Dietary lipid content influences the clinical and intestinal adaptive responses to islet transplantation in diabetic rats.

Nutrient absorption is increased in rats with streptozotocin-induced diabetes mellitus (DM). This intestinal adaptive response is modified by isocaloric manipulations of the dietary content of fatty acids, and separate studies have shown a normalization of the enhanced uptake of glucose and lipids when DM rats are treated with transplantation of 3000 syngeneic pancreatic islets of Langerhans. These studies were undertaken to test the hypothesis that modification of the type of fatty acids in the triglycerides in isocaloric semisynthetic diets (S, saturated fatty acids from beeftallow; or F, polyunsaturated fatty acids from fish oil) fed to DM recipients influences the ability of syngeneic transplanted islets to normalize the clinical indices of glycemic control and the intestinal adaptive response.

A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A.

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells.

The bacterial porin superfamily: sequence alignment and structure prediction.

The porins of Gram-negative bacteria are responsible for the 'molecular sieve' properties of the outer membrane. They form large water-filled channels which allow the diffusion of hydrophilic molecules into the periplasmic space. Owing to the strong hydrophilicity of their amino acid sequence and the nature of their secondary structure (beta strands), conventional hydropathy methods for predicting membrane topology are useless for this class of protein.

Optimal temperature in short-term hypothermic preservation of rat pancreas.

Rat pancreases were hypothermically preserved for 24 hr in University of Wisconsin preservation solution at specific temperatures (4, 7, 10, and 15 degrees C) to evaluate the effect of preservation temperature on islet yield and islet function. Following hypothermic storage, the loss of recoverable islets was highest in the 15 degrees C-preservation group, followed by that of the 4 degrees C group and significantly less in the 7 and 10 degrees C groups (p less than 0.05).

Fluorescence energy transfer distance measurements using site-directed single cysteine mutants. The membrane insertion of colicin A.

The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of insertion of a soluble protein into a lipid bilayer. The soluble structure is known from X-ray crystallography and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this work fluorescence spectroscopy was used to study the membrane-bound structure.

Membrane insertion of the pore-forming domain of colicin A. A spectroscopic study.

In order to gain some insight into the mechanism of insertion into membranes of the pore-forming domain of colicin A and the structure of its membrane-bound form, circular dichroism (in the near and far ultraviolet), fluorescence and ultraviolet spectroscopy experiments were carried out. Because the structure of the water-soluble form of this fragment has been determined by X-ray crystallography, these spectroscopic methods provided valuable information on the secondary structure and the environment of aromatic residues within the two forms of the peptide. These results strongly suggest that the pore-forming domain of colicin A does not undergo drastic unfolding upon insertion into membrane.

ompC mutants which allow growth on maltodextrins show increased channel size and greater voltage sensitivity.

Misra and Benson [(1988) J. Bacteriol. 170, 3611-3617] showed that point mutations in the ompC gene can allow Escherichia coli to grow on maltotriose in the absence of LamB.

A 136-amino-acid-residue COOH-terminal fragment of colicin A is endowed with ionophoric activity.

DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a 'STOP-Shine-Dalgarno-sequence-ATG' cassette encoding a termination codon, a Shine-Dalgano sequence and an initiation codon within the structural gene. This technique has been used to obtain a 137-amino-acid-residue pore-forming protein designated DA70C comprising the final 136-amino-acid-residue COOH-terminal of colicin A preceded by an NH2-terminal methionine. Da70C was correctly expressed but poorly released to the extracellular medium.

Colicins: prokaryotic killer-pores.

Colicins are plasmid-encoded protein antibiotics which kill bacteria closely related to the producing strain (generally Escherichia coli). The study of the function of colicins has revealed many features which reflect common targeting and translocation mechanisms with bacteriophages and toxins. Like many toxins, colicins are composed of structural domains specialized in one of the different steps of the activity, targeting, translocation and killing.

The voltage-dependent activity of Escherichia coli porins in different planar bilayer reconstitutions.

Because of conflicting results from differing techniques, the degree of voltage sensitivity of Escherichia coli porins in planar bilayers is still a matter of debate. In order to provide the first comparative study, OmpF porin was purified in three ways; firstly as native outer membrane vesicles, secondly as salt-extracted porin trimers in sodium dodecyl sulphate and thirdly as solubilised trimers extracted with octyl-polyoxyethylene (Octyl-POE). These methods represent the major approaches to porin isolation and purification.

The lipopeptide antibiotic A21978C has a specific interaction with DMPC only in the presence of calcium ions.

The A21978C group are lipopeptide antibiotics which kill Gram-positive bacteria only in the presence of calcium ions. The calcium requirement of the antibacterial activity of A21978C correlates well with an in vitro calcium-dependent insertion into phospholipid vesicles. In this paper the interaction of A21978C with phosphatidylcholine is investigated in mixed monomolecular films.

A comparative monomolecular film study of antibiotic A21978C homologues of various lipid chain length.

A21978C is a calcium-dependent lipopeptide antibiotic whose biological properties are modulated by changes in its lipid chain length. This article reports on the monolayer characteristics of this cyclic lipopeptide and of LY146032 a semi synthetic homologue. The equilibrium spreading pressure pi e increases linearly with the ionic concentration of the subphase and is higher with divalent cations.

Fluorescence indicates a calcium-dependent interaction between the lipopeptide antibiotic LY146032 and phospholipid membranes.

LY146032 is one of the A21978C family of calcium-dependent antibiotics. This paper reports on its interactions with membranes as studied by its intrinsic fluorescence. The Trp residue was found to have a low fluorescence yield because of Förster-type energy transfer to the kynurenine residue (Kyn) (epsilon = 5000 at 364 nm).

Conformational change on calcium binding by the lipopeptide antibiotic amphomycin. A C.D. and monolayer study.

The acidic linear lipopeptide amphomycin is a calcium dependent antibiotic which is thought to bind to carrier lipids such as dolichol monophosphate. The actual role of Ca++ is not definitely established and in this article we have examined the peptides interactions with a range of divalent cations. By CD we have shown that a conformational change is induced by Ca++, Sr++ and Ba++ but not by Mg++, Zn++, Cd++ or Gd+++.

Voltage gating in porin channels.

Data from experiments in which porin channels are reconstituted into planar bilayer membranes are reviewed for their relevance to porin channel gating in vivo. Contradictory evidence concerning voltage gating indicates that the different results may stem from the variety of purification techniques employed. The likelihood of voltage gating as a property of E.

The role of acyl chain character and other determinants on the bilayer activity of A21978C an acidic lipopeptide antibiotic.

An acidic lipopeptide A21978C has previously been shown to have a powerful antibiotic activity against Gram-positive organisms. Due to its ability to increase the K+ permeability of bacterial cells and its specific calcium requirement, which is similar to a previously described ionophore CDA, its effect on planar bilayer membranes has been studied. Although it produces significant increases in the conductivity of lipid bilayers it is shown that this alone cannot account for its in vivo activity.

Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins.

Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective.

A new channel-forming antibiotic from Streptomyces coelicolor A3(2) which requires calcium for its activity.

A recently discovered antibiotic (CDA; calcium-dependent antibiotic) of Streptomyces coelicolor A3(2) was found to be effective against a wide range of Gram-positive bacteria only in the presence of calcium ions. Producer and non-producer strains were identified and several media tested for their ability to support antibiotic production. The action of calcium was not simulated by any of the other cations tested.