Norcocaine: a pharmacologically active metabolite of cocaine found in brain

N-Demethylation of cocaine results in the production of norcocaine, which has been identified by gas chromatography-mass spectrometry in the brains of monkeys given repeated doses of cocaine. This metabolite is about as active as cocaine in inhibiting uptake of ³H-norepinephrine by synaptosomes prepared from rat brain. Other cocaine derivatives have been found to be relatively inactive in inhibiting uptake of this amine.     

Metal chelates in the storage and transport of neurotransmitters: interactions of Cu2+ with ATP and biogenic amines

Abstract— The thermodynamic stabilities of the coordinate binding of Cu²⁺ ion with adenosinetriphos-phate (ATP) and several biogenic amines have been determined in aqueous model systems in an attempt to examine the possible correlation between metal-amine binding and the in vivo affinities of the amines for granule-binding. In each of the ternary chelate systems consisting of Cu²⁺-ATP-amine (1:1:1), the Cu²⁺ ion is preferentially bound by ATP in the pH range 3–5 with a stability constant of Log K_ML= 517. In the pH range 5–8 each of the biogenic amines coordinates with Cu²⁺ -ATP chelate to form the respective ternary chelate. The nature and strength of binding of fourteen different amines with Cu²⁺-ATP have been evaluated on the basis of the stabilities of the ternary chelates. On the basis of the quantitative equilibrium data generated in this study, it appears that both pyrocatechol moiety and the ethanolamine side-chain of the catechol amines are involved in the coordination of copper. The metal-binding stabilities of the biogenic amines are then correlated with the molecular structure, donor basicities and the in vivo affinities of the amines for granule-binding in order to rationalize the possible involvement of metal chelates in the monoamine binding, storage and transport.     

Metal chelates in the storage and transport of neurotransmitters: interactions of metal ions with biogenic amines

Abstract— Equilibrium studies on the interaction of biogenic amines with iron (Fe²⁺ and Fe³⁺) and magnesium (Mg²⁺) were undertaken in an attempt to correlate the stabilities of metal-amine chelates and the reported granule-binding affinities of the biogenic amines. By means of potentiometric equilibrium measurements at 25°C and an ionic strength of 10 (KNO₃) the formation constants of the Fe²⁺ chelates with norepinephrine (NE) and adenosine-S-triphosphate (ATP) were determined. Possible structures were derived for the co-ordinate binding of Fe^{2 +} with NE. The interactions of Fe²⁺ and Fe²⁺-ATP with NE were investigated and the formation of Fe²⁺-NE-ATP (1:1:1) mixed ligand ternary chelate was proposed on the basis of the equilibrium data. Information obtained from chelation studies of Fe²⁺ with pyrocatechol and ethanolamine taken together with the data obtained on the Fe²⁺-NE system indicated that the binding of Fe²⁺ by NE was probably via the pyrocatechol moiety. Equilibrium constants for the binding of tyramine (TA) dopamine (DA) and NE with Mg²⁺ were also determined. The equilibrium data obtained on the Mg²⁺-amine systems indicated a correlation between the metal-amine binding affinities and the structure and amine-release (and storage) activities of the biogenic amines. A consideration of the stabilities of the Fe²⁺ and Mg²⁺ chelates together with the occurrence of these metal ions in synaptosomes suggests their possible involvement in the intra vesicular amine-binding and storage sites.     

Characterization and N-terminal sequence of a heparan sulphate proteoglycan synthesized by endothelial cells in culture.

We have isolated from the conditioned medium of an established endothelial cell line a heparan sulphate proteoglycan whose involvement in the inhibition of the extrinsic coagulation pathway was reported in previous studies [Colburn & Buonassisi (1982) Biochem. Biophys. Res. Commun. 104, 220-227]. The proteoglycan was purified by gel filtration and ion-exchange chromatography, and appears to be free of contaminating proteins as determined by polyacrylamide-gel electrophoresis of the radioiodinated protein core before and after removal of the glycosaminoglycan chains by treatment with heparitinase. By this procedure the Mr of the protein core was estimated to be 22000. The N-terminal end was sequenced up to amino acid 25. The 21st residue is likely to be glycosylated. Analysis of the purified proteoglycan by gel-filtration chromatography yielded Kd values of 0.2 for the whole molecule and 0.35 for the glycosaminoglycan chains. The structure that emerges from these data is that of a heparan sulphate proteoglycan characterized by a relatively small protein core and few glycosaminoglycan chains.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery.

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Phylogenetic relationships within phylum Rotifera: orders and genus Notholca

We investigated evolutionary relationships among orders in phylum Rotifera and among species in genus Notholca (Rotifera) by computing parsimonious cladograms. All of the most-parsimonious cladograms generated for the ordinal level confirm the view that class Monogononta, superclass Eurotatoria, and phylum Rotifera are monophyletic. Species within the genus Notholca were separated into six groups (clades), but some species have been defined based on highly variable characters not reliably studied using cladistics. Therefore, phenetic studies are warranted, especially for species possessing caudal processes.     

Effect of monensin on the sulfation of heparan sulfate proteoglycan from endothelial cells.

Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretory proteins at the level of trans-Golgi cisternae. Exposure of endothelial cells to monensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition is dose dependent and affects the ratio [35S]-sulfate/[3H]-hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular weight. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin-treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there is a specific decrease of the ones bearing a sulfate ester at the 6-position of the glucosamine moiety. All other biosynthetic steps were not affected by monensin. The results are indicative that monensin affects the hexosamine C-6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans-Golgi compartment.     

The use of capillary electrophoresis in a micropreparative mode: methods and applications.

The ability to collect sufficient quantities of analytes from capillary electrophoresis for subsequent analyses is demonstrated. Fractions collected have been analyzed using the following techniques: capillary electrophoresis, mass spectrometry, and protein sequencing. Fractions can be collected directly into small volumes of buffer or directly onto membrane surfaces. Relevant parameters such as capillary diameter, mass loading, and separation parameters are addressed.