Opioid Receptors of Neuroblastoma Cells Are in Two Domains of the Plasma Membrane that Differ in Content of G Proteins

Opioid receptors of NG 108-15 cell membranes are distributed in two membrane fractions sedimenting at 20,000 g (P2) and 200,000 g(P3). The number of receptors is identical in P2 and P3, but in P2 all sites are present in one high-affinity state (2 nM), whereas in P3 60% of these receptors display lower affinity (150 nM). Upon addition of GTP or pretreatment with pertussis toxin, 80% of the sites exist in low affinity in both P2 and P3. Therefore, the effect of GTP and pertussis toxin on agonist binding appears to be smaller in P2 than in P3. In contrast, sodium inhibits agonist binding in P2 and P3 to the same extent and with identical potency. Opioid-mediated stimulation of GTPase is much greater in P2 than in P3, whereas inhibition of adenylate cyclase does not differ in the two fractions. Using site-specific antibodies and pertussis toxin-catalyzed ADP-ribosylation, we found that the amount of G proteins in P3 is only 30-50% of that in P2. Treatment of intact cells with the hydrophilic protein-modifying agent sulfosuccinimido-biotin results in biotinylation of proteins from both fractions and in a similar reduction of opioid binding in P2 and P3. Likewise, exposure of intact cells to the alkylating opioid antagonist, chlornaltrexamine, produces identical degrees of receptor inactivation in P2 and P3. The rate of in vivo pertussis toxin-mediated modification of G proteins is not different in the two fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic Exposure of NG 108-15 Cells to Opiate Agonists Does Not Alter the Amount of the Guanine Nucleotide-Binding Proteins Gi and GO

We have characterized the pertussis toxin substrate in NG 108-15 cell membranes using site-specific antisera and ADP-ribosylation. Cell membranes contain two pertussis toxin-sensitive guanine nucleotide-binding protein alpha-subunits (G alpha) whose Rf values in gel electrophoresis coincide with those of G alpha o and G alpha i2. The total quantity of Gi and Go immunoreactivity amounted to 24.3 +/- 2.8 pmol/mg, whereas only 1.5 +/- 0.2 pmol/mg are capable of undergoing ADP-ribosylation catalyzed by pertussis toxin. Pretreatment of cells with the agonist [D-Ala2,D-Leu2]-enkephalin (DADLE) for 24 h and DADLE or morphine for 72 h did not alter the incorporation of ADP-ribose or the immunoreactive amount of Gi and Go subunits. However, pretreatment for 72 h with naloxone increased the incorporation of ADP-ribose without an apparent change in affinity or in the immunochemically determined protein levels of Gi and Go. This indicates that the process of down-regulation and desensitization of the delta-opioid receptor neither requires quantitative alterations in the levels of Gi and Go nor changes in the degree of coupling among their subunits. In contrast, chronic exposure to antagonists seems to alter the degree of precoupling between alpha- and beta-subunits of Gi and/or Go.     

A new transthyretin mutation associated with amyloid cardiomyopathy.

In transthyretin (TTR) a new mutation (TTR-Thr45) has been identified in a patient with familial amyloidosis characterized clinically by prominent cardiomyopathy and the absence of peripheral neuropathy. Comparative peptide mapping by high-performance liquid chromatography of the patient's plasma TTR together with normal TTR showed the presence of an abnormal tryptic peptide in the patient's TTR. The sequence of this peptide (peptide 6, residues 36-48) demonstrated the presence of a threonine-for-alanine substitution at position 45. This change can be explained by a single base change of adenine for guanine in the Ala-45 codon and was demonstrated directly by DNA sequence analysis of PCR-amplified exon 2 of the TTR gene; allele-specific oligonucleotide hybridization both in the patient and in fixed heart tissue from his aunt confirmed the base change. The TTR-Thr45 mutation is a new variant TTR found associated with cardiomyopathy.     

Gummivory and gut morphology in two sympatric callitrichids (Callithrix emiliae and Saguinus fuscicollis weddelli) from western Brazilian Amazonia.

A comparative analysis of the gastrointestinal tracts of wild-caught marmosets, Callithrix emiliae, and tamarins, Saguinus fuscicollis weddelli, was undertaken in order to evaluate the degree of specialisation for digestion of plant exudates. Compared to S.f. weddelli, C. emiliae exhibits a reduced small intestine and a relatively large, compartmentalised caecum in which gum is probably fermented. The apparent specialisation of the digestive tract in C. emiliae correlates with that of its dentition, which is adapted for gouging the bark of gum-producing plants. A similar degree of specialisation of the caecum is predicted for other marmosets (Callithrix spp. and Cebuella pygmaea).     

