Marine records reveal multiple phases of Toba's last volcanic activity.

The Indonesian Young Toba Tuff (YTT), classically dated around 74 ka BP, is considered as a short-lived explosive cataclysmic super-eruption. The huge amounts of ash and SO2 emitted are likely to have triggered a volcanic winter which accelerated the transition to the last glaciation, and may have induced a human genetic bottleneck. However, the global climatic impact of the YTT or its duration are hotly debated. The present work offers a new interpretation of the Toba volcanic complex eruptive history. Analysing the BAR94-25 marine core proximal to the Toba volcanic center and combining it with high-resolution tephrostratigraphy and δ18O stratigraphy, we show that the Toba complex produced a volcanic succession that consists of at least 17 distinct layers of tephra and cryptotephra. Textural and geochemical analyses show that the tephra layers can be divided in 3 main successive volcanic activity phases (VAP1 to VAP3) over a period of ~ 50 kyr. The main volcanic activity phase, VAP2, including the YTT, is likely composed of 6 eruptive events in an interval whose total duration is ~ 10 ka. Thus, we suggest that the eruptive model of the Toba volcano must be revised as the duration of the Toba volcanic activity was much longer than suggested by previous studies. The implications of re-estimating the emission rate and the dispersion of ashes and SO2 include global environmental reconstitutions, climate change modelling and possibly human migration and evolution.

Nanoscale trace metal imprinting of biocalcification of planktic foraminifers by Toba's super-eruption.

Bioactive metal releases in ocean surface water, such as those by ash falls during volcanic super-eruptions, might have a potentially toxic impact on biocalcifier planktic microorganisms. Nano-XRF imaging with the cutting-edge synchrotron hard X-ray nano-analysis ID16B beamline (ESRF) revealed for the first time a specific Zn- and Mn-rich banding pattern in the test walls of Globorotalia menardii planktic foraminifers extracted from the Young Toba Tuff layer, and thus contemporaneous with Toba's super-eruption, 74,000 years ago. The intra-test correlation of Zn and Mn patterns at the nanoscale with the layered calcareous microarchitecture, indicates that the incorporation of these metals is syngenetic to the wall growth. The preferential Mn and Zn sequestration within the incipient stages of chamber formation suggests a selective incorporation mechanism providing a resilience strategy to metal pollution in the test building of planktic foraminifers.

Tracking a CAD-ALK gene rearrangement in urine and blood of a colorectal cancer patient treated with an ALK inhibitor.

BACKGROUND: Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. PATIENTS AND METHODS: We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying a CAD-ALK translocation during treatment with an ALK inhibitor. RESULTS: Using a custom next generation sequencing panel we identified the genomic CAD-ALK rearrangement and a TP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of the CAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient's clinical course. Detection of the CAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identified ALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. CONCLUSION: We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.

Selective zircon accumulation in a new benthic foraminifer, Psammophaga zirconia, sp. nov.

Benthic foraminifera are single-celled eukaryotes that make a protective organic, agglutinated or calcareous test. Some agglutinated, single-chambered taxa, including Psammophaga Arnold, 1982, retain mineral particles in their cytoplasm, but the selective mechanism of accumulation is not clear. Here, we report the ability of a foraminiferal species to select and accumulate zircons and other heavy minerals in their cytoplasm. In particular, the use of Scanning Electron Microscope coupled with an Energy Dispersive X-ray microanalysis system (SEM-EDS) enabled a representative overview of the mineral diversity and showed that the analysed Psammophaga zirconia sp. nov. individuals contained dominantly crystals of zircon (51%), titanium oxides (27%), and ilmenite (11%) along with minor magnetite and other minerals. The studied specimens occur in the shallow central Adriatic Sea where the sediment has a content of zircon below 1% and of other heavy minerals below 4%. For that reason we hypothesize that: (i) P. zirconia may be able to chemically select minerals, specifically zircon and rutile; (ii) the chemical mechanism allowing the selection is based on electrostatic interaction, and it could work also for agglutinated foraminifera (whether for ingestion, like Xenophyophores, or incorporation in the test as in many other described taxa). In particular, this aptitude for high preferential uptake and differential ingestion or retention of zircon is reported here for the first time, together with the selection of other heavy minerals already described in members of the genus Psammophaga. They are generally counted among early foraminifera, constructing a morphologically simple test with a single chamber. Our molecular phylogenetic study confirms that P. zirconia is a new species, genetically distinctive from other Psammophaga, and occurs in the Adriatic as well as in the Black Sea.

