Pharmacological dissection of a neural pattern generator

Summary Injection of picrotoxin solutions into the pericardium of lobsters,Homarus americanus, to produce final, estimated blood concentrations of between 8×10^–8M and 4.5×10^–6M led to disruption of the normal motor output to the scaphognathite. The phase separation of the starts of the bursts in the D1 and D2 muscles (Young, 1975) was reduced on the average by 49%; that of L1 and L2 muscles by 16%. The durations of the D1 and L1 bursts increased by 94% and 48% respectively. The phase separation between the starts of the levator and depressor portions of the cycle was not altered. Whilst rhythmic activity persisted ventilatory rates were depressed, and reversals and ventilatory pauses continued to occur. The rate of reversals and their pattern were not greatly altered. Inexplicably, tonic, synchronous spikes occurred in all four groups of muscles, levator and depressor, during periods of severe disruption. It was concluded that in spite of this, the observations supported the proposals of Young (1975) that (a) recruitment of the D2 and L2 bursts normally is delayed partly by inhibition from D1 and L1 units respectively, and (b) termination of D1 and L1 bursts results normally from inhibition due to activity in D2 and L2 units respectively. The findings also indicated that the synaptic mechanism determining timing within the depressor and levator sessions differs fundamentally from that operating between sessions. One mechanism may be based on picrotoxin-sensitive, and the other on picrotoxin-insensitive inhibition.Abbreviations GABA gamma-aminobutyric acid - SG scaphognathite     

Overexpression inEscherichia coli,Folding, Purification, and Characterization of the First Three Short Consensus Repeat Modules of Human Complement Receptor Type 1

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system inEscherichia coliwas used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP–Sepharose. The oligomer was folded by one-step dilution in 20 m ethanolamine/1 m EDTA supplemented with 1 m GSH/0.5 m GSSG. The folded material was processed to a concentrated (>20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1–3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 × 110 Å. SCR(1–3) has an unusual CD spectrum exhibiting a broad maximum at 220–230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.     

Cholanoic acids and cholesterol 7-α-hydroxylase activity in human leucocytes

Evidence is presented for the identification of cholanoic acids and cholesterol 7α-hydroxylase activity in human leucocytes. Mononucleated cells contained most of the detectable cholanoic acids. Cholesterol 7α-hydroxylase activity was present only in the mononuclear cells. These data suggest that cholanoic acids in leucocytes may have originated by local biosynthesis. Because of their lipid solubilizing properties, the cholanoic acids might have a function in the phagocytic activity of leucocytes.     

Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants

A total of 530 strains of Staphylococcus aureus were isolated from the defeathering machinery of a chicken processing plant and from neck skin samples of carcasses at different stages of processing in two visits 4 weeks apart. Eleven different plasmid profiles were detected in the isolates, eight being common to both visits. The plasmid profiles of the strains forming the majority of the population on the freshly slaughtered birds were rarely present in the strains isolated from the pluckers (except at the entry to the first plucker) and were present in only a small proportion of the strains isolated from carcasses after plucking. However, the profiles from the strains isolated from the pluckers on both visits were different from those forming the majority of the population on the incoming birds but formed the major part of the carcass flora after plucking, suggesting that such strains were endemic. These strains were found as a small proportion of the isolates made from the incoming birds, suggesting that this was the route by which the endemic strains were introduced into the plant. Such endemic strains exhibited a clumping growth, even in liquid shake culture, which may have made it easier for them to become established on the pluckers and to resist cleaning and disinfection. This clumping phenotype was correlated with the presence of a 7.5-megadalton plasmid.     

Elution of interferon beta from blue sepharose by poly(vinylpyrrolidone)

Pre-equilibration of erythrocytes with the membrane-impermeable aldehyde, pyridoxal 5'-phosphate, for 30 min at 22 degrees C, prior to the addition of methyl acetimidate to the incubation mixture has been shown to prevent agglutination of acetamidinated cells which were resuspended in immune serum (Chao, T.L. and Berenfeld, M.R. (1981) J. Biol. Chem. 256, 5324-5326). This observation led to the possibility that the immune reaction, observed in some sickle cell anemia patients to reinfused cells which had been reacted with methyl acetimidate, could be prevented. The present communication further evaluates that reaction sequence and shows that while the pre-equilibration of cells with pyridoxal 5'-phosphate does protect membrane amines from reaction with methyl acetimidate, the protection is not extensive enough to prevent an immune response in a sickle cell anemia patient who had already been sensitized against acetamidinated cells. It is apparent that the design of antisickling agents which covalently modify hemoglobin must take into account protection of functional groups in the erythrocyte membrane, modification of which could produce an immunogenic response.