Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600

During Days 4 to 7 in ovo, beating of embryonic chick hearts becomes progressively more sensitive to inhibition by tetrodotoxin, an inhibitor of fast Na⁺ channels, and progressively less sensitive to inhibition by D600, an inhibitor of slow Ca2+/Na+ channels. The developmental change in tetrodotoxin sensitivity is not retained in heart cells cultured in monolayer. In contrast, the developmental change in D600 sensitivity is retained. Veratridine-stimulated ²²Na⁺ influx mediated by fast Na⁺ channels is inhibited by tetrodotoxin (K_i = 1.6 nM) in cells prepared from either 3-day or 12-day embryos. These results suggest that young embryonic hearts contain physiologically inactive Na⁺ channels. ²²Na⁺ influx mediated by slow Ca2+/Na+ channels is inhibited by D600 with a K_i of 40 nM for cells from 3-day hearts and 8 μM for cells from 12-day hearts. Beating of heart cells in aggregate cultures is also inhibited by D600. Aggregates which have reactivated after inhibition by tetrodotoxin are 10-fold more sensitive to inhibition by D600 than untreated controls. The results suggest that the primary developmental event is a change in slow Ca2+/Na+ channels which reduces their sensitivity to D600 and diminishes their ability to support beating without the activity of the fast Na⁺ channel.     

Itch and autophagy-mediated NF-κB activation contributes to inhibition of cathepsin D-induced sensitizing effect on anticancer drugs

Inhibition of cathepsin D (Cat D) sensitizes cancer cells to anticancer drugs via RNF183-mediated downregulation of Bcl-xL expression. Although NF-κB activation is involved in the upregulation of RNF183 expression, the molecular mechanism of NF-κB activation by Cat D inhibition is unknown. We conducted this study to investigate the molecular mechanism underlying Cat D-mediated NF-κB activation. Interestingly, Cat D inhibition-induced IκB degradation in an autophagy-dependent manner. Knockdown of autophagy-related genes (ATG7 and Beclin1) and lysosome inhibitors (chloroquine and bafilomycin A1) blocked IκB degradation via Cat D inhibition. Itch induced K63-linked ubiquitination of IκB and then modulated the protein stability of IκB by Cat D inhibition. Inhibition of Cat D-mediated Itch activation was modulated by the JNK signaling pathway, and phosphorylated Itch could bind to IκB, resulting in polyubiquitination of IκB. Additionally, inhibition of Cat D increased autophagy flux via activation of the LKB1-AMPK-ULK1 pathway. Therefore, our results suggested that Cat D inhibition activated NF-κB signaling via degradation of autophagy-dependent IκB, which is associated with the upregulation of RNF183, an E3 ligase of Bcl-xL. Cat D inhibition enhances TRAIL-induced apoptosis through Bcl-xL degradation via upregulation of RNF183.Subject terms: Chemotherapy, Ubiquitylation, Autophagy     

Involvement of poly(ADP-ribose) polymerase in paraptotic cell death of D. discoideum

Paraptosis is mediated by several proteins, poly(ADP-ribose) polymerase being one of them. D. discoideum lacks caspases thus providing a better system to dissect out the role of PARP in paraptosis. The cell death phenotype in unicellular eukaryote, D. discoideum is similar to the programmed cell death phenotype of multicellular animals. However, the events downstream to the death signal of PCD in D. discoideum are yet to be understood. Our results emphasize that oxidative stress in D. discoideum lacking caspases leads to PARP activation, mitochondrial membrane potential changes, followed by the release of apoptosis inducing factor from mitochondria. AIF causes large scale DNA fragmentation, a hallmark feature of paraptosis. The role of PARP in paraptosis is reiterated via PARP inhibition by benzamide, PARG inhibition by gallotannin and PARP down-regulation, which delays paraptosis. PARP, PARG and AIF interplay is quintessential in paraptosis of D. discoideum. This is the first report to establish the involvement of PARP in the absence of caspase activity in D. discoideum which could be of evolutionary significance and gives a lead to understand the caspase independent paraptotic mechanism in higher organisms.     

