Immunocytochemical demonstration ofγ-amino butyric acid and glutamic acid decarboxylase in R7 photoreceptors and C2 centrifugal fibres in the blowfly visual system

Summary Neurons within the compound eye of the flyCalliphora erythrocephala, suspected of containing gamma-aminobutyric acid were revealed immunocytochemically, using antibodies directed against gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). The GABA content within putative GABAergic neurons was increased by high affinity uptake of GABA and selective blocking of GABA metabolism with Gabaculine. Only neuronal populations which were labelled with the GABA as well as the GAD antibodies were presumed to be GABAergic. The first optic neuropil (lamina) exhibited two distinct GA-BAergic fibre populations amongst a larger population comprised of fourteen cell classes. One fibre population was formed by the axons of the photopic photoreceptors R7 which pass through the lamina and terminate in the second optic neuropil (the medulla). The identity of R7 was established from longitudinal and transverse sections of the retina where R7 can be unequivocally distinguished from the six scotopic photoreceptors R1-6 and the other photopic receptor, R8.The other fibre population matched the profiles in the lamina of terminals of efferent C2 neurons. These neurons project distally from beneath the medulla out to the lamina ganglionaris where each retinotopic unit (cartridge) contains a characteristic hook-like terminal arbor distally. We propose from these data that the photoreceptors R7 and the efferent C2 neurons use GABA as a neurotransmitter.     

Phototaxis in Drosophila: R1–6 input and interaction among ocellar and compound eye receptors

All 3 photoreceptor types in the compound eye of Drosophila can evoke positive phototaxis. Here we describe input from R1–6 receptors which are very sensitive. Previous reports in this series of studies described input from R7 and R8, the other less sensitive receptors. Here we studied fast-walking phototaxis using extremely dim stimuli. We also studied input from the simple ocellar eyes. Thus, we can now summarize a complete synthesis of inputs and interactions among all compound eye and ocellar receptor types. Receptor-deficient mutants were used to establish receptor-specific input. Two other findings are presented: (1) eye colour pigments affect the spectral sensitivity for phototaxis; and (2) the ocelli interact to facilitate input from the compound eye receptor types. Possible mechanisms of receptor interaction are discussed in the light of these findings of positive input from all photoreceptor types in Drosophila.     

Crosstalk between the ubiquitin–proteasome system and autophagy in a human cellular model of Alzheimer's disease

Alzheimer's disease is the most common progressive neurodegenerative disorder characterized by the abnormal deposition of amyloid plaques, likely as a consequence of an incorrect processing of the amyloid-β precursor protein (AβPP). Dysfunctions in both the ubiquitin–proteasome system and autophagy have also been observed. Recently, an extensive cross-talk between these two degradation pathways has emerged, but the exact implicated processes are yet to be clarified. In this work, we gained insight into such interplay by analyzing human SH-SY5Y neuroblastoma cells stably transfected either with wild-type AβPP gene or 717 valine-to-glycine AβPP-mutated gene. The over-expression of the AβPP mutant isoform correlates with an increase in oxidative stress and a remodeled pattern of protein degradation, with both marked inhibition of proteasome activities and impairment in the autophagic flux. To compensate for this altered scenario, cells try to promote the autophagy activation in a HDAC6-dependent manner. The treatment with amyloid-β₄₂ oligomers further compromises proteasome activity and also contributes to the inhibition of cathepsin-mediated proteolysis, finally favoring the neuronal degeneration and suggesting the existence of an Aβ₄₂ threshold level beyond which proteasome-dependent proteolysis becomes definitely dysfunctional.     

Interaction between αB-crystallin and the human 20S proteasomal subunit C8/α7

αB-Crystallin, a member of the small heat shock protein (sHsp) family, can bind unfolding proteins, but is unable to refold them. To fulfil its protective function in vivo it is therefore likely to interact with other cellular proteins. Here we report that αB-crystallin binds very specifically both in vitro and in vivo to C8/α7, one of the 14 subunits of the 20S proteasome. The C8/α7 protein forms heterogeneous complexes with αB-crystallin of about 540 kDa. However, no strong interaction between αB-crystallin and 20S proteasomes was observed. Since both proteins are localized in the cytoplasm, the interaction between αB-crystallin and C8/α7 subunit might affect the assembly of the proteasome complex or facilitate the degradation of unfolded proteins bound to αB-crystallin.     

