Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis

Background

Insect metamorphosis proceeds in two modes: hemimetaboly, gradual change along the life cycle; and holometaboly, abrupt change from larvae to adult mediated by a pupal stage. Both are regulated by 20-hydroxyecdysone (20E), which promotes molts, and juvenile hormone (JH), which represses adult morphogenesis. Expression of Broad-complex (BR-C) is induced by 20E and modulated by JH. In holometabolous species, like Drosophila melanogaster, BR-C expression is inhibited by JH in young larvae and enhanced in mature larvae, when JH declines and BR-C expression specifies the pupal stage.

Methods

Using Blattella germanica as a basal hemimetabolous model, we determined the patterns of expression of BR-C mRNAs using quantitative RT-PCR, and we studied the functions of BR-C factors using RNA interference approaches.

Results

We found that BR-C expression is enhanced by JH and correlates with JH hemolymph concentration. BR-C factors appear to be involved in cell division and wing pad growth, as well as wing vein patterning.

Conclusions

In B. germanica, expression of BR-C is enhanced by JH, and BR-C factors appear to promote wing growth to reach the right size, form and patterning, which contrast with the endocrine regulation and complex functions observed in holometabolous species.

General significance

Our results shed new light to the evolution from hemimetaboly to holometaboly regarding BR-C, whose regulation and functions were affected by two innovations: 1) a shift in JH action on BR-C expression during young stages, from stimulatory to inhibitory, and 2) an expansion of functions, from regulating wing development, to determining pupal morphogenesis.     

Biosynthesis and function of chondroitin sulfate

Background

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions.

Scope of review

Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo.

Major conclusions

Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes.

General significance

Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.     

PKC activation contributes to caspase-mediated eIF2α phosphorylation and cell death

Back ground

Stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2α), involved in translation, promotes cell suicide or survival. Since multiple signaling pathways are implicated in cell death, the present study has analyzed the importance of PKC activation in the stress-induced eIF2α phosphorylation, caspase activation and cell death in the ovarian cells of Spodoptera frugiperda (Sf9) and in their extracts.

Methods

Cell death is analyzed by flow cytometry. Caspase activation is measured by Ac-DEVD-AFC hydrolysis and also by the cleavage of purified recombinant PERK, an endoplasmic reticulum-resident eIF2α kinase. Status of eIF2α phosphorylation and cytochrome c levels are analyzed by western blots.

Results

PMA, an activator of PKC, does not promote cell death or affect eIF2α phosphorylation. However, PMA enhances late stages of UV-irradiation or cycloheximide-induced caspase activation, eIF2α phosphorylation and apoptosis in Sf9 cells. PMA also enhances cytochrome c-induced caspase activation and eIF2α phosphorylation in cell extracts. These changes are mitigated more efficiently by caspase inhibitor, z-VAD-fmk, than by calphostin, an inhibitor of PKC. In contrast, tunicamycin-induced eIF2α phosphorylation that does not lead to caspase activation or cell death is unaffected by PMA, z-VAD-fmk or by calphostin.

Conclusions

While caspase activation is a cause and consequence of eIF2α phosphorylation, PKC activation that follows caspase activation further enhances caspase activation, eIF2α phosphorylation, and cell death in Sf9 cells.

General significance

Caspases can activate multiple signaling pathways to enhance cell death.     

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells

Background

The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood.

Methods

In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis.

Results

Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis.

General significance

These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.     

O-GlcNAc modification affects the ATM-mediated DNA damage response

Background

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein.

Methods

The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM.

Results

Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress.

General significance

ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.     

Co-Sensitization to Silkworm Moth (Bombyx mori) and 9 Inhalant Allergens among Allergic Patients in Guangzhou,Southern China

Objectives

This study aimed to investigate the profile of sensitization to silkworm moth (Bombyx mori) and other 9 common inhalant allergens among patients with allergic diseases in southern China.

Methods

A total of 175 patients were tested for serum sIgE against silkworm moth in addition to combinations of other allergens: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Blattella germanica, Periplaneta americana, cat dander, dog dander, Aspergillus fumigatus and Artemisia vulgaris by using the ImmunoCAP system. Correlation between sensitization to silkworm moth and to the other allergens was analyzed.

Results

Of the 175 serum samples tested, 86 (49.14%) were positive for silkworm moth sIgE. With high concordance rates, these silkworm moth sensitized patients were concomitantly sensitized to Dermatophagoides pteronyssinus (94.34%), Dermatophagoides farinae (86.57%), Blomia tropicalis (93.33%), Blattella germanica (96.08%), and Periplaneta americana (79.41%). Moreover, there was a correlation in serum sIgE level between silkworm moth and Dermatophagoides pteronyssinus (r = 0.518), Dermatophagoides farinae (r = 0.702), Blomia tropicalis (r = 0.701), Blattella germanica (r = 0.878), and Periplaneta americana (r = 0.531) among patients co-sensitized to silkworm moth and each of these five allergens.

Conclusion

In southern Chinese patients with allergic diseases, we showed a high prevalence of sensitization to silkworm moth, and a co-sensitization between silkworm moth and other five common inhalant allergens. Further serum inhibition studies are warranted to verify whether cross-reactivity exists among these allergens.     

Amphotericin B induces trehalose synthesis and simultaneously activates an antioxidant enzymatic response in Candida albicans

Background

Enzymes involved in trehalose metabolism have been proposed as potential targets for new antifungals. To analyse this proposal, the susceptibility to Amphotericin B (AmB) of the C. albicans trehalose-deficient mutant tps1Δ/tps1Δ, was examined.

Methods

Determination of endogenous trehalose and antioxidant enzymatic activities as well as RT-PCR analysis in cells subjected to AmB treatments was performed.

Results

Exponential tps1Δ null cultures showed high degree of cell killing upon exposure to increasing AmB doses respect to CAI.4 parental strain. Reintroduction of the TPS1 gene restored the percentage of cell viability. AmB induced significant synthesis of endogenous trehalose in parental cells, due to the transitory accumulation of TPS1 mRNA or to the moderate activation of trehalose synthase (Tps1p) with the simultaneous deactivation of neutral trehalase (Ntc1p). Since tps1Δ/tps1Δ mutant cells are highly susceptible to acute oxidative stress, the putative antioxidant response to AmB was also measured. A conspicuous activation of catalase and glutathione reductase (GR), but not of superoxide dismutase (SOD), was observed when the two cell types were exposed to high concentrations of AmB (5 μg/ml). However, no significant differences were detected between parental and tps1Δ null strains as regards the level of activities.

Conclusions

The protective intracellular accumulation of trehalose together with the induction of antioxidant enzymatic defences are worthy mechanisms involved in the resistance of C. albicans to the fungicidal action of AmB.

General significance

The potential usefulness of trehalose synthesis proteins as an interesting antifungal target is reinforced. More importantly, AmB elicits a complex defensive response in C. albicans.     

Molecular basis of thermal stability in truncated (2/2) hemoglobins

Background

Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures.

Methods

We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position.

Results

The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible.

Conclusions

This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability.

General significance

These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins.     

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: Binding of substrate induces a second class of site with lowered affinity and catalytic activity

Background

Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P₂ and by high concentrations of its substrate Fru-1,6-P₂. The mechanism that produces substrate inhibition continues to be obscure.

Methods

Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P₂ and Fru-1,6-P₂ on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244.

Results

The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P₂ induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P₂, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a “stapler” that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition.

Conclusions

Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits.

General significance

Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.