Biomineralization of Schlumbergerella floresiana, a significant carbonate-producing benthic foraminifer.

Most foraminifera that produce a shell are efficient biomineralizers. We analyzed the calcitic shell of the large tropical benthic foraminifer Schlumbergerella floresiana. We found a suite of macromolecules containing many charged and polar amino acids and glycine that are also abundant in biomineralization proteins of other phyla. As neither genomic nor transcriptomic data are available for foraminiferal biomineralization yet, de novo-generated sequences, obtained from organic matrices submitted to ms blast database search, led to the characterization of 156 peptides. Very few homologous proteins were matched in the proteomic database, implying that the peptides are derived from unknown proteins present in the foraminiferal organic matrices. The amino acid distribution of these peptides was queried against the uniprot database and the mollusk uniprot database for comparison. The mollusks compose a well-studied phylum that yield a large variety of biomineralization proteins. These results showed that proteins extracted from S. floresiana shells contained sequences enriched with glycine, alanine, and proline, making a set of residues that provided a signature unique to foraminifera. Three of the de novo peptides exhibited sequence similarities to peptides found in proteins such as pre-collagen-P and a group of P-type ATPases including a calcium-transporting ATPase. Surprisingly, the peptide that was most similar to the collagen-like protein was a glycine-rich peptide reported from the test and spine proteome of sea urchin. The molecules, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses, included acid-soluble N-glycoproteins with its sugar moieties represented by high-mannose-type glycans and carbohydrates. Describing the nature of the proteins, and associated molecules in the skeletal structure of living foraminifera, can elucidate the biomineralization mechanisms of these major carbonate producers in marine ecosystems. As fossil foraminifera provide important paleoenvironmental and paleoclimatic information, a better understanding of biomineralization in these organisms will have far-reaching impacts.

The prospective white matter: an atypical neurogenic niche in the developing cerebellum.

Cerebellar GABAergic interneurons comprise heterogeneous phenotypes located at strategic levels of the local networks. Recent findings indicate that they all derive from a common population of multipotent progenitors whose fate choices are determined by instructive information provided by the PWM environment. Here we review about the atypical neurogenic strategy operated within such postnatal niche and we discuss possible instructive mechanisms governing interneuron specification and differentiation.

Acetyl-L-carnitine increases artemin level and prevents neurotrophic factor alterations during neuropathy.

Damages to the nervous system are the primarily cause of neuropathy and chronic pain. Current pharmacological treatments for neuropathic pain are not able to prevent or revert morphological and molecular consequences of tissue injury. On the other hand, many neurotrophins, like nerve growth factor (NGF), paired off restorative effects with hyperalgesia. Interestingly, the glial cell line-derived neurotrophic factors GDNF and Artemin (ARTN) seem to support neuron survival and to normalize abnormal pain behaviour. In the present research protein levels of NGF, GDNF and ARTN were evaluated in a rat model of peripheral neuropathy, the chronic constriction injury (CCI). NGF was increased by CCI in the ipsilateral dorsal root ganglia (DRG), in the spinal cord and in the periaqueductal grey matter (PAG). On the contrary, ARTN was decreased bilaterally in DRG, spinal cord and PAG. GDNF levels decreased in ipsilateral DRG, whereas the constriction did not modify its expression in the central nervous system districts. Repeated treatments with the antihyperalgesic and neuroregenerative compound acetyl-l-carnitine (ALCAR; 100 mgkg(-1) i.p. twice daily for 15 days) was able to prevent the increase of NGF levels. In conditions of pain relief ALCAR normalized peripheral and central alterations of GDNF and ARTN levels. Characteristically, sham animals that underwent the same ALCAR treatment, showed increased levels of ARTN both in the DRG and in the spinal cord. These data offer a new point of view on the mechanism of the antihyperalgesic as well as the neuroprotective effect of ALCAR.