Direct molecular interactions between Beclin 1 and the canonical NFκB activation pathway

General (macro)autophagy and the activation of NFκB constitute prominent responses to a large array of intracellular and extracellular stress conditions. The depletion of any of the three subunits of the inhibitor of NFκB (IκB) kinase (IKKα, IKKβ, IKKγ/NEMO), each of which is essential for the canonical NFκB activation pathway, limits autophagy induction by physiological or pharmacological triggers, while constitutive active IKK subunits suffice to stimulate autophagy. The activation of IKK usually relies on TGFβ-activated kinase 1 (TAK1), which is also necessary for the optimal induction of autophagy in multiple settings. TAK1 interacts with two structurally similar co-activators, TAK1-binding proteins 2 and 3 (TAB2 and TAB3). Importantly, in resting conditions both TAB2 and TAB3 bind the essential autophagic factor Beclin 1, but not TAK1. In response to pro-autophagic stimuli, TAB2 and TAB3 dissociate from Beclin 1 and engage in stimulatory interactions with TAK1. The inhibitory interaction between TABs and Beclin 1 is mediated by their coiled-coil domains (CCDs). Accordingly, the overexpression of either TAB2 or TAB3 CCD stimulates Beclin 1- and TAK1-dependent autophagy. These results point to the existence of a direct molecular crosstalk between the canonical NFκB activation pathway and the autophagic core machinery that guarantees the coordinated induction of these processes in response to stress.     

Substrate recognition in selective autophagy and the ubiquitin–proteasome system

Dynamic protein turnover through regulated protein synthesis and degradation ensures cellular growth, proliferation, differentiation and adaptation. Eukaryotic cells utilize two mechanistically distinct but largely complementary systems — the 26S proteasome and the lysosome (or vacuole in yeast and plants) — to effectively target a wide range of proteins for degradation. The concerted action of the ubiquitination machinery and the 26S proteasome ensures the targeted and tightly regulated degradation of a subset of commonly short-lived cellular proteins. Autophagy is a distinct degradation pathway, which transports a highly heterogeneous set of cargos in dedicated vesicles, called autophagosomes, to the lysosome. There the cargo becomes degraded and its molecular building blocks are recycled. While general autophagy randomly engulfs portions of the cytosol, selective autophagy employs dedicated cargo adaptors to specifically enrich the forming autophagosomes for a certain type of cargo as a response to various intra- or extracellular signals. Selective autophagy targets a wide range of cargos including long-lived proteins and protein complexes, organelles, protein aggregates and even intracellular microbes. In this review we summarize available data on cargo recognition mechanisms operating in selective autophagy and the ubiquitin–proteasome system (UPS), and emphasize their differences and common themes. Moreover, we derive general regulatory principles underlying cargo recognition in selective autophagy, and describe the system-wide crosstalk between these two cellular protein degradation systems. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Climate change and coevolution in the cuckoo–reed warbler system

The evolution of traits in hosts may be influenced by their parasites and vice versa and a coevolutionary arms race often develops between the two. As part of such an arms race, the common cuckoo mimics the eggs of its hosts to avoid egg rejection. Traits related to this arms race may also be influenced by climatic conditions, such as temperature, affecting, for example, food availability and, thus, female condition and egg size (therefore may reflect Bergmann’s rule or the resource rule). The potential interaction between coevolution and climate has rarely been studied. We investigated whether egg and body size of cuckoos and reed warblers from Britain and Denmark had undergone change between 1868 and 1956, and whether such changes were correlated with climatic factors. Cuckoo egg size decreased during the studied period while warbler egg size remained stable. Hence, cuckoo and warbler eggs have become more similar in size over time. Cuckoo egg volume decreased with increasing annual precipitation, but annual precipitation decreased over time. Warbler egg volume increased with spring temperatures (which could not reflect Bergmann’s rule, but may support the resource rule). Hence, it seems that the measured climatic indices did not affect cuckoo egg size but may in part affect warbler egg size. Therefore, the decrease in cuckoo egg size may be the result of the coevolutionary arms race. Body and egg sizes in the cuckoos were negatively correlated whereas warbler body and egg sizes were uncorrelated, suggesting that selection probably acted on egg size directly and not via selection on body size. Taken together, these findings may indicate that climate change, the coevolutionary arms race, or both, affected egg sizes. It is suggested that drawing conclusions regarding the arms race without taking into account other selective pressures (e.g., climate) may confound conclusions regarding parasite-host systems.     

Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

Angiotensin II (AngII), a peptide hormone released by adipocytes, can be catabolized by adipose angiotensin-converting enzyme 2 (ACE2) to form Ang(1–7). Co-expression of AngII receptors (AT₁ and AT₂) and Ang(1–7) receptors (Mas) in adipocytes implies the autocrine regulation of the local angiotensin system upon adipocyte functions, through yet unknown interactive mechanisms. In the present study, we reveal the adipogenic effects of Ang(1–7) through activation of Mas receptor and its subtle interplays with the antiadipogenic AngII-AT1 signaling pathways. Specifically, in human and 3T3-L1 preadipocytes, Ang(1–7)-Mas signaling promotes adipogenesis via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways, and Ang(1–7)-Mas antagonizes the antiadipogenic effect of AngII-AT₁ by inhibiting the AngII-AT₁-triggered MAPK kinase/ERK pathway. The autocrine regulation of the AngII/AT₁-ACE2-Ang(1–7)/Mas axis upon adipogenesis has also been revealed. This study suggests the importance of the local regulation of the delicately balanced angiotensin system upon adipogenesis and its potential as a novel therapeutic target for obesity and related metabolic disorders.     

Chemical interaction between the larva of a dipteran parasitoid and its coleopteran host: a case of exploitation of the communication system during the searching behaviour?

The robber fly Mallophora ruficauda is one of the principal apicultural pests in the Pampas region of Argentina. As adults, the flies prey on honey bees and other insects; while, as larvae, they parasitize scarab beetle larvae. Females of M. ruficauda lay eggs away from the host in tall grasses. After being dispersed by the wind, larvae drop to the ground, where they dig in search of their hosts. It is known that second instar larvae of M. ruficauda exhibit active host searching behaviour towards its preferred host, third instar larva of Cyclocephala signaticollis, using host-related chemical cues. Furthermore, previous works show that these chemical cues are produced in the posterior body half of hosts. However, the precise anatomical origin of these cues and whether they mediate any behaviour of C. signaticollis larvae remains yet unknown. In order to determine the precise origin of the chemical cue, we carried out olfactometer assays with different stimuli of extracts of the posterior C. signaticollis body half. Additionally, we tested whether C. signaticollis is attracted to any of the same extracts as in the previous experiments. We found that both second instar of M. ruficauda and third instar of C. signaticollis are attracted to extracts of the fermentation chamber (proctodeum). This is the first report of attraction of conspecific larvae in scarab beetles. We discuss a possible case of system communication exploitation in an immature parasitoid-host system.     

Immuno-localization of type-IV collagen in the blood-gas barrier and the epithelial–epithelial cell connections of the avian lung

The terminal respiratory units of the gas exchange tissue of the avian lung, the air capillaries (ACs) and the blood capillaries (BCs), are small and rigid: the basis of this mechanical feature has been highly contentious. Because the strength of the blood-gas barrier (BGB) of the mammalian lung has been attributed to the presence of type-IV collagen (T-IVc), localization of T-IVc in the basement membranes (BM) of the BGB and the epithelial–epithelial cell connections (E-ECCs) of the exchange tissue of the lung of the avian (chicken) lung was performed in order to determine whether it may likewise contribute to the strength of the BGB. T-IVc was localized in both the BM and the E-ECCs. As part of an integrated fibroskeletal scaffold on the lung, T-IVc may directly contribute to the strengths of the ACs and the BCs.     

Matrix metalloproteinases in the adult brain physiology: a link between c-Fos,AP-1 and remodeling of neuronal connections?

Matrix metalloproteinases (MMPs), together with their endogenous inhibitors (TIMPs) form an enzymatic system that plays an important role in a variety of physiological and pathological conditions. These proteins are also expressed in the brain, especially under pathological conditions, in which glia as well as invading inflammatory cells provide the major source of the MMP activity. Surprisingly little is known about the MMP function(s) in adult neuronal physiology. This review describes available data on this topic, which is presented in a context of knowledge about the MMP/TIMP system in other organs as well as in brain disorders. An analysis of the MMP and TIMP expression patterns in the brain, along with a consideration of their regulatory mechanisms and substrates, leads to the proposal of possible roles of the MMP system in the brain. This analysis suggests that MMPs may play an important role in the neuronal physiology, especially in neuronal plasticity, including their direct participation in the remodeling of synaptic connections-a mechanism pivotal for learning and memory.     

Docosahexaenoic acid induces the degradation of HPV E6/E7 oncoproteins by activating the ubiquitin–proteasome system

The oncogenic human papillomavirus (HPV) E6/E7 proteins are essential for the onset and maintenance of HPV-associated malignancies. Here, we report that activation of the cellular ubiquitin–proteasome system (UPS) by the omega-3 fatty acid, docosahexaenoic acid (DHA), leads to proteasome-mediated degradation of E6/E7 viral proteins and the induction of apoptosis in HPV-infected cancer cells. The increases in UPS activity and degradation of E6/E7 oncoproteins were associated with DHA-induced overproduction of mitochondrial reactive oxygen species (ROS). Exogenous oxidative stress and pharmacological induction of mitochondrial ROS showed effects similar to those of DHA, and inhibition of ROS production abolished UPS activation, E6/E7 viral protein destabilization, and apoptosis. These findings identify a novel role for DHA in the regulation of UPS and viral proteins, and provide evidence for the use of DHA as a mechanistically unique anticancer agent for the chemoprevention and treatment of HPV-associated tumors.     