Inhibitory effects of oostatic hormone on ovarian maturation and ecdysteroid production in diptera

Extracts from post-vitellogenic ovaries of Musca domestica, Aedes atropalpus and Drosphila melanogaster were examined for the ability to inhibit the vitellogenic phase of growth in newly emerged A. atropalpus and D. melanogaster adult females. All extracts were inhibitory in A. atropalpus. D. melanogaster females were less sensitive than A. atropalpus females, only showing inhibition at a two-fold greater concentration of M. domestica extract. The titre of oostatic hormone in A. atropalpus ovaries correlated with the decline in ecdysteroid synthesis by these ovaries. A. atropalpus ovaries containing post-vitellogenic follicles were shown to inhibit ovarian ecdysteroid synthesis in vitro by vitellogenic ovaries of both A. atropalpus and D. melanogaster. D. melanogaster ovaries were not capable of eliciting such an inhibition in vitro, at least with the numbers of ovaries utilized in our experiments.     

Susceptibility of Agrobacterium tumefaciens Strains to Two Agrocin-Producing Agrobacterium Strains

Sixty-five strains and isolates of Agrobacterium tumefaciens representing each of the known biotypes, were tested for in vitro and in vivo susceptibility to the agrocin-producing strains Agrobacterium radiobacter 84 and A. tumefaciens D286. No biotype 3 strain was susceptible to the effects of either of the agrocinogenic strains in vitro. On datura and tobacco, the best inhibition of tumor formation was obtained when the agrocinogenic strains were applied to wounds 24 h before the pathogens and by the concomitant application of agrocin producer and pathogen at cell ratios of 10:1 or 3:1; inhibition of infection tended to decrease progressively as the cell ratio decreased from 10:1 to 3:1 to 1:1. Generally, strain 84 was superior to D286 in inhibiting tumor formation. A combined cell suspension of 84 and D286 was as effective as 84 alone. The overall pattern of inhibition of tumor formation by biotype 1 and 2 pathogens resistant to the agrocinogenic strains in vitro was similar to that obtained with strains that were susceptible in vitro.     

Inhibition of photosynthetic electron transport by palmitoleic acid is partially correlated to loss of thylakoid membrane proteins

Palmitoleic acid (16:1^Δ9), a monounsaturated fatty acid, is found to inhibit electron transport. Inhibition occurs rapidly (within 30 s). The oxidizing side of photosystem (PS) II is ~ 90% inactivated, whereas no inhibition occurs on the reducing side of the PSII complex. PSI activity is ∼ 65% inhibited. Inhibition of electron transport is not correlated to lipid peroxidation. 16:1 causes the loss of proteins from the thylakoid membrane which is exacerbated by the light. The loss of five proteins, viz. plastocyanin (PC), manganese stabilizing protein (MSP), cytochrome f (Cyt f), D1 and D2 is examined. The proteins are found to be lost in the following order: PC (< 1 min), MSP (~5 min), Cyt f (~10 min), D1 (~60 min) and D2 (~60 min). The timing of the loss of a PSI associated protein, PC, overlaps with that of the inhibition of PSI. Also, preventing the loss of PC with spermine (spm) treatment can partially protect PSI from 16:1 inhibition. Thus, the results suggest that the loss of PC is the cause of PSI inhibition. On the other hand, the loss of MSP from the oxygen-evolving complex is not likely to be the cause of the 16:1 inhibition on the oxidizing side of PSII, as the MSP loss occurs later than the inhibition. Also, increased retention of MSP with spm treatment does not relieve the 16:1 inhibition of PSII. Instead, inhibition of PSII by 16:1 is prevented by Mn²⁺ treatment, suggesting that loss of Mn²⁺ ions is the cause of PSII inhibition.     

