae Virulence Gene

Neisseria gonorrhoeae is the causative agent of gonorrhea and an obligate pathogen of humans. The Opa proteins of these bacteria are known to mediate attachment and internalization by host cells, including neutrophils. The Opa protein repertoire of a typical N. gonorrhoeae isolate is encoded on ∼11 genes distributed throughout the chromosome and is subject to stochastic changes in expression through phase variation. Together, these characteristics make Opa proteins a critical yet unpredictable aspect of any experimental investigation into the interaction of N. gonorrhoeae with host cells. The goal of this study was to identify novel virulence factors of N. gonorrhoeae by assessing the contribution of a set of uncharacterized hydrogen peroxide-induced genes to bacterial survival against neutrophil-mediated killing. To this end, a strain harboring an engineered mutation in the NGO0322 gene was identified that exhibited increased sensitivity to neutrophil-mediated killing, enhanced internalization by neutrophils, and the ability to induce high levels of neutrophil-generated reactive oxygen species. Each of these phenotypes reverted to near wild-type levels following genetic complementation of the NGO0322 mutation. However, after immunoblot analysis of Opa proteins expressed by the isogenic parent, mutant, and genetically complemented strains, it was determined that phase variation had resulted in a disparity between the Opa profiles of these strains. To determine whether Opa phase variation, rather than NGO0322 mutation, was the cause of the observed neutrophil-related phenotypes, NGO0322 function was investigated in N. gonorrhoeae strains lacking all Opa proteins or constitutively expressing the OpaD variant. In both cases, mutation of NGO0322 did not alter survival of gonococci in the presence of neutrophils. These results demonstrate the importance of controlling for the frequent and random variation in Opa protein production by N. gonorrhoeae when investigating host cell interactions.     

Elevated zeaxanthin bound to oligomeric LHCII enhances the resistance of Arabidopsis to photooxidative stress by a lipid-protective, antioxidant mechanism

The xanthophyll cycle has a major role in protecting plants from photooxidative stress, although the mechanism of its action is unclear. Here, we have investigated Arabidopsis plants overexpressing a gene encoding beta-carotene hydroxylase, containing nearly three times the amount of xanthophyll cycle carotenoids present in the wild-type. In high light at low temperature wild-type plants exhibited symptoms of severe oxidative stress: lipid peroxidation, chlorophyll bleaching, and photoinhibition. In transformed plants, which accumulate over twice as much zeaxanthin as the wild-type, these symptoms were significantly ameliorated. The capacity of non-photochemical quenching is not significantly different in transformed plants compared with wild-type and therefore an enhancement of this process cannot be the cause of the stress tolerant phenotype. Rather, it is concluded that it results from the antioxidant effect of zeaxanthin. 80-90% of violaxanthin and zeaxanthin in wild-type and transformed plants was localized to an oligomeric LHCII fraction prepared from thylakoid membranes. The binding of these pigments in intact membranes was confirmed by resonance Raman spectroscopy. Based on the structural model of LHCII, we suggest that the protein/lipid interface is the active site for the antioxidant activity of zeaxanthin, which mediates stress tolerance by the protection of bound lipids.     

Redistribution of polycations bound to lymphocytes

Summary The redistribution of surface-bound polycations on human lymphocytes and mouse-spleen lymphocytes was studied by fluorescence microscopy. A redistribution pattern after incubation with protamine, polylysine and DEAE-dextran resembling patching and capping processes was observed both at 20°C and at 4°C. The interaction between polycations and the cell surface is considered to represent non-specific binding to the membrane proteins. The redistribution process is regarded as precipitation processes. This is in accordance with the finding that no effect on the binding and redistribution of polycations was noticed in the presence of NaN₃, vinblastine sulphate, vincristine sulphate, colchichine, cytochalasin B or trypsin treatment. When incubation periods longer than 5 min. were employed beginning internalization of flourescent material is observed. Interaction with the nuclear membrane similarly resulted in rearrangements and capping.Sponsored by the Danish Cancer Society.     