Are there symplastic connections between the endosperm and embryo in some angiosperms?—a lesson from the Crassulaceae family

It is believed that there is symplastic isolation between the embryo (new sporophyte) and the endosperm (maternal-parental origin tissue, which nourishes the embryo) in angiosperms. However, in embryological literature there are rare examples in which plasmodesmata between the embryo suspensor and endosperm cells have been recorded (three species from Fabaceae). This study was undertaken in order to test the hypothesis that plasmodesmata between the embryo suspensor and the endosperm are not so rare but also occur in other angiosperm families; in order to check this, we used the Crassulaceae family because embryogenesis in Crassulaceae has been studied extensively at an ultrastructure level recently and also we tread members of this family as model for suspensor physiology and function studies. These plasmodesmata even occurred between the basal cell of the two-celled proembryo and endosperm cells. The plasmodesmata were simple at this stage of development. During the development of the embryo proper and the suspensor, the structure of plasmodesmata changes. They were branched and connected with electron-dense material. Our results suggest that in Crassulaceae with plasmodesmata between the endosperm and suspensor, symplastic connectivity at this cell-cell boundary is still reduced or blocked at a very early stage of embryo development (before the globular stage). The occurrence of plasmodesmata between the embryo suspensor and endosperm cells suggests possible symplastic transport between these different organs, at least at a very early stage of embryo development. However, whether this transport actually occurs needs to be proven experimentally. A broader analysis of plants from various families would show whether the occurrence of plasmodesmata between the embryo suspensor and the endosperm are typical embryological characteristics and if this is useful in discussions about angiosperm systematic and evolution.     

Effect of division of cortico — Subcortical connections on spontaneous unit activity in the lateral geniculate body and visual cortex

In acute experiments on cats the cortical projection connections of one hemisphere were divided, and the principal characteristics of spontaneous unit activity were studied in the lateral geniculate bodies (LGB) and visual cortex (area 17). After the operation the same types of spontaneous activity were found in these structures as in the intact structures. However, the number of spontaneously active cells in the structures on the side of the operation was considerably reduced. In the isolated visual cortex there was a redistribution of the relative percentage of neurons with spontaneous activity in the various layers: these cells were most numerous in layers IV–V, whereas in the normal cortex they are more numerous in layers III–IV. The mean firing rate of all types of cells was reduced in the isolated cortex. In LGB on the side of the operation a relative decrease in the number of cells with a high firing rate was observed.Institute of Experimental Medicine, Academy of Medical Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 4, No. 1, pp. 47–53, January–February, 1972.     

Lipolytic and adenyl-cyclase-stimulating activity of glucagon1–6: comparison with glucagon derivatives chemically modified in the 7–29 sequence

Glucagon_1–6 has a maximum lipolytic activity (L_max) in the rat adipocyte which is 66% of that of glucagon. The N-guanidyl derivative, modified at Lys₁₂ , has about the same L_max as glucagon_1–6. Modifying the carboxyl groups of glucagon with glycinamide or removing the COON-terminal residues with cyanogen bromide reduces L_max to less than 25% of the level of glucagon. The potency of each of these analogs (A₅₀) in M is as follows: glucagon 6×10^–3; glucagon^1–6 2 ×10^–2; N-guanidyl glucagon 9×10^–3; glycinamide glucagon 10^–2; cyanogen bromide peptide of glucagon 2 ×10^–1. The ability of all of the glucagon analogs to stimulate adenyl cyclase was somewhat less than their tipolytic activities with the exception of the glycin-amide derivative and the cyanogen bromide peptide, which were slightly more active in stimulating adenyl cyclase than in lipolysis. Glucagon_1–6 is much more potent in stimulating adipocyte than liver adenyl cyclase.     

Interactome of signaling networks in wheat: the protein–protein interaction between TaRAR1 and TaSGT1

RAR1 and SGT1 are required for development and disease resistance in plants. In many cases, RAR1 and SGT1 regulate the resistance (R)-gene-mediated defense signaling pathways. Lr21 is the first identified NBS-LRR-type R protein in wheat and is required for resistance to the leaf rust pathogen. The Lr21-mediated signaling pathways require the wheat homologs of RAR1, SGT1, and HSP90. However, the molecular mechanisms of the Lr21-mediated signaling networks remain unknown. Here I present the DNA and protein sequences of TaRAR1 and TaSGT1, and demonstrate for the first time a direct protein-protein interaction between them.