A novel GFP-based approach for screening biocontrol microorganisms in vitro against Dothistroma septosporum

Dothistroma septosporum is the causal agent of Dothistroma needle blight of pine trees. A novel green fluorescent protein (GFP)-based screening method was developed to assess the potential of microorganisms for biocontrol of Dothistroma. The screen utilizes GFP expression as an indicator of metabolic activity in the pathogen and hygromycin resistance selection to determine if the interaction is fungistatic or fungicidal. Results suggested that six of eight Trichoderma isolates tested have the potential to control Dothistroma in vitro, via a fungicidal action. Because D. septosporum produces a broad-spectrum toxin, dothistromin, the inhibition of Trichoderma spp. by D. septosporum was determined by growth rate measurements compared to controls. Inhibition of the Trichoderma spp. ranged from no inhibition to 30% inhibition and was influenced by the assay medium used. The GFP screening method was also assessed to determine if it was suitable for screening bacteria as potential biocontrol candidates. Although a method involving indirect-contact had to be used, two of four Bacillus strains showed antagonistic activity against D. septosporum in vitro, via a fungistatic interaction. The four bacterial strains inhibited D. septosporum growth by 14.0 to 39.8%. This GFP-based method represents a novel approach to screening fungi and bacteria for antagonistic activity.     

Synthesis and biological evaluation of tubulysin D analogs related to stereoisomers of tubuvaline

The synthesis and biological evaluation of stereoisomers in tubulysin D are described. The stereoselective synthesis of all possible stereoisomers of C-11 and C-13 positions in tubulysin D was achieved by employing 1′-epi-Tuv-Me, 3′-epi-Tuv-Me, and ent-Tuv-Me and their biological properties were evaluated. It is clear that the stereochemistries of the C-11 and C-13 positions in tubulysin D have no practical impact on the inhibition of tubulin polymerization but play a role in the potent antiproliferative activities.     

Antibody-mediated growth inhibition of Plasmodium falciparum: relationship to age and protection from parasitemia in Kenyan children and adults

Background

Antibodies that impair Plasmodium falciparum merozoite invasion and intraerythrocytic development are one of several mechanisms that mediate naturally acquired immunity to malaria. Attempts to correlate anti-malaria antibodies with risk of infection and morbidity have yielded inconsistent results. Growth inhibition assays (GIA) offer a convenient method to quantify functional antibody activity against blood stage malaria.

Methods

A treatment-time-to-infection study was conducted over 12-weeks in a malaria holoendemic area of Kenya. Plasma collected from healthy individuals (98 children and 99 adults) before artemether-lumefantrine treatment was tested by GIA in three separate laboratories.

Results

Median GIA levels varied with P. falciparum line (D10, 8.8%; 3D7, 34.9%; FVO, 51.4% inhibition). The magnitude of growth inhibition decreased with age in all P. falciparum lines tested with the highest median levels among children <4 years compared to adults (e.g. 3D7, 45.4% vs. 30.0% respectively, p = 0.0003). Time-to-infection measured by weekly blood smears was significantly associated with level of GIA controlling for age. Upper quartile inhibition activity was associated with less risk of infection compared to individuals with lower levels (e.g. 3D7, hazard ratio = 1.535, 95% CI = 1.012–2.329; p = 0.0438). Various GIA methodologies had little effect on measured parasite growth inhibition.

Conclusion

Plasma antibody-mediated growth inhibition of blood stage P. falciparum decreases with age in residents of a malaria holoendemic area. Growth inhibition assay may be a useful surrogate of protection against infection when outcome is controlled for age.     

Vitamins A & D inhibit the growth of mycobacteria in radiometric culture

Background

The role of vitamins in the combat of disease is usually conceptualized as acting by modulating the immune response of an infected, eukaryotic host. We hypothesized that some vitamins may directly influence the growth of prokaryotes, particularly mycobacteria.