The neuropathy-protective agent acetyl-L-carnitine activates protein kinase C-gamma and MAPKs in a rat model of neuropathic pain.

The gamma isoform of protein kinase C (PKCgamma) is an injury-activated intracellular modulator that boosts neuronal activity in algesic and neuroregenerative signalling pathways. Acetyl-L-carnitine (ALCAR), a physiological compound with role in bioenergetic functions, shows an antihyperalgesic effect and at the same time can exert neuroregenerative and neuroprotective effects. Aimed to explore the link between pain and neuroregeneration, the effect of ALCAR treatment (100 mg kg(-1) i.p. twice daily for 15 days) on PKCgamma and mitogen-activated protein kinases (MAPKs) expression has been evaluated in CCI (chronic constriction injury) rats. The sciatic nerve and the lumbar tract of the spinal cord were processed to evaluate the levels of the phosphorylated form of PKCgamma, ERK 1,2, SAP/JNK, p-38 and c-Jun; furthermore, the mRNA expression of the early genes c-Jun and c-Fos has been investigated. Fifteen days after injury, the analysis in the sciatic nerves highlighted a bilateral increase of the activated forms of PKCgamma, ERK 1,2 and SAP/JNK, whereas c-Jun showed an increase only ipsilaterally. ALCAR completely prevented mechanical hyperalgesia and provoked in the nerve a c-Jun increment only. In the lumbar tract of the spinal cord, higher levels of activated PKCgamma, ERK 1,2, p38, SAP/JNK and c-Jun proteins were detected in the ipsilateral side in respect of sham. ALCAR was able to stimulate this expression profile. At the transcriptional level c-Jun mRNA was increased in the ipsilateral side of spinal cord of CCI saline-treated rats, whereas c-Fos mRNA was unchanged. ALCAR had a stimulatory effect on both these early genes. These findings may represent a different approach in the study of the complex balance between pain and neuroregeneration and could constitute the basis for developing new disease modifying agents in the treatment of neuropathic pain.

Supraspinal role of protein kinase C in oxaliplatin-induced neuropathy in rat.

Oxaliplatin is a platinum-based chemotherapy drug characterized by the development of a painful peripheral neuropathy which is reproduced in rodent animal models with features observed in humans. Our focus was to explore the alterations of intracellular second messengers at supraspinal level in oxaliplatin-induced mechanical hyperalgesia. In our experiments, chronic administration of oxaliplatin to rats induced mechanical hyperalgesia which lasted for many days. When the hyperalgesic rats were submitted to paw pressure test in the presence of selective PKC inhibitor Calphostin C supraspinally administered, hyperalgesic effect could be reversed showing that PKC activity in supraspinal brain regions is needed. Concurrently, oxaliplatin chronic treatment induced a specific upregulation of gamma isoforms of PKC and increased phosphorylation of gamma/epsilon PKC isoforms within thalamus and PAG. Phosphorylation was reversed when PKC activity was inhibited by Calphostin C. Distinct PKC-activated MAPK pathways, including p38MAPK, ERK1/2 and JNK, were investigated in chronic oxaliplatin rat. A dramatic phosphorylation increase, Calphostin C sensitive, could be observed in thalamus and PAG for p38MAPK. These data show that, in oxaliplatin-induced neuropathy, enhanced mechanical nociception is strictly correlated with increased phosphorylation of specific intracellular mediators in PAG and thalamus brain regions pointing to a role of these supraspinal centers in oxaliplatin-induced neuropathic pain mechanism.

