Some Mutations Affecting Neural or Muscular Tissues Alter the Physiological Components of the Electroretinogram in Drosophila

Mutants displaying generalized behavioral defects and one mutant having an enzyme deficiency were examined for electroretinogram (ERG) defects. Mutations in nine genes were examined that cause ERG defects. Two, para^ts4 and slrp^D, cause reversibly temperature dependent loss of the off-transients in the ERG. stn^C and Tyr-2 cause loss of the on and off-transients. The transient defect in Tyr-2 mapped close to a site shown to affect tyrosinase activity in this strain. Mutations bas, rex and sesD delay recovery from the prolonged depolarization afterpotential. The visual defects of mutations elav^jl and nbA^EE171 are not complemented by lethal mutations, which, presumably, affect other tissues.     

The History of the Prolonged Depolarizing Afterpotential (PDA) and Its Role in Genetic Dissection of Drosophila Phototransduction

Abstract: In invertebrate photoreceptors, the photopigment exhibits a long-lived and physiologically active photoproduct, called metarhodopsin (M). The long life of invertebrate M implies that under physiological conditions, M and the original pigment state rhodopsin, R, are in photoequilibrium. In many invertebrates, the absorption spectra of R and M states are different, allowing large photopigment conversion between R and M states. These net pigment molecules conversions between R and M are the basis of the prolonged depolarizing afterpotential (PDA) phenomenology, which is the main subject of this review. A large net conversion of R to M disrupts phototransduction termination at the photopigment level, which in turn results in sustained excitation long after the light is turned off. Throughout this period, the photoreceptors are partially desensitized and are insensitive (or less sensitive) to subsequent test lights. In Drosophila, the PDA tests the maximal capacity of the photoreceptor cell to maintain excitation for an extended period and is strictly dependent on the presence of high concentrations of rhodopsin and the transient receptor potential (TRP) channels. Therefore, it detects even minor defects in rhodopsin or TRP biogenesis and easily scores deficient replenishment of phototransduction components, which results in temporary desensitization of the phototransduction process. Indeed, the introduction and use of PDA to screen for phototransduction-defective Drosophila mutants by Pak and colleagues yielded a plethora of new and most interesting visual mutants. Remarkably, to this day, the PDA mutants that Pak and his colleagues isolated are the main source of mutants for analysis of the Drosophila visual system.     

Phosphoinositide metabolism in Drosophila phototransduction: a coffee break discussion leads to 30 years of history

The aim of this review is to summarize the history of Dr. Yoshiki Hotta and his collaborators' contributions to the research field of Drosophila phototransduction. The electroretinogram-defective mutants reported in 1970 by Dr. Hotta and Dr. Seymour Benzer in the article entitled "Genetic dissection of the Drosophila nervous system by means of mosaics" have attracted the interest of many researchers, and have been used as a great tool to dissect the mechanisms underlying phototransduction. The early collaboration of Dr. Hotta with the group of Dr. Tohru Yoshioka, who was studying the roles of phosphoinositides in the nervous system biochemically, combined biochemical and genetic approaches to phototransduction-defective no receptor potential A (norpA) and retinal degeneration A (rdgA) mutants, which led to the hypothesis that phosphoinositide metabolism regulates phototransduction in Drosophila. This was proven later by the identification of the norpA and rdgA mutant genes, which encode phospholipase C and diacylglycerol kinase, respectively. Thus the collaboration of Dr. Hotta and Dr. Yoshioka laid the foundation of our understanding of the role of phosphoinositide metabolism in Drosophila phototransduction. In addition, a collaboration carried out with the group of Dr. Kazushige Hirosawa on the ultrastructural analyses of retinal degeneration mutants, rdgA and rdgB, led to the discovery of the subcellular membrane organelle called submicrovillar cisternae, which is involved in the phosphoinositide metabolism. In this review, the authors will summarize these results, which were inspired by Dr. Hotta's insights.     

