Passive release of monoclonal antibodies from hybridoma cells

Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. This release is promoted at room temperature. Passively released MCAB is indistinguishable from that released by actively growing cells, as judged by SDS-polyacrylamide gel electrophoresis. The significance of these observations in relation to the continuous culture of hybridoma cells is discussed.Maximum MCAB content of TB/C3 hybridoma cells is about 55pg per cell, any additional MCAB produced is secreted.Abbreviations MCAB monoclonal antibodies - PBS phosphate buffered saline - RT room temperature - SDS sodium dodecyl sulphate     

Stage-specific cuticular proteins of Ostertagia circumcincta and Ostertagia ostertagi

In this study we have shown that NHS-biotin and I¹²⁵-streptavidin can detect cuticular polypeptides of Ostertagia spp. The labelled polypeptide profile of intact nematodes is simple compared to the profile obtained by labelling homogenates. None of the major internal polypeptides are labelled and the subset of proteins labelled in intact nematodes appears to be mainly surface associated. The results presented here demonstrate that NHS-biotin may be used as a reagent for the analysis of surface polypeptides. The surface polypeptide profiles of the five major developmental stages (L1, L2, L3, L4 and adult) of Ostertagia circumcincta show a series of stage-specific molecules with no polypeptides common to all stages, indicating that the cuticle is a dynamic structure which changes throughout the life cycle. Similarity comparison of Ostertagia ostertagi L3 and L4 stage surface profiles showed that each stage is clearly distinct; comparison of these stages between the two species shows an overall similarity.     

A tubulo-cisternal endoplasmic reticulum system in the potassium transporting marginal cells of the stria vascularis and effects of the ototoxic diuretic ethacrynic acid

Summary Sections of metal impregnated tissue and freeze-fracture have been used to examine intracellular membrane systems in marginal cells of the stria vascularis in mammalian cochleae. A continuous network of elements of the smooth endoplasmic reticulum was revealed. Notable features of this system were a series of flattened cisternae just inside and parallel with the lateral plasma membrane in continuity with an apical network of tubules, cisternae and sheets oriented in parallel with the luminal membrane. The whole system was closely associated with mitochondria. These characteristics suggest that the potassium transporting marginal cells possess a tubulo-cisternal endoplasmic reticulum (TER) like that found in many sodium transporting epithelial cells. The lateral elements of the TER dilated, appearing like vacuoles, and opened to the lateral extracellular space in response to the effects of ethacrynic acid. This diuretic impairs ion transport in the stria vascularis. It is suggested that the TER in marginal cells is involved in the transport of ions and fluid from the cell to the intercellular space when ion balance is disturbed and may play a role in cell volume regulation.This work was supported by the Medical Research CouncilPart of this work was presented at the 18th Workshop on Inner Ear Biology, Montpellier, September, 1981     

Factors restricting maximal preservation of neuronal glycogen after perfusion fixation with dimethyl sulfoxide and iodoacetic acid in Bouin's solution. Histochemical observations in the brain of the Netherlands dwarf rabbit

Thirty seconds after an initial intracardial epinephrine injection, deeply anesthetized animals are perfused consecutively with saline, Bouin's and 100% ethanol solutions, each containing 1% or 5% DMSO (Me2SO) and 0.01 M iodoacetic acid. In the Netherlands dwarf rabbit and the guinea pig, a maximal preservation of dimedone PAS-stainable, saliva-digestible glycogen is achieved, without signs of polarization of glycogen, in many neuronal and neuroglial cells occupying either brain stem nuclei or occasionally narrow perivascular zones. Tentatively, these results are ascribed to a combined effect of (a) the alleged capacity of DMSO to accelerate fixation and to suppress activation of adenylate cyclase and (b) the rapid action of Bouin's solution so that the glycogen particles become instantaneously enclosed in situ in a skeleton of coagulated proteinaceous elements. The paradoxical over-all reduction in preservation of neuronal and astrocytic glycogen may be associated either with a demonstrable loss of the fixative into the peripheral vasculature, because of contrary actions of DMSO and epinephrine, or with a transvascular passage of epinephrine resulting in neuronal glycogenolysis where the blood-brain barrier is absent or affected by DMSO. Other defects are the occurrence of myriad pericapillary foci of inadequate tissue preservation, rare petechial hemorrhages, post mortem fat emboli, and ubiquitous Buscaino plaques. Despite these adverse results preventing utilization of this technique in systematic histochemical investigations on neuronal glycogen, remarkable qualitative characteristics such as the neurons' capacity to store glycogen throughout their perikarya have been revealed.     