Neuropathy-induced apoptosis: protective effect of physostigmine.

Traumatic, infectious, metabolic, and chemical noxa to the nervous system are the etiology of a crippling disease generally termed neuropathy. Motor disorders, altered sensibility, and pain are the pathognomonic traits. Cellular alterations induced by this chronic pathology include mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. Energy imbalance can compromise the maintenance of mitochondrial membrane potential, furthering the release of cytochrome C and the subsequent cleavage and activation of caspases. Chronic constriction injury (CCI) of the rat sciatic nerve is a neuropathy model able to induce a strong mitochondrial impairment with a consequent apoptotic induction. In this model, the acetylcholinesterase inhibitor physostigmine is administered at 0.125 mg/kg i.p. (twice per day) starting from the operation and for 15 days after. The cholinergic activation reduces cytosolic levels of cytochrome C, suggesting an improved stability of the mitochondrial membrane, and the expression level of the active caspase 3 fragments (19, 16 kDa) is reduced significantly with respect to saline treatment. Accordingly, physostigmine impairs caspase 3 protease activity. In fact, the target of the activated caspase 3, the 89-kDa PARP fragment, is significantly less expressed in the ligated nerve of physostigmine-treated rats, reaching levels that are comparable to those in the contralateral unligated nerve. Finally, this natural acetylcholinesterase inhibitor reduces DNA fragmentation both in the proximal and in the distal parts of the nerve. This protection correlates with the induction of XIAP. Therefore, apoptosis, central to tissue degeneration, is prevented by repeated physostigmine treatment of CCI animals.

An antidepressant behaviour in mice carrying a gene-specific InsP3R1, InsP3R2 and InsP3R3 protein knockdown.

Evidence has accumulated for the involvement of Ca(2+) in the pathophysiology of mood disorders. Elevations in both resting and stimulated intracellular Ca(2+) levels in patients with affective disorders have been reported. The role of inositol-1,4,5-trisphosphate receptors (InsP3Rs), which allow mobilization of intracellular Ca(2+) stores, was, then, investigated in the mouse forced swimming test. InsP3R antagonists (heparin, xestospongin C) as well as an inositol monophosphatase inhibitor (LiCl) showed an antidepressant activity of intensity comparable to clinically used antidepressants. InsP3Rl, InsP3R2 and InsP3R3 knockdown mice were obtained to investigate the role of InsP3R isoforms. We generated mice carrying a cerebral knockdown of InsP3Rl, InsP3R2 and InsP3R3 proteins by administering antisense oligonucleotides complementary to the sequence of InsP3Rl, InsP3R2 and InsP3R3. These antisense-treated mice showed a specific InsP3R protein level reduction in the mouse cerebral cortex and hippocampus, demonstrated by immunoblotting, immunoprecipitation and immunocytochemistry experiments. Knockdown mice for each InsP3R isoforms showed an antidepressant behaviour and the induced phenotype was reversible disappearing 7 days after the end of the treatment. The absence of impairment of locomotor activity and spontaneous mobility in InsP3R knockdown mice was revealed. These results indicate the involvement of the InsP3R-mediated pathway in the modulation of depressive conditions and may be useful for the development of new therapeutical strategies for the treatment of mood disorders.

Type 1 and type 3 ryanodine receptors are selectively involved in muscarinic antinociception in mice: an antisense study.