Detection of virulence factors in culturable Escherichia coli isolates from water samples by DNA probes and recovery of toxin-bearing strains in minimal o-nitrophenol-beta-D-galactopyranoside-4-methylumbelliferyl-beta-D-g luc uronide media.

A total of 449 Escherichia coli isolates in treated and raw water sources were submitted to DNA-DNA hybridization using seven different DNA probes to detect homology to sequences that code for Shiga-like toxins I and II; heat-stabile and heat-labile toxins, adherence factors EAF and eae, and the fimbrial antigen of entero-hemorrhagic E. coli. Fifty-nine (13%) of the isolates demonstrated homology with one or more specific DNA probes. More than 50% of the isolates in treated water were not recovered in MMO-4-methylumbelliferyl-beta-D-glucuronide media designed for detection of this indicator.     

Serine/threonine protein phosphatases in Dictyostelium discoideum: no evidence for type I activity.

Extracts from Dictyostelium discoideum contain type 2A and 2C serine/threonine-specific protein phosphatases with properties very similar to those from mammals according to their sensitivity to okadaic acid and to their dependence for divalent cations. In contrast, no type 1 protein phosphatase is found at any time of development, neither in the cytosolic nor in the particulate fraction, using glycogen phosphorylase a, casein, histone or the non-proteinous 4-Methylumbelliferyl phosphate as substrates. Both type 2A and 2C protein phosphatase activities remain constant throughout the development cycle.     

Transport of groundwater-borne nutrients from watersheds and their effects on coastal waters

Anthropogenic activities on coastal watersheds increase nutrient concentrations of groundwater. As groundwater travels downslope it transports these nutrients toward the adjoining coastal water. The resulting nutrient loading rates can be significant because nutrient concentrations in coastal groundwaters may be several orders of magnitude greater than those of receiving coastal waters. Groundwater-borne nutrients are most subject to active biogeochemical transformations as they course through the upper 1 m or so of bottom sediments. There conditions favor anaerobic processes such as denitrification, as well as other mechanisms that either sequester or release nutrients. The relative importance of advective vs. regenerative pathways of nutrient supply may result in widely different rates of release of nutrients from sediments. The relative activity of denitrifiers also may alter the ratio of N to P released to overlying waters, and hence affect which nutrient limits growth of producers. The consequences of nutrient (particularly nitrate) loading include somewhat elevated nutrient concentrations in the watercolumn, increased growth of macroalgae and phytoplankton, reduction of seagrass beds, and reductions of the associated fauna. The decline in animals occurs because of habitat changes and because of the increased frequency of anoxic events prompted by the characteristically high respiration rates found in enriched waters.     

Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water

M orais , P.V. & D a C osta , M.S. 1990. Alterations in the major heterotrophic bacterial populations isolated from a. still bottled mineral water. Journal of Applied Bacteriology 69 , 750–757.
The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9–12 months. The plate counts in R₂A medium incubated at 22 and 37C were low initially, increasing to 10⁴-10⁵ cfu/ml within a few days of bottling. The number of bacteria recovered at 22C from PVC bottles was fairly constant during the storage period, but the population isolated at 37C decreased markedly after storage for 1 year. The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis . Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.     

Adenylyl Cyclases: mRNA and Characteristics of Enzyme Activity in Three Areas of Brain

Abstract: Arachidonic acid and oleoylacetylglycerol enhance depolarization-evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca²⁺-dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca²⁺ ionophore ionomycin. The Ca²⁺ dependence of this effect was established, as removal of Ca²⁺ eliminated evoked release and the lipid-dependent potentiation. Also, Ca²⁺ channel blockers attenuated ionomycin- and KCI-evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca²⁺, it may not involve an enhancement of evoked Ca²⁺ accumulation, because ionomycin-dependent glutamate release was potentiated under conditions that did not increase ionomycin-induced Ca²⁺ accumulation. Also, the facilitation may not depend on inhibition of K⁺ efflux, because enhanced release was observed in the presence of increasing concentrations of 4-aminopyridine and diazoxide did not reduce the lipid-dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid-dependent facilitations of both KCI- and ionomycin-evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid-dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca²⁺ and involves multiple presynaptic effects, some of which depend on protein kinase C.