Eosinophil leukocyte degranulation in response to serum-opsonized beads: C5a and platelet-activating factor enhance ECP release, with roles for protein kinases A and C

Background Eosinophils have a typical content of granule-bound, cytotoxic, cationic proteins which may. when released to the external milieu, play roles i n diseases such as asthma and parasitic infestation. Therefore, we have investigated possible mechanisms by which their release is regulated in eosinophils.
Methods The enzyme-linked immunosorhent assay (ELISA) was used to detect released eosinophil cationic protein (ECP). Release of ECP was induced by serum-opsonized, nonphagocytosable Sephadex beads (SOS). Results The complement fragment C5a and platelet-activating factor (PAF) were found to enhance ECP release in response to SOS in a dose-dependent fashion, and, contrary to previous reports, they were not found to act as secretagogues themselves on eosinophils in suspension. The role of protein kinase C (PKC) in eosinophil degranulation has been controversial. We found that ECP release induced by SOS was inhibited by the PKC inhibitors staurosporine and calphostin C. Activation of protein kinase A (PKA). by raising cAMP, also inhibited ECP release. Furthermore, pertussis toxin decreased ECP release on opsonized beads, indicating the involvement of pertussis-toxin-sensitive G proteins.
Conclusions C5a. and PAF enhance granule release, rather than acting as secretagogues themselves. PKC and PKA have opposing roles in the regulation of ECP release in response to SOS.     

Andrology: Modulation of prostaglandin synthesis in mammalian sperm acrosome reaction

Exogenous arachidonic acid induces the acrosome reaction andthe production of the prostaglandins PGE₂ and PGF₂ in bovinespermatozoa. Exogenous PGE₂ also induces the acrosome reactionand PGF₂ synthesis. To understand better the role of PGE₂ inthe induction of PGF₂ synthesis through modulation of phospholipaseA₂, inhibitors of this enzyme were used. The effects of PGE₂were blocked by phospholipase A₂ inhibitors and this inhibitionwas reversed by addition of arachidonic acid. These data indicatethat PGE₂ activates phospholipase A₂ to produce arachidonicacid. To determine whether protein kinase C modulates phospholipaseA₂ activity in this process, staurosporin, an inhibitor of proteinkinase C, was used. The effect of PGE₂ on PGF₂ production isinhibited by staurosporin and this inhibition was reversed byaddition of arachidonic acid indicating that protein kinaseC is involved in phospholipase A₂ activation. The effect ofexogenous arachidonic acid or PGE₂ on the acrosome reactionis blocked by lipoxygenase inhibitors but not by inhibitorsof cyclo-oxygenase, indicating that lipoxygenase products areinvolved in the mechanism of the acrosome reaction. The presenteddata shed light on the cross-talk between cyclo-oxygenase andlipoxygenase and their involvement in the sperm acrosome reaction.It is suggested that cyclo-oxygenase products modulate the activityof lipoxygenase which is a key enzyme in the mechanism leadingto the acrosome reaction. Stimulation of cyclo-oxygenase tosynthesize PGE₂ activates phospholipase A₂ to release arachidonicacid which is the substrate for lipoxygenase activity. Thus,the role of cyclo-oxygenase products is probably to reactivatethe phospholipase A₂ which was initially activated via the signaltransduction cascade.     

Measuring learning in individual flies is not necessary to study the effects of single-gene mutations inDrosophila: A reply to Holliday and Hirsch

Holliday & Hirsch (this issue) now agree that Quinnet al. (1974) have demonstrated learning [inDrosophila] with group data, and their inability to identify individual differences (IDs) in performance does not invalidate their conclusion that some individuals in the population must have learned. However, they consider it important, if not necessary, to show that anindividual fly has learned. In response to Holliday and Hirsch, this paper discusses why it is not necessary to measure learning in individual fruit flies before searching for underlying biochemical mechanisms.     

Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes

Background Activation of human basophils results in the release of many different mediators and the expression of new cell surface proteins. The markers CD63 and CD203c have been used in recent years to assess basophil activation but there have been many studies that demonstrate that expression of these markers can be dissociated from histamine release. Objective To determine the signal transduction requirements for CD203c and CD63 expression. Methods The current study began by exploring the dependency of CD203c and CD63 expression on protein kinase C (PKC) using known selective inhibitors of PKC. Results Between 30 and 300 nm , Ro‐31‐8220 and bisindoylmaleimide II (Bis II) had no effect on formyl–met–leu–phe‐ or anti‐IgE‐induced CD63 or CD203c but enhanced IgE‐mediated expression of CD63 by an average of 15‐fold at concentrations >1 μm . These results led to the suggestion that these inhibitors altered the normal pathways of degranulation (by a non‐PKC dependent mechanism), shifting the normal presence of piecemeal degranulation to the process termed anaphylactic degranulation (AND). Morphological studies demonstrated that concentrations of Ro‐31‐8220 and Bis II>1 μm dramatically increased the presence of degranulation sacs, a morphological feature of AND. Conclusion It is proposed that CD63 expression results from only the AND form of histamine release. Cite this as: D. MacGlashan Jr, Clinical & Experimental Allergy, 2010 (40) 1365–1377.     