The importance of an intracellular calcium content increase to obtain cholinergic antinociception was demonstrated. The physiological and pathological role of ryanodine receptors (RyRs), receptors involved in the mobilization of intracellular calcium stores, at the CNS level is poorly understood. The aim of the present study was, therefore, to investigate the role of supraspinal endoplasmic type 1, 2 and 3 RyR subtypes in muscarinic antinociception in conditions of acute thermal (hotplate test) and inflammatory (abdominal constriction test) pain. In the absence of isoform selective RyR antagonists, types 1, 2 and 3 RyR knockdown mice were obtained. Western blotting experiments were performed to quantify the RyR isoform protein levels in knockdown mice demonstrating a selective protein level reduction in knockdown animals. I.c.v. pretreatment with an antisense oligonucleotide (aODN) against type 1 or type 3 RyR prevented cholinergic antinociception in the hotplate test shifting to the right of the physostigmine dose-response curve. This antagonistic effect disappeared 7 days after the end of the aODN administration. Conversely, the physostigmine analgesia remained unmodified in type 2 RyR knockdown mice. Similar results were obtained in the abdominal constriction test. Mice undergoing aODN treatments showed neither alteration of animals' gross behavior nor locomotor impairment (rota-rod and hole board tests). These results elucidate the intracellular mechanism underlying muscarinic antinociception. A selective involvement of RyR1 and RyR3 in supraspinal muscarinic analgesia was demonstrated whereas RyR2 appears not to play an essential role in acute thermal and inflammatory pain.

Cerebral abscesses and necrotic cerebral tumours: differential diagnosis by perfusion-weighted magnetic resonance imaging.

PURPOSE: This study was undertaken to evaluate the usefulness of perfusion-weighted imaging (PWI) in the differential diagnosis of ring-enhancing cerebral lesions, including abscesses, high-grade gliomas and metastases. MATERIALS AND METHODS: Nine cerebral abscesses (five pyogenic, four from Toxoplasma gondii), ten glioblastomas and five cerebral metastases in 19 patients were studied with gadolinium-enhanced magnetic resonance imaging, diffusion-weighted imaging (DWI) including calculation of mean apparent diffusion coefficient (ADC) of the lesion core, and PWI. At PWI, the mean of the maximum regional cerebral blood volume (rCBV) was calculated in the gadolinium-enhancing peripheral solid areas and compared with that of the contralateral normal-appearing white matter [ratio=rCBV (lesion)/rCBV (contralateral normal-appearing white matter)]. RESULTS: DWI achieved the differential diagnosis in all cases except for the four Toxoplasma abscesses. At PWI, the mean ratio of the rCBV of the capsular portion was 0.72+/-0.08 (range 0.60-0.82) in the pyogenic abscesses, 0.84+/-0.07 (range 0.75-0.91) in the Toxoplasma abscesses, 4.45+/-1.5 (range 2.9-8.0) in the high-grade gliomas and 3.58+/-0.68 (range 3.28-4.27) in the metastases. CONCLUSIONS: PWI seems to be useful in the differential diagnosis of ring-enhancing cerebral lesions. High rCBV values in the peripheral areas appear to indicate the possibility of a necrotic tumour, whereas low values tend to indicate an abscess.

Facial nerve schwannoma presenting as a mass in the middle cranial fossa: typical neuroradiological patterns for a preoperative diagnosis.

Facial nerve schwannoma is a rare primary neurogenic tumour that may originate anywhere along the VII(th) nerve course. The clinical presentation is highly dependent on the location of the lesion along the nerve course and this makes the pre-operative diagnosis difficult without radiologic examination. The most common presentation is facial palsy and even though tumours are responsible for only 5% of facial palsies, if a patient does not recover within six months a complete work-up for neoplasm is recommended. On the basis of clinical presentation and imaging characteristics radiologists should try to make a preoperative diagnosis, to help in the patient's management and possibly to plan the surgical approach. We describe the case of a successful preoperative diagnosis of facial nerve schwannoma. The aim is to describe the main CT and MRI findings which may help the radiologist to establish a correct differential diagnosis.

Knockdown of the type 2 and 3 inositol 1,4,5-trisphosphate receptors suppresses muscarinic antinociception in mice.