Methods

The effect of four fat-soluble vitamins was studied in radiometric Bactec® culture. The vitamins were A (including a precursor and three metabolites,) D, E and K. We evaluated eight strains of three mycobacterial species (four of M. avium subspecies paratuberculosis (MAP), two of M. avium and two of M. tb. complex).

Principal Findings

Vitamins A and D cause dose-dependent inhibition of all three mycobacterial species studied. Vitamin A is consistently more inhibitory than vitamin D. The vitamin A precursor, β-carotene, is not inhibitory, whereas three vitamin A metabolites cause inhibition. Vitamin K has no effect. Vitamin E causes negligible inhibition in a single strain.

Significance

We show that vitamin A, its metabolites Retinyl acetate, Retinoic acid and 13-cis Retinoic acid and vitamin D directly inhibit mycobacterial growth in culture. These data are compatible with the hypothesis that complementing the immune response of multicellular organisms, vitamins A and D may have heretofore unproven, unrecognized, independent and probable synergistic, direct antimycobacterial inhibitory activity.     

Upregulation of IFN-β induced by Sema4D-dependent partial Erk1/2 inhibition promotes NO production in microglia

Interactions between Sema4D and its receptors, PlexinB1 and CD72, induce various functions, including axon guidance, angiogenesis, and immune activation. Our previous study revealed that Sema4D is involved in the upregulation of nitric oxide production in microglia after cerebral ischemia. In this study, we investigated the underlying mechanisms of the enhancement of microglial nitric oxide production by Sema4D. Primary microglia expressed PlexinB1 and CD72, and cortical microglia expressed CD72. Sema4D promoted nitric oxide production and slightly inhibited Erk1/2 phosphorylation in microglia. Partial Erk1/2 inhibition enhanced microglial nitric oxide production. Inhibition of Erk1/2 phosphorylation induced the expression of Ifn-β mRNA, and IFN-β promoted nitric oxide production in microglia. In the ischemic cortex, the expression of Ifn-β mRNA was downregulated by Sema4D deficiency. These findings indicated that the enhancement of nitric oxide production by Sema4D is involved in partial Erk1/2 inhibition and upregulation of IFN-β.     

Vitamin D Inhibits Human Immunodeficiency Virus Type 1 and Mycobacterium tuberculosis Infection in Macrophages through the Induction of Autophagy

Low vitamin D levels in human immunodeficiency virus type-1 (HIV) infected persons are associated with more rapid disease progression and increased risk for Mycobacterium tuberculosis infection. We have previously shown that 1α,25-dihydroxycholecalciferol (1,25D3), the active form of vitamin D, inhibits HIV replication in human macrophages through the induction of autophagy. In this study, we report that physiological concentrations of 1,25D3 induce the production of the human cathelicidin microbial peptide (CAMP) and autophagic flux in HIV and M. tuberculosis co-infected human macrophages which inhibits mycobacterial growth and the replication of HIV. Using RNA interference for Beclin-1 and the autophagy-related 5 homologue, combined with the chemical inhibitors of autophagic flux, bafilomycin A₁, an inhibitor of autophagosome-lysosome fusion and subsequent acidification, and SID 26681509 an inhibitor of the lysosome hydrolase cathepsin L, we show that the 1,25D3-mediated inhibition of HIV replication and mycobacterial growth during single infection or dual infection is dependent not only upon the induction of autophagy, but also through phagosomal maturation. Moreover, through the use of RNA interference for CAMP, we demonstrate that cathelicidin is essential for the 1,25D3 induced autophagic flux and inhibition of HIV replication and mycobacterial growth. The present findings provide a biological explanation for the benefits and importance of vitamin D sufficiency in HIV and M. tuberculosis-infected persons, and provide new insights into novel approaches to prevent and treat HIV infection and related opportunistic infections.     