Early Signals in the Mitogenic Response of Swiss 3T3 Cells: A Comparative Study of Purified PDGF Homodimers

Abstract

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB. Two distinct receptor subunits, α and β, have been identified which bind either all three isoforms of PDGF (α) or PDGF-BB only (β). Here, we have compared the effect of purified PDGF homodimers on the early intracellular signaling events and mitogenesis in Swiss 3T3 cells, which possess equivalent numbers of the α and β subunits. Both PDGF-AA and PDGF-BB stimulated receptor phosphorylation, inositol phosphate formation, activation of protein kinase C, calcium mobilization, EGF receptor transmodulation, sodium uptake, arachidonic acid release, cyclic AMP accumulation, and c-fos induction in a comparable, dose-dependent manner (half-maximal values for all these responses were in the 2–10 ng/ml range for both homodimers). At high concentrations of PDGF (> 10 ng/ml), the BB homo-dimer effect on early membrane and cytosolic signals was 20–30% greater than PDGF-AA, reflecting the greater number of available binding sites for PDGF-BB. DNA synthesis studies indicated that PDGF-AA and PDGF-BB were potent mitogens for Swiss 3T3 cells, displaying identical dose-response effects. Moreover, the mitogenic activities of both homodimers were equally potentiated in the presence of insulin. These results indicate that both PDGF-AA and PDGF-BB stimulate the full complement of molecular responses required for the synergistic interactions mediating long-term mitogenesis. We conclude that α and β receptor subunits do not differ in their ability to transduce PDGF-mediated signals leading to DNA synthesis in Swiss 3T3 cells.     

Basolateral transport of glutarate in proximal S2 segments of rabbit kidney: kinetics of the uptake process and effect of activators of protein kinase A and C

 The kinetics of tubular glutarate uptake, the coupling of glutarate to p-aminohippurate (PAH) transport and the effect of activators of protein kinase A and C on glutarate uptake were studied using isolated S₂ segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of [¹⁴C]glutarate reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s glutarate uptake measurements were performed. In a first set of experiments it could be shown that preloading proximal S₂ segments with glutarate (10^–3 M) stimulated [³H]PAH uptake indicating that glutarate may be a substrate of the PAH /dicarboxylate exchanger. The kinetic data revealed a K _m value of 0.62 mM and a V _max value of 84.1 pmol nl^–1min^–1 for tubular [¹⁴C]glutarate uptake across the basolateral cell membrane. In contrast to basolateral PAH transport (previous studies from this laboratory), tubular 30 s [¹⁴C]glutarate uptake was not affected by either the phorbol ester phorbol 12-myristate 13-acetate (PMA, 10^–7 M), an activator of protein kinase C, or by the membrane-permeant analogues of cAMP, dibutyryl cyclic AMP (db-cAMP, 10^–4 M) and 8-bromoadenosine 3′,5′-cyclic monophosphate (Br-cAMP, 10^–4 M). The results indicate that the protein kinases A and C have no function in the regulation of the renal basolateral dicarboxylate transporter. This finding agrees well with the structural feature of the recently cloned rabbit renal dicarboxylate transporter which does not contain any putative phosphorylation sites for protein kinase C or cAMP-dependent kinase. Received: 30 December 1997 / Received after revision and accepted: 25 February 1998     

Platelet‐derived growth factor receptors expressed in response to injury of differentiated vascular smooth muscle in vitro: effects on Ca2+ and growth signals

Vascular smooth muscle cells (VSMCs) in the intact vascular wall are differentiated for contraction, whereas the response to vascular injury involves transition towards a synthetic phenotype, with increased tendency for proliferation. Platelet‐derived growth factor (PDGF) is thought to be important for this process. We investigated expression and functional coupling of PDGF receptors (PDGFRs) α and β in rat tail arterial rings kept in organ culture, in order to capture early events in the phenotypic transition. In freshly dissected rings no PDGFR immunoreactivity was found in medial VSMCs, whereas PDGFR α was detected in nerve fibres. After organ culture for 1–4 days PDGFR α and β as well as phospholipase Cγ2 (PLCγ2), known to couple to PDGFR, were expressed in VSMCs within 100 μm of the cut ends. Calponin, a marker for the contractile phenotype, was decreased near the injured area, suggesting that cells were in transition towards synthetic phenotype. In these cells, which showed functional Ca²⁺‐release from the sarcoplasmic reticulum, PDGF‐AB (100 ng mL^–1) had no effect on [Ca²⁺]_i, whereas cultured VSMCs obtained from explants of rat tail arterial rings responded to PDGF‐AB with an increase in [Ca²⁺]_i. However, PDGFR within the cultured rings coupled to growth signalling pathways, as PDGF‐AB caused a tyrphostin AG1295‐sensitive activation of extracellular signal‐regulated kinases 1 and 2 and of [³H]‐thymidine incorporation. Thus, early expression of PDGFR in VSMC adjacent to sites of vascular injury coincides with signs of dedifferentiation. These receptors couple to growth signalling, but do not activate intracellular Ca²⁺ release.     

