Acetylcholine receptors in normal and denervated rat diaphragm muscle. I. Purification and interaction with [125I]-alpha-bungarotoxin.

Acetylcholine receptors have been purified from junctional regions of normal rat diaphragm muscle and from extrajunctional regions of denervated diaphragm. The reaction of purified receptors with [122I]-alpha-bungarotoxin has been investigated by kinetic methods. The toxin-receptor complexes dissociated in a biphasic manner at 35 degrees with a rapidly dissociating component (t1/2 = 4 hr) and a slowly dissociating component (t1/2 is greater than or equal to 100 hr). The association reaction between toxin and receptor did not obey simple second-order kinetics but could be analyzed in terms of two classes of binding sites corresponding to the two rates of dissociation. This treatment of the data allowed derivation of association rate constants for the two sites. Value obtained for the dissociation constants were 3.7 times 10(-10) and less than or equal to 0.4 times 10(-10) M for the junctional receptor and 1.7 times 10(-10) and is less than or equal to 0.2 times 10(-10) M for the extrajunctional receptor. In each case it is the more tightly binding component that associates and dissociates more slowly. Receptors present in crude preparations were comparable to purified receptors in their reaction with [125I-alpha-bungarotoxin. The validity of the two site model is discussed in relation to the kinetic studies.     

Acetylcholine receptors in normal and denervated rat diaphragm muscle. II. Comparison of junctional and extrajunctional receptors.

Acetylcholine (ACh) receptors have been purified separately from normal rat diaphragm muscle (junctional receptors) and from extrajunctional regions of denervated diaphragm (extrajunctional receptors) in order to compare their properties. The toxin-receptor complexes of the two receptors were indistinguishable by gel filtration and by zone sedimentation in sucrose gradients, and showed identical precipitation curves with rabbit antiserum to the eel ACh receptor. Both toxin-receptor complexes bind concanavalin A and are therefore probably glycoproteins. Low concentrations of d-tubocuratine (dTC) were more effective in decreasing the rate of toxin binding to junctional than to extrajunctional receptors. The apparent dissociation constant for dTC binding to the junctional receptor was 4.5 X 10 minus 8 M, whereas the value for the extrajunctional receptor was 5.5 X 10 minus 7 M. When the complexes were analyzed by isoelectric focusing, the junctional complex focused at approximately 0.15 pH unit lower than the extrajunctional complex. This result was also found with crude preparations of receptor. We conclude that junctional and extrajunctional receptors are similar but distinct molecules. The properties of receptors present in neonatal diaphragm muscle were also examined and found to be similar to those of receptors in denervated muscle, as shown by dTC inhibition and isoelectric focusing.     

Oscillation phenomena in black lipid membranes induced by a single alamethicin pore

In this paper we show how alamethicin (a small cyclic peptide of molecular weight 1691) can produce voltage oscillations in black lipid membranes and how a nonactin-alamethicin oscillator can be constructed. Alamethicin alone induces oscillations only with an applied bias current, but with nonactin and appropriate salt solutions oscillations occur with no bias current. Both kinds of oscillations can be quantitatively understood in terms of the known properties of alamethicin and nonactin and both depend on the statistical nature of the formation of pores in the membrane by alamethicin.     

Methyl galactosidase activity: an alternative evolutionary destination for the ebgA0 gene.

Previous studies (Campbell et al., 1973; Hall and Hartl, 1974; Hall and Hartl, 1975) have shown that the ebgA0 gene, whose product does not hydrolyze lactose may evolve so that its product does hydrolyze lactose; i.e., lactase activity is one evolutionary destination of the ebgA0 gene. Beginning with a strain that synthesizes ebgA0 gene product constitutively and grows extremely slowly (doubling time, 30 to 50 h) on methyl-beta-D-galactopyranoside (MG), a derivative was selected capable of growth on MG at a moderate rate (doubling time, 5.9 h). Genetic evidence is presented showing that the gene that permits growth on MG is an allele of ebgA. A comparison among strains bearing several alleles of ebgA shows that the new allele, termed ebgAmg, synthesizes a product specific for MG and thus represents a true alternative evolutionary destination for the ebgA0 gene.     

Agonal phase,ischaemic times,and renal vascular abnormalities and outcome of cadaver kidney transplants.

A retrospective study of 250 cadaver kidney transplants was carried out to determine the effects of the agonal period, the warm and cold ischaemic times, and the use of kidneys with vascular anomalies on the primary success and failure and the subsequent level of function of the transplants. Kidneys with vascular anomalies or from non-ventilated donors had a primary failure rate of over 30%, whereas those with normal vasculature or from ventilated donors had a rate of 17%. An initial warm ischaemic time of more than 60 minutes was associated with a primary failure rate of 57% and a cold ischaemic time of over 550 minutes with a primary failure rate of 47%. The interrelationship between the warm and cold ischaemic times in the primary success or failure of the transplants was examined and criteria defined for selecting potentially viable cadaver kidneys for transplantation, as follows: (1) The donor should be (a) ventilated, (b) aged 6-50 years, and (c) have normal ante-mortem renal function and have secreted more than 1-5 1 of urine in the 24 hours before death (or an equivalent volume if the urinary output was recorded for less than 24 hours before death); (2) the kidney should have normal renal vasculature enabling single arterial and venous anastomoses to be performed; (3) kidneys with I.W.I.T.s of longer than 60 minutes should not be used; (4) for kidneys with I.W.I.T.s of less than 20 minutes the C.I.T. is not critical but should not exceed 12 hours; (5) for kidneys with I.W.I.T.s of 20-60 minutes the C.I.T. should not exceed 450 minutes.     

Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Optimization of recombinant aminolevulinate synthase production in<Emphasis Type="Italic"> Escherichia coli</Emphasis> using factorial design

The production of recombinant Rhodobacter sphaeroides aminolevulinate (ALA) synthase was optimized in two strains of Escherichia coli: the wild-type strain MG1655, and a ptsG mutant AFP111. The effects of initial succinate, glucose and isopropyl--d-thiogalactopyranoside (IPTG) concentrations and the time of induction on enzyme activity were studied. One-way analysis was used to approximate the optimal ranges for these factors, followed by a full factorial design to quantify the effects of each factor and the interactions between the factors. Initial succinate, glucose, and IPTG concentration were observed to be the key factors affecting ALA synthase activity with the optimal levels determined to be above 6 g/l succinate, 0 g/l glucose, and 0.10 mM IPTG. ALA synthase activity was generally lower with AFP111 than with MG1655, and the effect of these three key factors was also lower with AFP111 than with MG1655. Based on the full factorial design results, a fermentation was completed that yielded 296 mU/mg protein with a final ALA concentration of 5.2 g/l (39 mM).