Potentiation of arginine-induced glucagon secretion by adenosine

Adenosine and the synthetic adenosine agonists 2-chloroadenosine and N⁶-(L-2-phenylisopropyl)-adenosine were tested for effects on hormone secretion from the rat isolated perfused pancreas. These nucleosides, at concentrations of 5 μM, markedly potentiated both phases of arginine-induced glucagon release; the two synthetic agonists were more effective than adenosine. In the absence of arginine, each of the nucleosides induced a transient burst of glucagon. In contrast, adenosine and both synthetic agonists inhibited arginine-induced insulin secretion to varying degrees and caused only negligible insulin release when perfused without arginine. The adenosine antagonist 8-($\text{p}$-sulfophenyl)-theophylline prevented the actions of adenosine on hormone release from the pancreas. Our data suggest that adenosine potentiation of arginine-induced glucagon release may be mediated via adenosine receptors on alpha cell membranes; such a mechanism could provide an important endogenous control over glucagon secretion.     

Comparative properties of ferredoxins from a marine and freshwater species of Porphyridium

Ferredoxins were isolated from the freshwater red alga Porphyridium aerugineut, and from Porphyridium cruentum, a related marine species. A sin     

Fast atom bombardment: A new mass spectrometric method for peptide sequence analysis

We have studied a selection of peptides using a new mass spectrometric ionisation technique - fast atom bombardment (FAB). We define the fragmentation pathways observed and comment on the utility in sequence analysis. A simple acetylation experiment is shown to aid rapid sequence assignment.     

Vascular smooth muscle mitochondria: Magnesium content and transport

Endogenous magnesium content and magnesium transport of isolated bovine vascular smooth muscle mitochondria were studied. Mitochondria isolated from atherosclerotic bovine arteries contained two to three times as much magnesium (178 nmol/mg of mitochondrial protein) as those isolated from normal arteries (67 nmol/mg of mitochondrial protein). Electron-opaque granules were visible in the unstained unfixed mitochondria and could be shown with electron probe analysis to consist of magnesium, calcium, and phosphorus. At concentrations of external Mg²⁺ from 0 to 6 mm, the vascular smooth muscle mitochondria exhibited respiratory substrate-supported release of Mg²⁺ as studied with metallochromic indicator, eriochrome blue, using dual-wavelength spectrophotometry. The maximal velocity of energized release (3 nmol of Mg²⁺/s/mg of mitochondrial protein) was observed at 4 mm external Mg²⁺ and the half-maximal transport occurred at 0.5 mm.     

The amino acid sequence of chick skin collagen α1-CB7: The presence of a previously unrecognized triplet

Because alignment of the amino acid sequences of chick skin collagen α2-CB3 (1) with the relevant portion of chick skin collagen α1-CB7 (2) suggested that a Gly-X-Y triplet may have been missed in the latter, the peptide TM-2, produced by tryptic digestion of maleylated α1-CB7, was reinvestigated. Cleavage by trypsin at the unblocked lysine at position 18, and isolation of the resulting COOH-terminal peptide, showed this to be a 15-residue peptide containing a previously unrecognized Gly-Pro-Hyp triplet. Sequencing of the peptide showed this to occupy positions 4 through 6, or 56 through 58 of α1-CB7. The latter thus has 271 instead of 268 residues, and the α1[I] chain 1055 instead of 1052.