Community-Level Analysis of psbA Gene Sequences and Irgarol Tolerance in Marine Periphyton

This study analyzes psbA gene sequences, predicted D1 protein sequences, species relative abundance, and pollution-induced community tolerance in marine periphyton communities exposed to the antifouling compound Irgarol 1051. The mechanism of action of Irgarol is the inhibition of photosynthetic electron transport at photosystem II by binding to the D1 protein. The metagenome of the communities was used to produce clone libraries containing fragments of the psbA gene encoding the D1 protein. Community tolerance was quantified with a short-term test for the inhibition of photosynthesis. The communities were established in a continuous flow of natural seawater through microcosms with or without added Irgarol. The selection pressure from Irgarol resulted in an altered species composition and an inducted community tolerance to Irgarol. Moreover, there was a very high diversity in the psbA gene sequences in the periphyton, and the composition of psbA and D1 fragments within the communities was dramatically altered by increased Irgarol exposure. Even though tolerance to this type of compound in land plants often depends on a single amino acid substitution (Ser₂₆₄→Gly) in the D1 protein, this was not the case for marine periphyton species. Instead, the tolerance mechanism likely involves increased degradation of D1. When we compared sequences from low and high Irgarol exposure, differences in nonconserved amino acids were found only in the so-called PEST region of D1, which is involved in regulating its degradation. Our results suggest that environmental contamination with Irgarol has led to selection for high-turnover D1 proteins in marine periphyton communities at the west coast of Sweden.     

Reversal of the effects of a night interruption in lemna by inhibitors of ribonucleic Acid synthesis

The inhibition of flowering of Lemna perpusilla Torr. strain 6746 caused by a light break can be partially reversed by treatment with actinomycin D or 2-thiouracil. Actinomycin D is most active in reversing the response to a light break if the inhibitor is present in the fronds at the time the light break is administered.     

A Saccharomyces cerevisiae-based bioassay for assessing pesticide toxicity

This study evaluates the toxic effect of three pesticides (Azoxystrobin, Cymoxanil, and Diuron) on the yeast Saccharomyces cerevisiae for the development of a new bioassay based on inhibition of S. cerevisiae metabolic activity at the level of adenosine-5-triphosphate (ATP) synthesis, as compared with two different toxicity tests based on inhibition of Daphnia magna mobility (NF EN ISO 6341) and inhibition of Vibrio fisheri activity (NF EN ISO 11348). The S. cerevisiae bioassay is cheaper and 96 times faster than the D. magna toxicity bioassay, but has lower sensitivity. It is as fast as the V. fisheri bioassay and more sensitive. Thus, this new toxicity test can be proposed for rapid detection of pesticide residues in environmental samples as a complement to the more expensive and time-consuming D. magna toxicity test.     

In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors

We evaluated 22 different host and non-host plant protease inhibitors (PIs) for in vivo inhibition of Helicoverpa armigera gut pro- and proteinases, and their biological activity against the pod borer, H. armigera, the most important pest of agriculture and horticultural crops worldwide. In vitro activation of H. armigera gut pro-proteinases (HaGPPs) in larvae fed on non-host plant PIs showed significant in vivo inhibition of HaGPPs activation in solution as well as in gel assays. The larvae fed on diet incorporated with Datura alba ness PIs showed highest inhibition of HaGPPs, followed by Psophocarpus tetragonolobus. Non-host plant PIs from Pongamia pinnata, Mucuna pruriens, Capsicum annuum, and Nigela sativa showed maximum inhibitory potential towards HaGPs in vivo, and also exhibited moderate level of inhibition of pro-proteinases. However, some of non-host plant PIs, such as those from Penganum harmala and Solanum nigrum, and the principal host plant PIs, viz., Cicer arietinum and Cajanus cajan did not inhibit HaGPP activity. Pro-proteinase level increased with the growth of the larvae, and maximum HaGPP activity was observed in the fifth-instars. Larvae fed on diets with D. alba ness PIs showed greater inhibition of HaGPPs as compared to the larvae fed on diets with P. tetragonolobus. Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases. Larval growth and development were significantly reduced in the larvae fed on the non-host plant PIs, of which D. alba ness resulted in highest stunted growth of H. armigera larvae. The in vivo studies indicated that non-host plant PIs were good candidates as inhibitors of the HaGPs as well as HaGPPs. The PIs from the non-host plants can be expressed in genetically engineered plants to confer resistance to H. armigera.     