The involvement of central endoplasmic inositol 1,4,5-trisphosphate receptors (IP3R) in muscarinic antinociception was investigated in the mouse hot plate test. Selective knockdown of type 1, 2 and 3 IP3R was obtained by means of an antisense oligonucleotide (aODN) strategy. A selective IP3R protein level reduction of approximately 30-50% produced by aODN administration for each receptor subtype investigated was demonstrated by Western blotting experiments. I.c.v. pretreatment with an aODN complementary to the sequence of the type 2 IP3R (0.1-3 nmol per mouse i.c.v.) prevented the antinociception induced by physostigmine (0.15 mg kg(-1) s.c.) and oxotremorine (60 microg kg(-1) s.c.). Similarly, an aODN against type 3 IP3R (0.1-3 nmol per mouse i.c.v.) antagonized cholinergic antinociception. A shift to the right of the physostigmine dose-response curve was obtained after anti-type 2 IP3R2 and anti-type 3 IP3R treatments. Conversely, pretreatment with an aODN complementary to the sequence of type 1 IP3R (0.1-5 nmol per mouse i.c.v.) did not modify the antinociception induced by physostigmine and oxotremorine. Mice undergoing treatment with aODNs did not show any impairment of the locomotor activity, spontaneous motility and exploratory activity as revealed by the rota-rod and hole board tests. These results indicate a selective involvement of type 2 and 3 IP3R in central muscarinic antinociception in mice.

Protective effect of acetyl-L-carnitine on the apoptotic pathway of peripheral neuropathy.

Peripheral neuropathies are widespread disorders induced by autoimmune diseases, drug or toxin exposure, infections, metabolic insults or trauma. Nerve damage may cause muscle weakness, altered functionalities and sensitivity, and a chronic pain syndrome characterized by allodynia and hyperalgesia. Pathophysiological mechanisms related to neuropathic disease are associated with mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. In a model of peripheral neuropathy, obtained by the loose ligation of the rat sciatic nerve (CCI), we describe a nerve apoptotic state that encompasses the release of cytochrome C in the cytosol, the activation of caspase 3, and the fragmentation of the genome. Animal treatment with acetyl-L-carnitine (ALCAR), but not with L-carnitine (L-Carn) or Gabapentin, prevents apoptosis induction. ALCAR reduces cytosolic cytochrome C and caspase 3 active fragments expression in a significant manner with respect to saline treatment. Accordingly, ALCAR treatment impairs caspase 3 protease activity, as demonstrated by reduced levels of cleaved PARP. Finally, ALCAR decreases the number of piknotic nuclei. This protection correlates with the induction of X-linked inhibitor apoptosis protein (XIAP). Taken together these results show that CCI is a valuable model to investigate neuropathies-related apoptosis phenomena and that ALCAR is able to prevent regulated cell death in the damaged sciatic nerve.

Epidemiology of burns at the Italian Red Cross Hospital in Baghdad.

This study describes the epidemiology and patterns of moderate to severe burn injuries (%BSA>or=20-90) treated at the Italian Red Cross Hospital in Baghdad, Iraq, during the medical mission of the tenth Italian Red Cross Contingent, from 3 April to 19 May 2004. Burn injuries sustained by members of the civilian population admitted to the Burns Unit, irrespective of age and sex, are analysed (n=48). Differences in risk distribution between different segments of the population are reported and typical injury patterns are identified. The six injury patterns identified are labelled "child scaldings", "domestic accidents", "unsafe working conditions", "suicide attempts", "injuries among children in role-play" and "war related injuries". The results show that burn injury incidence is particularly high for women and children. There is a pronounced difference between genders after adulthood (age 15 and up). The mortality rate is higher in females. Most of the burns occurred in the home: a public health education campaign might help reduce the incidence of these injuries.

Alpha-2 agonists induce amnesia through activation of the Gi-protein signalling pathway.

