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Biomedical subjects

B L Kagan

Publications and source records attributed to B L Kagan.

At least 37 records · Page 2Linked to original sources
PubMed1996

Pore formation by the cytotoxic islet amyloid peptide amylin.

Amylin is a 37-amino acid cytotoxic constituent of amyloid deposits found in the islets of Langerhans of patients with type II diabetes. Extracellular accumulation of this peptide results in damage to insulin-producing beta cell membranes and cell death. We report here that at cytotoxic concentrations, amylin forms voltage-dependent, relatively nonselective, ion-permeable channels in planar phospholipid bilayer membranes. Channel formation is dependent upon lipid membrane composition, ionic strength, and membrane potential. At 1-10 microM, cytotoxic human amylin dramatically increases the conductance of lipid bilayer membranes, while non-cytotoxic rat amylin does not. We suggest that channel formation may be the mechanism of cytotoxicity of human amylin.

Amyloid↗
PubMed1996

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.

Amino Acid Sequence↗
PubMed1995

Conformation and ion channel activity of lymphotoxin at neutral and low pH.

Lymphotoxin (LT or TNF-beta), a T cell-derived lymphokine with partial homology to TNF-alpha, was found to bind to dimyristoylphosphatidylcholine vesicles in a pH-dependent manner: binding increased with decreasing pH. Binding was not limited to surface association with phospholipid head groups because studies with a photoreactive membrane-restricted probe revealed protein penetration of the hydrocarbon core of the bilayer. Intramembranous photolabeling of the trimeric form of LT demonstrated maintenance of quaternary structure upon bilayer insertion. The efficiency of insertion was greatly enhanced with gel-phase bilayers compared with fluid phase bilayers even though the binding efficiency was much lower. Hence, binding and insertion represent two distinct physical processes. Intrinsic fluorescence and dye binding assays showed that the acid-facilitated membrane interactions stemmed from acid-induced changes in protein conformation. The acquisition of hydrophobic characteristics through these conformational changes supplies a physical explanation for LT conversion from a water-soluble form to a membrane-embedded structure. Moreover, the use of vesicle-embedded LT to prepare planar bilayers vs the addition of soluble LT subsequent to bilayer formation demonstrated that LT exhibits channel activity and that low pH-induced membrane insertion precedes and is distinct from expression of voltage-dependent ion gating. LT's ability to associate intimately with lipid vesicles and form ion channels mirrors the behavior of TNF-alpha. Thus, although LT and TNF-alpha are secreted by different cell types, the conservation of membrane binding, insertion, and channel-forming activities suggests a functional role in response induction.

Anilino Naphthalenesulfonates↗
PubMed1995

Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.

Animals↗
PubMed1994

Channel formation in planar lipid bilayers by a neurotoxic fragment of the beta-amyloid peptide.

Alzheimer's disease (AD) pathology is characterized by plaques, tangles, and neuronal cell loss. The main constituent of plaques is beta-amyloid peptide (A beta), a 39-42 residue peptide which has been linked to disruption of calcium homeostasis and neurotoxicity in vitro. We demonstrate that a neurotoxic fragment of A beta, A beta (25-35) spontaneously inserted into planar lipid membranes to form weakly selective, voltage dependent, ion-permeable channels. We suggest that channel formation may be involved in the pathogenesis of AD and that A beta (25-35) may be the active channel forming segment.

Amino Acid Sequence↗
PubMed1994

Defensins: a family of antimicrobial and cytotoxic peptides.