Joy Leads to Overconfidence,and a Simple Countermeasure

Overconfidence has been identified as a source of suboptimal decision making in many real-life domains, with often far-reaching consequences. This study identifies a mechanism that can cause overconfidence and demonstrates a simple, effective countermeasure in an incentive-compatible experimental study. We observed that joy induced overconfidence if the reason for joy (an unexpected gift) was unrelated to the judgment task and if participants were not made specifically aware of this mood manipulation. In contrast, we observed well-calibrated judgments among participants in a control group who were in their resting mood. Furthermore, we found well-calibrated judgments among participants who received the joyful mood induction together with questions that forced them to reflect on their current mood and the (ir)relevance of its cause to our judgment tasks. Our findings suggest that being aware of one’s positive mood and the reason for that mood may effectively reduce overconfidence for a short period.     

Olfactory Bulbectomy Leads to the Development of Epilepsy in Mice

There is a clear link between epilepsy and depression. Clinical data demonstrate a 30–35% lifetime prevalence of depression in patients with epilepsy, and patients diagnosed with depression have a three to sevenfold higher risk of developing epilepsy. Traditional epilepsy models partially replicate the clinical observations, with the demonstration of depressive traits in epileptic animals. Studies assessing pro-epileptogenic changes in models of depression, however, are more limited. Here, we examined whether a traditional rodent depression model—bilateral olfactory bulbectomy—predisposes the animals towards the development of epilepsy. Past studies have demonstrated increased neuronal excitability after bulbectomy, but continuous seizure monitoring had not been conducted. For the present study, we monitored control and bulbectomized animals by video-EEG 24/7 for approximately two weeks following the surgery to determine whether they develop spontaneous seizures. All seven bulbectomized mice exhibited seizures during the monitoring period. Seizures began about one week after surgery, and occurred in clusters with severity increasing over the monitoring period. These results suggest that olfactory bulbectomy could be a useful model of TBI-induced epilepsy, with advantages of relatively rapid seizure onset and a high number of individuals developing the disease. The model may also be useful for investigating the mechanisms underlying the bidirectional relationship between epilepsy and depression.     

Enhancement of Swimming Speed Leads to a More-Efficient Chemotactic Response to Repellent

Negative chemotaxis refers to the motion of microorganisms away from regions with high concentrations of chemorepellents. In this study, we set controlled gradients of NiCl₂, a chemorepellent, in microchannels to quantify the motion of Escherichia coli over a broad range of concentrations. The experimental technique measured the motion of the bacteria in space and time and further related the motion to the local concentration profile of the repellent. Results show that the swimming speed of bacteria increases with an increasing concentration of repellent, which in turn enhances the drift velocity. The contribution of the increased swimming speed to the total drift velocity was in the range of 20 to 40%, with the remaining contribution coming from the modulation of the tumble frequency. A simple model that incorporates receptor dynamics, including adaptation, intracellular signaling, and swimming speed variation, was able to qualitatively capture the observed trend in drift velocity.     

Sequence conservation of light-harvesting and stress-response proteins in relation to the three-dimensional molecular structure of LHCII

The structure of pea light-harvesting complex LHCII determined to 3.4 Å resolution by electron crystallography (Kühlbrandt, Wang and Fujiyoshi (1994) Nature 367: 614–621) was examined to determine the relationship between structural elements and sequence motifs conserved in the extended family of light-harvesting antennas (Chl a/b, fucoxanthin Chl a/c proteins) and membrane-intrinsic stress-induced proteins (ELIPs) to which LHCII belongs. It is predicted that the eukaryotic ELIPs can bind at least four molecules of Chl. The one-helix prokaryotic ELIP of Synechococcus was modelled as a homodimer based on the high degree of conservation of residues involved in the interactions of the first (B) and third (A) helices of LHCII.Abbreviations CAB Chl a/b-binding - ELIP early light-inducible protein - FCP fucoxanthin-Chl a/c protein - Lut1, Lut2 lutein molecules 1 and 2