Immunoaffinity purification of the lipid transfer protein complex directly from human plasma

The human cholesteryl ester (CE) and triglyceride (TG) exchange protein (denoted LTC or lipid transfer complex) was isolated in a single step from plasma using immunoaffinity batch extraction. Antibodies were raised against two preparations of conventionally purified LTC. LTC-I and LTC-II (purified 20,000-fold and 3500-fold, respectively) were used as immunogens. The antiLTC antibodies were isolated by anion-exchange chromatography and coupled to Affi-Gel 10. Chromatography of plasma on antiLTC Affi-Gel removed all of the CE and TG transfer activity. Moreover, LTC prepared from both antiLTC-I and antiLTC-II-Affi-Gel matrices were identical when analyzed by sodium dodecyl sulfate-polyacrylamide gel LTC electrophoresis. LTC exhibited two protein bands of Mr (apparent) 67,000 and 58,000 and a broad, faintly staining region at greater than 150,000. Analysis of LTC by immunoblotting indicated that both antiLTC-I and antiLTC-II antibodies recognized the same LTC proteins. Isoelectric focussing of LTC gave two pI values, 5.2 and 8.7. These data suggest that LTC is a complex of specific proteins and perhaps lipid. Specific CE and TG exchange activities of immunoaffinity-purified LTC were comparable, although the activities were low with respect to that of the antigen used to generate antiLTC-I. This is not due to contamination of LTC by albumin, lecithin:cholesterol acyltransferase, or apolipoproteins AI, AII, B, CIII, D, or E.     

A new internal perfusion method for transport studies in squid giant axons

A new internal perfusion method has been developed which allows control of the internal solute composition in squid axons. The superiority of this technique compared to the old perfusion methods is shown by the experiments performed which have reproduced, both qualitatively and quantitatively, the Na+ and Ca2+ fluxes observed in intact and dialyzed axons. Compared with the internal dialysis, the perfusion method has the advantage that the permeability barrier give by the porous capillary has been eliminated. This allows the introduction into the axon of solutes with very high molecular weight, at the same time that a fast and reliable internal control can be achieved.     

Enhanced activation of a T cell line specific for acetylcholine receptor (AChR) by using anti-AChR monoclonal antibodies plus receptors

Immune complex-mediated regulation of the immune response has been studied by using T cell lines and monoclonal antibodies (MAb), both specific for the acetylcholine receptor (AChR). Rat T lymphocytes bearing the W3/25 phenotype and specific for AChR from Torpedo californica have been propagated in vitro for nearly 1 yr. These T cells proliferate in response to optimal concentrations of AChR presented by irradiated syngeneic thymus cells. At suboptimal concentrations of antigen there is little activation of the T cell line. We report here that the addition of small amounts of anti-AChR MAb produces dramatic stimulation of the T cell lines at suboptimal doses of AChR. Enhanced activation depends on the isotype and not the fine specificity of the MAb that are used. The observed phenomenon is antigen specific, and in fact, the immune complexes may actually suppress the proliferative response of irrelevant T cells to some extent. The MAb plus antigen are rapidly bound to the surface of the antigen-presenting cell, which we have shown is the dendritic cell.     

Olfactory deprivation in pigeons: examination of methods applied in homing experiments

1. Effectiveness of three methods of olfactory deprivation or impairment was tested by means of unconditioned cardiac acceleration in response to odorous stimuli. 2. Occlusion of nostrils reduced stimulus intensity to approx. 20-30% of the level in unimpeded state. Capability of stimulus quantification remained unimpaired. 3. Bilateral olfactory nerve section and backward bending of the nerve stumps irreversibly eliminated responses to weaker stimuli. With higher concentrations of the odorant, pigeons responded at a reduced level, most likely due to trigeminal reception. 4. Spraying the nasal cavities with an anaesthetic largely abolished sensitivity to odorous stimuli. However, its effect was quite variable depending on the kind of application (which cannot be fully standardized). The time course of effectiveness is shown. 5. The relevance of these findings to experiments on pigeon homing is discussed. Conclusions on involvement of unspecified non-olfactory stimuli can only be drawn if methods are applied that reliably isolate the birds from airborne environmental odours.     

Intramuscular loading dose of quinine for falciparum malaria: pharmacokinetics and toxicity.

In a study of intramuscular injection of quinine eight adults with moderately severe falciparum malaria resistant to chloroquine were treated with quinine dihydrochloride, being given a loading dose of 20 mg salt (16.7 mg base)/kg followed by three or four eight hourly maintenance doses of 10 mg salt (8.3 mg base)/kg injected into the anterior thigh. All patients responded to treatment. Fever and parasite clearance times (mean (SD) 60 (23) h and 53 (22) h respectively) were comparable with those obtained with intravenous quinine. The mean peak plasma quinine concentration of 11.0 mg/l (34.4 mu mol/l) [corrected] was reached a median of five hours after administration of the loading dose. In all patients plasma quinine concentrations exceeded the high minimum inhibitory concentration for Plasmodium falciparum malaria prevalent in Thailand within four hours of the start of treatment but did not cause toxicity other than mild cinchonism. When intravenous infusion is not possible an intramuscular quinine loading dose is an effective means of starting treatment in patients with moderately severe falciparum malaria who cannot swallow tablets.     

Androgenic stimulation of endocytosis, amino acid and hexose transport in mouse kidney cortex involves increased calcium fluxes

Testosterone was previously shown to induce an early (less than 1 min) receptor-dependent stimulation of endocytosis, hexose and amino acid transport in mouse kidney cortex (Koenig, H., Goldstone, A. and Lu, C.Y. (1982) Biochem. Biophys. Res. Commun. 104, 165-172). Testosterone (10(-8) M) has now been found to stimulate rapidly (less than 30 s) the influx and efflux of 45Ca2+ in cortex slices. Testosterone also decreased mitochondrial 45Ca and augmented soluble 45Ca, indicating a mobilization of intracellular calcium. Incubation of cortex slices in calcium-free medium without or with 2.5 mM EGTA decreased basal endocytosis, hexose and amino acid transport and blocked the hormonal response. 100 microM verapamil blocked the hormonal response without affecting basal transport. The calcium ionophore A23187 rapidly stimulated endocytosis, hexose and amino acid transport. These data indicate that androgenic stimulation of membrane transport functions involves an increased influx of extracellular calcium and a mobilization of intracellular calcium. Increased cytosolic Ca2+ is probably the regulatory signal for these transport processes.     

Recombinant human insulin.

Insulin is a well-characterized peptide that can be produced by recombinant DNA technology for human therapeutic use. A brief overview of insulin production from both traditional mammalian pancreatic extraction and recombinant bacterial and yeast systems is presented, and detection techniques, including electrophoresis, are reviewed. Analytical systems for insulin separation are principally based on reversed-phase chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin, proinsulin, and insulin. Process-scale separation is a multistep process and includes ion exchange, reversed-phase, and size exclusion chromatography. Advantages and/or disadvantages of various separation approaches, as described by the numerous literature references on insulin purification, are presented.