AII amacrine neurons of the rat retina show diurnal and circadian rhythms of parvalbumin immunoreactivity

We investigated parvalbumin immunoreactivity (PA-IR) in the retinas of rats maintained on a 12:12 h light:dark cycle, or after being placed in constant darkness for 24–72 h. Retinas were harvested at zeitgeber and circadian times 02:00, 06:00, 10:00, 14:00, 18:00 and 22:00 h. PA-IR was found primarily in retinal amacrine cells of the AII subtype. In a light/dark cycle, PA-IR showed a clear rhythm, with a low near zeitgeber time (ZT) 10:00 h and a peak near ZT 18:00 h. The ratio of immunofluorescence intensities at these timepoints was >15-fold. When animals were kept in complete darkness for 1–3 days, the rhythm of PA-IR was still preserved, but was progressively reduced in amplitude. The rhythm of PA-IR inferred from immunohistochemical data was confirmed by Western blots. We conclude that PA-IR in the rat retina shows an underlying circadian rhythm that is enhanced by cyclic light. The regulation may involve translocation of the protein between cell compartments and/or new protein synthesis.This study was supported by an OTKA grant (T 34160), NIH grants NS 37919 (R.S.) and ET 03570, NSF grant IBN-96418886 (R.S.), and grants from the Helen Hoffritz Charitable Trust and Research to Prevent Blindness, Inc. R.G. was also in receipt of a János Bolyai fellowship     

Immunocytochemical localization of ornithine decarboxylase in cultured murine cells

Summary Antiserum elicited to ornithine decarboxylase (ODC) purified from murine RAW 264 macrophage-like cells has been employed to localize ODC in cultured murine cells. The antiserum immunoprecipitated 100% of the ODC activity from the cultured cells. The specificity of the antiserum was demonstrated by the immunoprecipitation from ³⁵S-methionine metabolically-labeled cell extracts of a single protein which migrated upon SDS-gel electrophoresis coincident with authentic ODC. Indirect immunofluorescence experiments were performed on paraformaldehyde-fixed RAW 264 cells and JB6 epidermal cells using the rabbit anti-ODC antiserum and FITC-conjugated goat anti-rabbit IgG. Little immunofluorescence was apparent in non-stimulated cells. Intense immunofluorescence was detectable in stimulated cells at times of peak cellular ODC activity. Antigenically-reactive ODC was localized diffusely in the cytoplasm and was absent in the nuclei of RAW 264 cells, whereas in the JB6 cells the immunodetectable enzyme protein was localized in a punctate pattern in both the cytoplasm and nucleoplasm and was absent in the nucleolus. The appearance and disappearance of immunoreactive ODC in both cell types after stimulation was consistent with the alterations in ODC activity.     

The relevance of various tests for the study of specificity in immunocytochemical staining: a review

Factors determining the specificity of immunocytochemical (ICC) tissue stainings as well as the various tests to study these factors are discussed. Since every specificity test only deals with particular aspects of the ICC procedure, a practical sequence of known test methods is proposed, which enables the determination of the specificity of the ICC tissue staining and, after possibly needed antiserum purification steps, may result in a monospecific staining. It is made clear that such a sequence has always to include a tissue-spectrum affinity test, in which the spectrum of tissue antigens is controlled for antibody binding. A variety of such tests, consisting of separation of tissue compounds, fixation, and ICC detection, are discussed as well as their pros and cons with respect to their predictability for the actual serum specificity in the tissue section.     