The post-receptorial mechanism of the amnesic action of the alpha2-agonists clonidine and guanabenz was investigated in the mouse passive avoidance test. Animals were i.c.v. injected with pertussis toxin (PTX) or with antisense oligonucleotides, complementary to the sequence of the alpha-subunit mRNA of Gi1, Gi2, Gi3, Go1 and Go2 proteins. The administration of PTX (0.25 microg per mouse i.c.v.) reversed the amnesia induced by both alpha2-agonists. Similarly, anti-Gialpha1 (6.25-12.5 microg per mouse i.c.v.), anti-Gialpha3 (3.12-12.5 microg per mouse i.c.v.), anti-Goalpha1 (12.5-25 microg per mouse i.c.v.) antagonised the detrimental effect induced by clonidine and guanabenz. By contrast, pretreatment with anti-Gialpha2 (3.12-25 microg per mouse i.c.v.) and anti-Goalpha2 (12.5-25 microg per mouse i.c.v.) never modified the impairment of memory processes induced by the alpha2-agonists. At the highest effective doses, none of the compounds used impaired motor coordination (rota rod test), nor modified spontaneous motility and inspection activity, (hole board test). These results indicate the involvement of Gi1, Gi3, and Go1, but not Gi2 and Go2, protein subtypes in the transduction mechanism responsible for the induction of amnesia by clonidine and guanabenz.

Diphenhydramine-induced amnesia is mediated by Gi-protein activation.

The effect of the i.c.v. administration of antisense oligodeoxynucleotides directed against the alpha subunit of different Gi-proteins (anti-Gialpha(1), anti-Gialpha(2), anti-Gialpha(3), anti-Goalpha(1), anti-Goalpha(2)) on the amnesia induced by the H(1)-antihistamine diphenhydramine (20 mg kg(-1) s.c.) was evaluated in the mouse passive avoidance test. Pretreatment with anti-Gialpha(1) (12.5-25 microg per mouse i.c.v.) and anti-Gialpha(2) (25 microg per mouse i.c.v.), administered 24 and 18 h before test, prevented antihistamine-induced amnesia. By contrast, pretreatment with an anti-Gialpha(3) (25 microg per mouse i.c.v.), anti-Goalpha(1) (25 microg per mouse i.c.v.) and anti-Goalpha(2) (25 microg per mouse i.c.v.) did not modify the detrimental effect induced by diphenhydramine. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous motility and inspection activity, as revealed by the hole board test. These results suggest the important role played by the Gi(1)- and Gi(2)-protein pathway in the transduction mechanism involved in the impairment of memory processes produced by the H(1)-antihistamine diphenhydramine.

Anti-mu opioid antiserum against the third external loop of the cloned mu-opioid receptor acts as a mu receptor neutral antagonist.

The region from the third external loop to the C terminus of MOR-1 appeared to be critical to the selective binding of MOR-1 ligands as DAMGO and morphine to MOR-1. To study the pharmacological properties of the third extracellular loop an antibody was raised in rabbits against the sequence 304-316 which is unique to MOR-1 and includes the third external loop; the anti-MOR-1 antibody was affinity purified against the immunogen sequence and characterized by [3H]DAMGO and Western blotting; [3H]DPDPE binding assay remained unchanged in the presence of the antibody. Anti-MOR-1 IgG was characterized as a neutral antagonist in Chinese hamster ovary (CHO) cells hyperexpressing constitutively active MOR-1s; in fact, anti-MOR-1 IgG completely reversed the inhibition induced by the MOR-1 agonist endomorphin1, endomorphin2, DAMGO and morphine on forskolin stimulated cyclic AMP (cAMP) accumulation and attenuated both the action of the selective MOR-1 agonist DAMGO to increase [35S]GTPgammaS binding and the action of the MOR-1 inverse agonist beta-chlornaltrexamine (CNA) to decrease [35S]GTPgammaS binding. Radioligand binding assay using membrane suspensions from CHO cells hyperexpressing MOR-1 revealed a significant decreased binding affinity and capacity of all the tested MOR-1 selective ligands after preincubation with anti-MOR-1 IgG. Therefore, the third extracellular loop of MOR-1 appeared to be a key element for the binding of MOR-1 ligands.