Molecular and functional characterization of polymorphisms in the secreted phospholipase A2 group X gene: relevance to coronary artery disease

Among secreted phospholipases A2 (sPLA2s), human group X sPLA2 (hGX sPLA2) is emerging as a novel attractive therapeutic target due to its implication in inflammatory diseases. To elucidate whether hGX sPLA2 plays a causative role in coronary artery disease (CAD), we screened the human PLA2G10 gene to identify polymorphisms and possible associations with CAD end-points in a prospective study, AtheroGene. We identified eight polymorphisms, among which, one non-synonymous polymorphism R38C in the propeptide region of the sPLA2. The T-512C polymorphism located in the 5′ untranslated region was associated with a decreased risk of recurrent cardiovascular events during follow-up. The functional analysis of the R38C polymorphism showed that it leads to a profound change in expression and activity of hGX sPLA2, although there was no detectable impact on CAD risk. Due to the potential role of hGX sPLA2 in inflammatory processes, these polymorphisms should be investigated in other inflammatory diseases. Claire Perret and Ikram Jemel contributed equally to this paper.     

Food Hypersensitivity in Mexican Adults at 18 to 50 Years of Age: A Questionnaire Survey

Purpose

There is limited epidemiological evidence of food hypersensitivity (FH) in the adult population. We aimed to determine the prevalence of FH in Mexican adults, their clinical features and to establish common food involved in its appearance.

Methods

We designed a cross-sectional study using a fixed quota sampling; 1,126 subjects answered a structured survey to gather information related to FH.

Results

The prevalence of FH in adults was 16.7% (95% CI, 14.5% to 18.8%), without statistical significant differences related to gender (women, 17.5% and men, 15.9%) or residential location. The most common clinical manifestations in adults with FH were oral allergy syndrome (70 of 1,126) and urticaria (55 of 1,126). According to category, fruits and vegetables were the most frequent foods to trigger FH (6.12%) and were individually related to shrimp (4.0%), and cow milk (1.5%). Adults under age 25 had a higher frequency of FH (OR, 1.39; 95% CI, 1.01 to 1.91, P <0.001). Personal history of any atopic disease was significantly associated with FH (P <0.0001).

Conclusions

The prevalence of FH is relatively high in Mexican adults, and FH is significantly associated with atopic diseases.     

The effect of heliox treatment in a rat model of focal transient cerebral ischemia

At nerve terminals G protein coupled receptors modulate neurotransmitter release probability. We recently showed that prolonged activation of metabotropic glutamate receptor 7, mGlu7 receptor, potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein, the hydrolysis of phosphatidylinositol (4,5)-bisphosphate, and the subsequent activation of the non-kinase diacylglycerol binding protein Munc13-1 which primes synaptic vesicle for exocytosis at the active zone. Here we found that inhibitors of diacylglycerol metabolism (diacylglycerol kinase inhibitor II and diacylglycerol lipase inhibitor RHC80267) remarkably reduce the time of mGlu7 receptor stimulation required for glutamate release potentiation in mice cerebrocortical nerve terminals. We conclude that changes in diacylglycerol levels at nerve terminals control the efficiency of the exocytotic release machinery.     

Role of spinal metabotropic glutamate receptor subtype 5 in the development of tolerance to morphine-induced antinociception in rat

Prolonged intrathecal (i.t.) administration of morphine results in tolerance to morphine-induced antinociception. We found that co-administration of selective metabotropic glutamate receptor subtype 5 antagonist MPEP with morphine could suppress the loss of morphine-induced antinociception and inhibit the development of tolerance to morphine-induced antinociceptive effect. Whereas, the specific metabotropic glutamate receptor subtype 5 agonist CHPG does the opposite. As the activation of NMDA receptor after chronic morphine administration has been verified, we suppose there is an enhanced activation of mGluR5 during the development of tolerance to morphine-induced antinociception. Activation of mGluR5 may mobilize the release of intracellular Ca²⁺ and activate PKC, leading to morphine-induced antinociception suppression. We conclude that mGluR5 contributes to the development of tolerance to morphine-induced antinociception after chronic morphine exposure.     