Activation of Adenylyl Cyclase by Preincubation of Rat Cerebral-Cortical Membranes Under Phosphorylating Conditions: Role of ATP, GTP, and Divalent Cations

Abstract: The effects of preincubation under phosphorylating conditions on adenylyl cyclase activity were studied in preparations containing synaptic membranes from rat cerebral cortex. Preincubation of the membranes with 2 mM ATP and 10 mM MgCl₂ resulted in a 50% increase of adenylyl cyclase activity which withstood sedimentation and washing. This activation was maximal after 5 min of preincubation, was reversed after longer preincubations, and paralleled the time course of endogenous phosphorylation-dephosphorylation of proteins observed under these conditions. The activation showed a critical requirement for Mg²⁺ ions and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral-cortical membranes with adenosine-5′-0-(3-thiophosphate) (ATPγS), but this activation was not reversed by prolonged preincubation times. The activation by ATPγS was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATPγS to guanosine-5′-0-(3-thiophosphate) (GTPγS), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 μ.M GTP and 10 mM MgCl₂ also produced a 50% activation of adenylyl cyclase which withstood sedimentation and washing and was reversed by longer preincubations. Endogenous phosphorylation of specific protein components in the membranes during the preincubation was examined by including radioactively labeled nucleoside thiophosphates in the preincubation medium. Incorporation of ³⁵S from [³⁵S]ATPγS into a protein component with apparent M_r of 54,000 daltons (54K) correlated significantly with the activation of adenylyl cyclase by ATPγS. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [³⁵S]ATPγS. Endogenous activity utilizing [γ-³²P]GTP as a phosphate donor also preferentially phosphorylated the 54K protein band. These results support previous suggestions that protein phosphorylation plays a role in the regulation of adenylyl cyclase activity. Among the numerous membrane-bound phosphoproteins in rat brain, we have identified a specific protein component with an apparent M_r of 54,000 daltons as the most likely candidate for involvement in this mode of regulation. This 54K protein, which is a principal substrate for a GTP-preferring protein kinase activity in brain membranes, can now be at the focus of investigations attempting to demonstrate a direct role for protein phosphorylation in adenylyl cyclase regulation.     

Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F,Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α₁β₁ possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.     

The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2a Decreases Receptor Expression and Prevents Gi-mediated Inhibition of Adenylyl Cyclase Activity

The corticotropin-releasing factor receptor type 2a (CRF_2(a)R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF₁R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF_2(a)R in comparison with the CRF₁R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G_s protein, G_i-mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF₁R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.     

Delivery of antibody-captured proteins into living cells using PTD-fused protein A

Protein transduction domain (PTD)-mediated protein delivery into animal cells is a useful technique for regulating cellular functions. Proteins captured by antibodies were delivered into living cells using an antibody/PTD-fused protein A complex. As a model protein, fluorescent-modified antibodies, captured by their respective primary antibody, were analyzed by fluorescence-activated cell sorting (FACS) which showed that the fluorescent-modified antibodies were directly delivered into cells. Peroxidase, captured by its specific antibody, was also delivered into cells and retained its activity.     

Allosteric Activation of Bordetella pertussis Adenylyl Cyclase by Calmodulin: MOLECULAR DYNAMICS AND MUTAGENESIS STUDIES*

Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg³³⁸, Asn³⁴⁷, and Asp³⁶⁰) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn³⁴⁷; ACm1A), two (Arg³³⁸ and Asp³⁶⁰; ACm2A), or three residues (Arg³³⁸, Asn³⁴⁷, and Asp³⁶⁰; ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3A·C-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.     

Stimulation of Postsynaptic and Inhibition of Presynaptic Adenylyl Cyclase Activity by Metabotropic Glutamate: Receptor Activation

