Characterization of bimodal cell death of insect cells in a rotating‐wall vessel and shaker flask

In previous publications, we reported the benefits of a high‐aspect rotating‐wall vessel (HARV) over conventional bioreactors for insect‐cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker‐flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first‐order term. Cell‐cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell–cell interactions, waste accumulation, and mass transport. These studies may benefit insect‐cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 14–26, 1999.     

Pharmacological Profile of the “Triple” Monoamine Neurotransmitter Uptake Inhibitor, DOV 102,677

Summary 1. The molecular and behavioral pharmacology of DOV 102,677 is characterized.2. This characterization was performed using radioligand binding and neurotransmitter uptake assays targeting the monoamine neurotransmitter receptors. In addition, the effects of DOV 102,677 on extracellular neurotransmitter levels were investigated using in vivo microdialysis. Finally, the effects of DOV 102,677 in the forced swim test, locomotor function, and response to prepulse inhibition was investigated.3. DOV 102,677 is a novel, “triple” uptake inhibitor that suppresses [³H]dopamine (DA), [³H]norepinephrine (NE) and [³H]serotonin (5-HT) uptake by recombinant human transporters with IC₅₀ values of 129, 103 and 133 nM, respectively. Radioligand binding to the dopamine (DAT), norepinephrine (NET), and serotonin (SERT) transporters is inhibited with k _i values of 222, 1030, and 740 nM, respectively. DOV 102,677 (20 mg/kg IP) increased extracellular levels of DA and 5-HT in the prefrontal cortex to 320 and 280% above baseline 100 min after administration. DA levels were stably increased for the duration (240 min) of the study, but serotonin levels declined to baseline by 200 min after administration. NE levels increased linearly to a maximum of 348% at 240 min post-dosing. Consistent with these increases in NE levels, the density of β-adrenoceptors was selectively decreased in the cortex of rats treated with DOV 102,677 (20 mg/kg per day, PO, 35 days).4. DOV 102,677 dose-dependently reduced the amount of time spent immobile by rats in the forced swim test, a model predictive of antidepressant activity, with a minimum effective dose (MED) of 20 mg/kg and a maximal efficacy comparable to imipramine. This decrease in immobility time did not appear to result from increased motor activity. Further, DOV 102,677 was as effective as methylphenidate in reducing the amplitude of the startle response in juvenile mice, without notably altering motor activity.5. In summary, DOV 102,677 is an orally active, “balanced” inhibitor of DAT, NET and SERT with therapeutic versatility in treating neuropsychiatric disorders beyond depression.     

Formamidines interact withDrosophila octopamine receptors,alter the flies' behavior and reduce their learning ability

Summary We have studied the effect of formamidines onDrosophila melanogaster. Low concentrations of formamidines are toxic to adultDrosophila. A mutant with reduced cAMP synthesis displays increased resistance to the toxin. Formamidines also reduce viability ofDrosophila eggs and retard imago eclosion. At sublethal concentrations, formamidines markedly affect the flies' behavior. Upon injection, the compounds increase muscle activity. Upon feeding, formamidines induce motor excitation, reduce phototaxis and impair olfactory learning without affecting the ability to recognize an olfactory cue. In vitro, two formamidines were found to inhibit octopamine-stimulated adenylate cyclase without affecting the basal activity of the enzyme, while a third one was found to stimulate adenylate cyclase; this stimulation was blocked by phentolamine and to a lesser degree by propranolol, thus resembling the effect of octopamine. The binding of [³H]octopamine toDrosophila head membranes was also inhibited. Taken together, our results indicate that formamidines interact with octopaminergic systems inDrosophila, exert both peripheral and central effects in the fly, and could be used to dissect the roles of octopamine in development and behavior, including behavioral plasticity. The results also suggest that formamidines could be used to select mutants in aminergic transmission and in the cAMP cascade.Abbreviations CDMF chlordimeform - DMPF N,N-dimethyl-N2-(2,4-dimethylphenyl) formamidine     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver, fetal liver, cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123.