Inhibition of chloroplast development by tentoxin

Light-dependent chloroplast development in detached pea shoots was measured in terms of chlorophyll synthesis and the synthesis of Fraction 1 protein. Both synthetic processes were inhibited more than 90% by the fungal metabolite, tentoxin (1 or 10 μg/ml). These results place Pisum sativum in the class of tentoxin-sensitive higher plants. Tentoxin, actinomycin D, lincomycin, D-threo-chloramphenicol and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were compared in their ability to inhibit RNA and protein synthesis by isolated pea chloroplasts. Energy for the synthetic reactions was supplied either by light or by added ATP. Only CCCP gave the same pattern of inhibition as tentoxin, i.e. inhibition of both RNA and protein synthesis in the light-driven system but no inhibition in the ATP-driven system. It is concluded that chloroplast developmental processes are inhibited by tentoxin through the inhibition of photophosphorylation.     

Enhanced aniline degradation by Desulfatiglans anilini in a synthetic microbial community with the phototrophic purple sulfur bacterium Thiocapsa roseopersicina

Desulfatiglans anilini is a sulfate-reducing bacterium (SRB) capable of oxidizing aniline, although growth and aniline turnover rates are slow, making it difficult to analyze the metabolism of the strain. Therefore, this study was designed to investigate the effect of sulfide on growth of D. anilini cultures, in order to improve its growth and aniline turnover rates, and study the biochemical mechanisms of sulfide inhibition. Hydrogen sulfide was found to inhibit growth of D. anilini, regardless of whether the strain was grown with aniline or phenol, and complete inhibition was observed at 20 mM hydrogen sulfide. For improving the growth of D. anilini with aniline, the sulfide-consuming phototrophic bacterium Thiocapsa roseopersicina was co-cultured in a synthetic microbial community with D. anilini using a co-cultivation device that continuously removed hydrogen sulfide from the culture. The doubling time of D. anilini with aniline was 15 days in the co-cultivation device, compared to 26 days in the absence of a sulfide-oxidizing partner. Moreover, the aniline degradation rate was significantly increased by a factor of 2.66 during co-cultivation of D. anilini with T. roseopersicina. The initial carboxylation reaction during aniline degradation was measured in cell-free extracts of D. anilini with carbon dioxide (CO₂) as a co-substrate in the presence of aniline and ATP. The effects of hydrogen sulfide on this aniline carboxylating system and on phenylphosphate synthase activity for phenol activation were studied, and it was concluded that hydrogen sulfide severely inhibited these enzyme activities.     

Discovery of dihydrobenzofuran neolignans from Rubus ideaus L. with enantioselective anti-Aβ1–42 aggregation activity

Four new dihydrobenzofuran neolignans 1a/1b and 2a/2b were isolated from the fruit of Rubus ideaus. 1a/1b and 2a/2b as two pairs of enantiomers were separated on a chiral chromatographic column. Their structures were determined using a suite of techniques including 1D and 2D NMR, HRESIMS, together with theoretical electronic circular dichroism (ECD) calculation. All compounds were evaluated for their inhibition of self-induced Aβ_1–42 aggregation. Compounds 1b and 2a exhibited optimal Aβ_1–42 aggregation inhibition capability, with an inhibition potency of 81.6% and 83.4% at 20 μM, respectively. Additionally, molecular docking was performed to identify the possible factor responsible for the enantioselectivity in the anti-Aβ_1–42 aggregation activity.