Abstract: To determine the subcellular distribution of cyclic AMP-coupled metabotropic glutamate receptors (mGluRs), the effects of glutamate agonists on adenylyl cyclase activity were examined using two hippocampal membrane preparations. These were synaptosomes (SY), which are composed of presynaptic terminals, and synaptoneurosomes (SN), which are composed of both pre-and postsynaptic elements. In SY, a water-soluble analogue of forskolin (7β-forskolin) increased enzyme activity ˜ 10-fold at the highest concentration tested. The selective metabotropic receptor agonist (1S,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3 R -ACPD) inhibited enzyme activity as did glutamate and quisqualate. l -Amino-4-phosphobutanoate ( l -AP4) had no effect on enzyme activity at any concentration tested. The metabotropic receptor antagonist l -2-amino-3-phosphopropionic acid ( l -AP3) was not effective in the SY in antagonizing the agonist-induced decreases in adenylyl cyclase activity by glutamate or 1S,3 R -ACPD. It was, however, effective at antagonizing quisqualate-induced decreases in enzyme activity. In SN, at the highest concentration tested, 7β-forskolin produced a 60-fold increase in adenylyl cyclase activity. As was observed in SY, glutamate decreased adenylyl cyclase activity in SN. In contrast, 1S,3 R -ACPD, quisqualate, and l -AP4 increased adenylyl cyclase activity. In the SN, l -AP3 was ineffective in antagonizing any agonist-induced increases (1S,3 R -ACPD, l -AP4, and quisqualate) or decreases (glutamate) in adenylyl cyclase activity. The data suggest that postsynaptic metabotropic glutamate receptor activation results in stimulation of adenylyl cyclase activity, whereas inhibition of this enzyme appears to be mediated at least partly through presynaptic mechanisms.     

Persistence of the Interaction of Calmodulin with Adenylyl Cyclase: Implications for Integration of Transient Calcium Stimuli

Abstract: Ca²⁺/calmodulin-sensitive adenylyl cyclase plays a role in several forms of synaptic plasticity and learning. To understand how cellular signals from neuronal activity during behavioral stimuli might be integrated by adenylyl cyclase, we have characterized the response of type I adenylyl cyclase to transient Ca²⁺ stimuli. Stimulation by a several second Ca²⁺ stimulus is delayed, rising to a peak after the Ca²⁺ stimulus has ended. We attempted to identify the site of the persistent Ca²⁺ signal that enabled adenylyl cyclase stimulation to increase after free Ca²⁺ had declined. Free calmodulin itself displayed no persistent activation by Ca²⁺ and was unable to activate adenylyl cyclase if exposed to low Ca²⁺ solution <1 s before reaching adenylyl cyclase. In contrast, activation of the calmodulin-adenylyl cyclase complex persisted for seconds after Ca²⁺ stimulus. Activation decayed with a time constant of 6 or 13 s depending on assay conditions. These results suggest that the calmodulin-adenylyl cyclase complex can serve as a site of cellular memory for a Ca²⁺ transient that has ended even before adenylyl cyclase is fully activated.     

A new tool for plant cell biology: in vivo antibody uptake in plant protoplasts

We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34^cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.     

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson''s disease. Acute activation of Gαi/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of Gαi/o-linked receptors including D₂ dopamine and μ opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gbγ signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384 well format would facilitate future studies. Using two D₂ dopamine receptor cellular models (i.e. CHO-D_2L and HEK-AC6/D_2L), we have converted our 48-well sensitization assay (>20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Adenylyl Cyclase Activity in Postmortem Human Brain: Evidence of Altered G Protein Mediation in Alzheimer's Disease

The effects of agonal status, postmortem delay, and age on human brain adenylyl cyclase activity were determined in membrane preparations of frontal cortex from a series of 18 nondemented subjects who had died with no history of neurological or psychiatric disease. Basal and guanosine 5'-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were not significantly reduced over an interval from death to postmortem of between 3 and 37 h and were also not significantly different between individuals dying with a long terminal phase of an illness and those dying suddenly. Basal and aluminum fluoride-stimulated enzyme activities showed a negative correlation with increasing age of the individual. In subsequent experiments, basal and guanosine 5'-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were compared in five brain regions from a series of eight Alzheimer's disease and seven matched nondemented control subjects. No significant differences were observed between the groups for either basal activity or activities in response to forskolin stimulation of the catalytic subunit of the enzyme. In contrast, enzyme activities in response to stimulation with guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride were significantly reduced in preparations of neocortex and cerebellum from the Alzheimer's disease cases compared with the nondemented controls. Lower guanosine 5'-O-(3-thiotriphosphate)-, but not aluminum fluoride-, stimulated activity was also observed in preparations of frontal cortex from a group of four disease controls compared with nondemented control values. The disease control group, which contained Parkinson's disease and progressive supranuclear palsy patients, showed increased forskolin-stimulated activity compared with both the nondemented control and the Alzheimer's disease groups. These findings indicate a widespread impairment of G protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain, which occurs in the absence of altered enzyme catalytic activity and which is unlikely to be the result of non-disease-related factors associated with the nature of terminal illness of individuals.     