The cholesterol to phospholipid ratio in mitochondria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochondria is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane. In mitochondria from cholesterol-enriched liver and hepatomas, there occurs a decrease in extent of hypoosmotic and phosphate-induced swelling and a decrease of conformational changes linked to energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholipid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformational changes. No significant modifications of these parameters were found in fetal liver mitochondria. Cholesterol content does not influence K+ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K+ uptake, total mitochondrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42-48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochondrial water content. Water content is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is significantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspensions during K+ uptake. The higher the ratio, the lower the extent and initial rate of absorbance decrease.     

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver,fetal liver,cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123

The choleterol to phospholipid ratio in mitochodria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochonda is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane.In mitochondria from cholesterol-enriched liver and hepatomas, there occur a decrease in extent of hypoosmotic and phosphate-induced sweeling and decrease of conformational linked energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholopid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformation changes. No significant modifications of these parameters were found in fetal liver mitochondria.Cholesterol content does not influence K⁺ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K⁺ uptake, total mitochodrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42–48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochobdrial water content. Water content. Water is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is signifacantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspension during K⁺ uptake. The higher the ratio, the lower the extent and and initial rate of absorbance decrease.     

Floristic distinctiveness and endemic richness of woody plants highlight the biodiversity value of the herriza among all Mediterranean heathlands

Background: Heathlands are relatively abundant in the landscape of the western Mediterranean region, especially in the Strait of Gibraltar region, where it is locally known as herriza. They are associated with a mild Mediterranean climate regime and with acid, nutrient-poor soils. They harbour a high plant diversity, often viewed as a consequence of the transition between European Atlantic heathland and Mediterranean sclerophyllous shrubland floras.

Aims: To determine whether species-rich Mediterranean heathlands, including the herriza, constitute distinct heathland formations rather than transitional vegetation units between Atlantic heathlands and Mediterranean garrigue shrublands.

Methods: We quantified species richness, endemism and analysed the β-diversity of the woody component of Mediterranean heathland communities throughout its geographic range, with special emphasis on the Strait of Gibraltar region.

Results: Mediterranean heathlands, including the herriza, are not transitional communities between Atlantic heathlands and Mediterranean shrublands. Woody species richness and, particularly, endemic richness was the highest in the herriza.

Conclusions: The high biodiversity values of the herriza are a likely consequence of the ecological singularity of the Strait of Gibraltar region and its known role as a glacial refugium. Despite its treeless feature, the herriza deserves special recognition and protection from both in its European and North African extension.     

Fungal dual-domain LysM effectors undergo chitin-induced intermolecular,and not intramolecular,dimerization

Chitin is a homopolymer of β-(1,4)-linked N-acetyl-D-glucosamine (GlcNAc) and a major structural component of fungal cell walls. In plants, chitin acts as a microbe-associated molecular pattern (MAMP) that is recognized by lysin motif (LysM)-containing plant cell surface-localized pattern recognition receptors (PRRs) that activate a plethora of downstream immune responses. To deregulate chitin-induced plant immunity and successfully establish infection, many fungal pathogens secrete LysM domain-containing effector proteins during host colonization. The LysM effector Ecp6 from the tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum can outcompete plant PRRs for chitin binding because two of its three LysM domains cooperate to form a composite groove with ultra-high (pM) chitin-binding affinity. However, most functionally characterized LysM effectors contain only two LysMs, including Magnaporthe oryzae MoSlp1, Verticillium dahliae Vd2LysM, and Colletotrichum higginsianum ChElp1 and ChElp2. Here, we performed modeling, structural, and functional analyses to investigate whether such dual-domain LysM effectors can also form ultra-high chitin-binding affinity grooves through intramolecular LysM dimerization. However, our study suggests that intramolecular LysM dimerization does not occur. Rather, our data support the occurrence of intermolecular LysM dimerization for these effectors, associated with a substantially lower chitin binding affinity than monitored for Ecp6. Interestingly, the intermolecular LysM dimerization allows for the formation of polymeric complexes in the presence of chitin. Possibly, such polymers may precipitate at infection sites to eliminate chitin oligomers, and thus suppress the activation of chitin-induced plant immunity.

Fungal LysM effectors composed of two LysM domains bind chitin via intermolecular LysM dimerization, leading to polymers that may precipitate to eliminate chitin from infection sites to prevent the activation of host immune receptors.