A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera

G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution.     

A Prospective Study to Compare Clinical Outcomes of Allergic Rhinitis Between Older and Younger Adults: A Potential Effect of Depression in Older Patients

Although younger patients with allergic rhinitis (AR) have been successfully treated with pharmacotherapy, there are no definitive data on treatment outcomes in older patients with AR. We performed a prospective study of 51 older adults with AR (aged over 65 years) and 101 younger AR patients (aged from 19 to 40 years) to compare clinical outcomes between the 2 groups and to evaluate the impact of depressed mood on treatment outcomes in older AR patients. Changes in total symptom scores (TSS), rhinitis-specific quality of life questionnaire (RQLQ) results, rhinitis control assessment test (RCAT) results and visual analog scale (VAS) scores were evaluated after 4-week treatment according to the Allergic Rhinitis and its Impact on Asthma (ARIA) guideline, and the severity of depressed mood was assessed by using the geriatric depression scale. After 4-week treatment, younger AR patients had greater improvements in clinical scores compared with older adults; differences in least squares mean changes from baseline in older patients vs. younger patients were 1.71 (P = 0.004) for TSS, 10.84 (P < 0.001) for RQLQ, 0.80 (P = 0.275) for RCAT, and 8.60 for VAS score (P = 0.061). Multiple logistic regression analysis showed that the severity of depressed mood was independently associated with severe chronic upper airway disease (adjusted odds ratio, 1.385; P = 0.004). Our results suggest that older AR patients are less responsive to standard treatment compared with younger AR patients and that depressed mood is strongly associated with the increased risk of uncontrolled AR in older AR patients.     

Class II MHC cytoplasmic domain-mediated signaling in B cells: A tail of two signals

In addition to their role in antigen presentation, class II MHC molecules also transmit signals to B lymphocytes. Class II MHC-mediated signals initiate a range of events in B cells, including induction of cell surface proteins, initiation of cell-cycle progression/proliferation, activation of or protection from apoptosis, and antigen-dependent plasma cell differentiation. Although various transmembrane signaling proteins associate with class II MHC molecules, the class II MHC cytoplasmic domains are essential for signals leading to increased intracellular cAMP and activation of protein kinase C (PKC). Although truncation and mutagenesis studies have provided considerable information about the cytoplasmic domain sequences required, how class II MHC molecules elicit cAMP and PKC activation is not known. Further, appropriate T-dependent B cell responses require intact cAMP and PKC signaling, but the extent to which class II MHC signals are involved is also unknown. This review details our current knowledge of class II MHC cytoplasmic domain signaling in B cells with an emphasis on the likely importance of class II MHC signals for T-dependent antibody responses.     

RACK1 has the nerve to act: structure meets function in the nervous system

The receptor for activated protein kinase C 1 (RACK1) is an intracellular adaptor protein. Accumulating evidence attributes to this member of the tryptophan-aspartate (WD) repeat family the role of regulating several major nervous system pathways. Structurally, RACK1 is a seven-bladed-beta-propeller, interacting with diverse proteins having distinct structural folds. When bound to the IP3 receptor, RACK1 regulates intracellular Ca2+ levels, potentially contributing to processes such as learning, memory and synaptic plasticity. By binding to the NMDA receptor, it dictates neuronal excitation and sensitivity to ethanol. When bound to the stress-induced acetylcholinesterase variant AChE-R, RACK1 is implicated in stress responses and behavior, compatible with reports of RACK1 modulations in brain ageing and in various neurodegenerative diseases. This review sheds new light on both the virtues and the variety of neuronal RACK1 interactions and their physiological consequences.     

Calcium-permeable acid-sensing ion channel in nociceptive plasticity: A new target for pain control

The development of chronic pain involves increased sensitivity of peripheral nociceptors and elevated neuronal activity in many regions of the central nervous system. Much of these changes are caused by the amplification of nociceptive signals resulting from the modulation and altered expression of specific ion channels and receptors in the central and peripheral nervous system. Understanding the processes by which these ion channels and receptors are regulated and how these mechanisms malfunction may lead to new treatments for chronic pain. Here we review the contribution of the Ca²⁺-permeable acid-sensing ion channel (ASIC_Ca) in the development and persistence of chronic pain, and the potential underlying mechanisms. Accumulating evidence suggests that ASIC_Ca represents an attractive new target for developing effective therapies for chronic pain.