一种改良型的免疫印迹法

目的：蛋白免疫印迹法是现代生物实验过程中运用最为广泛的实验技术,常规的免疫印迹法在应用过程中存在很多弊端,如浪费抗体等,因此非常有必要探索出一种新型的免疫印迹法,本文旨在探索一种能够节约抗体的免疫印迹实验方法。方法：将8只SD大鼠随机分常规组和改进组两组,每组4只,活取视网膜组织,进行组织匀浆、蛋白定量,取不同蛋白总量的匀浆变性液进行β-Tubulin的免疫印迹实验,比较两组之间β-Tubulin的蛋白表达量之间是否存在显著性差异,实验需重复三次。结果：不同总量蛋白的免疫印迹显示两组之间β-Tubulin的表达量并无显著性差异。但是,相比常规方法,改进法使用的抗体量更少,条带更容易检测得到。结论：改进后的免疫印迹法能有效的节约抗体,操作方便,实用性强。     

Activation of Type II Adenylyl Cyclase by the Cloned μ-Opioid Receptor: Coupling to Multiple G Proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ-opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ-opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ-opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ-selective agonist, [d -Ala², N-Me-Phe⁴,Gly⁵-ol]enkephalin, to stimulate cyclic AMP accumulation in a dose-dependent manner. The opioid-induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin-sensitive G_i proteins, because it was abolished completely by the toxin. Possible coupling between the μ-opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid-induced response became pertussis toxin-insensitive and was enhanced significantly upon co-expression with the α subunit of G_z, whereas those of G_q, G₁₂, or G₁₃ inhibited the opioid response. When pertussis toxin-sensitive G protein α subunits were tested under similar conditions, all three forms of α_i and both forms of α_o were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co-expression of α subunits reflects a functional coupling between α subunits and the μ-opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ-opioid receptor can couple to G_i1–3, G_o1–2, and G_z, but not to G_s, G_q, G₁₂, G₁₃, or G_t.     

Bimodal Opioid Regulation of Cyclic AMP Formation: Implications for Positive and Negative Coupling of Opiate Receptors to Adenylyl Cyclase

Abstract: A μ-selective opiate receptor agonist, sufentanil, can either increase or decrease the stimulated formation of cyclic AMP (cAMP) in the myenteric plexus. The direction of the opioid modulation of this second messenger depends on the concentration of opioid used. Low doses of opioid enhance, whereas higher concentrations inhibit, the magnitude of cAMP that is formed in response to electrical stimulation. Opioids exert this dual regulation on only stimulated cAMP formation. Basal levels are not affected. Opioid facilitation and inhibition of stimulated cAMP formation are blocked by naloxone, indicating mediation by opiate receptors. Because all experiments were conducted in the presence of a phosphodiesterase inhibitor, it is highly unlikely that opioid regulation of stimulated cAMP formation is due to changes in the rate of its degradation. Positive and negative coupling of μ-opiate receptors to adenylyl cyclase is the most plausible explanation for the bimodal opioid effects on cAMP content. The marked parallel between the current observations and the previously reported bimodal opioid regulation of evoked enkephalin release is consistent with the hypothesis that adenylyl cyclase is one biochemical substrate for the bimodal opiate receptor-coupled regulatory mechanism governing the stimulated release of this opioid peptide.