Defensins are antimicrobial and cytotoxic peptides that contain 29-35 amino acid residues, including 6 invariant cysteines that form 3 intramolecular disulfide bonds. They constitute more than 5% of the total cellular protein of human and rabbit neutrophils (PMN), and are also produced by rabbit lung macrophages and by murine and human small intestinal Paneth cells. Defensins exerted antimicrobial effects in vitro against many Gram-positive and Gram-negative bacteria, fungi, mycobacteria and some enveloped viruses, and were cytotoxic to a wide range of normal and malignant targets, including cells resistant to TNF-alpha and NK-cytolytic factor. Human and rabbit defensins formed voltage-sensitive channels in a variety of planar lipid bilayers when a negative voltage of approximately 70-90 mV was applied to the contralateral side. These channels showed modest anion selectivity and their formation was strongly influenced by defensin concentration. Although most other channel-forming peptides have prominent alpha-helical domains, the structure of defensin molecules is primarily composed of antiparallel beta-sheets. Studies with various prokaryotic and eukaryotic cells provided convincing evidence that defensins killed these targets by forming voltage-regulated channels in the susceptible cell's membrane. The broad spectrum of defensin-susceptible targets and the abundance of defensins in specialized host defense cells of the blood, lungs and intestinal tract suggest that defensins could play a significant role in innate immunity to infection and neoplasia.

Amino Acid Sequence↗
PubMed1993

Activation and mechanism of Clostridium septicum alpha toxin.

Clostridium septicum produces a single lethal factor, alpha toxin (AT), which is a cytolytic protein with a molecular mass of approximately 48 kDa. The 48 kDa toxin was found to be an inactive protoxin (ATpro) which could be activated via a carboxy-terminal cleavage with trypsin. The cleavage site was located approximately 4 kDa from the carboxy-terminus. Proteolytically activated ATpro had a specific activity of approximately 1.5 x 10(6) haemolytic units mg-1. The trypsin-activated toxin (ATact) was haemolytic, stimulated a prelytic release of potassium ions from erythrocytes which was followed by haemoglobin release, induced channel formation in planar membranes and aggregated into a complex of M(r) > 210,000 on erythrocyte membranes. ATpro did not exhibit these properties. ATact formed pores with a diameter of at least 1.3-1.6 nm. We suggest that pore formation on target cell membranes is responsible for the cytolytic activity of alpha toxin.

Amino Acid Sequence↗
PubMed1992

Formation of ion-permeable channels by tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF, cachectin), a protein secreted by activated macrophages, participates in inflammatory responses and in infectious and neoplastic disease states. The mechanisms by which TNF exerts cytotoxic, hormonal, and other specific effects are obscure. Structural studies of the TNF trimer have revealed a central pore-like region. Although several amino acid side chains appear to preclude an open channel, the ability of TNF to insert into lipid vesicles raised the possibility that opening might occur in a bilayer milieu. Acidification of TNF promoted conformational changes concordant with increased surface hydrophobicity and membrane insertion. Furthermore, TNF formed pH-dependent, voltage-dependent, ion-permeable channels in planar lipid bilayer membranes and increased the sodium permeability of human U937 histiocytic lymphoma cells. Thus, some of the physiological effects of TNF may be elicited through its intrinsic ion channel-forming activity.

Cell Membrane Permeability↗
PubMed1991

Inositol 1,4,5-trisphosphate directly opens diphtheria toxin channels.

Inositol 1,4,5-trisphosphate (IP3) is a soluble second messenger, which acts by cooperatively releasing Ca2+ into the cytosol from non-mitochondrial stores, probably by activating Ca(2+)-permeable channels. We demonstrate that submicromolar concentrations of IP3 can directly open the Ca(2+)-permeable diphtheria toxin channel, and that this occurs by IP3 binding to a specific site on the toxin protein. This provides a model for IP3-induced Ca2+ release and suggests that IP3-induced channel opening may play a role in diphtheria intoxication and in protein translocation across membranes.

Binding Sites↗
PubMed1991

Amphipathic beta structure of a leucine-rich repeat peptide.

Long tandem arrays of a characteristic leucine-rich repeat motif on the order of 24 amino acids in length have been found in the primary structure of an increasing number of proteins. The most striking feature of these repeats is an amphipathic sequence, with leucine as the predominant hydrophobic residue. Based on this amphipathic sequence and the function of the proteins in which they have been found, the repeats have been proposed to be involved in protein-protein and protein-lipid interactions. As a step toward elucidating the structure and biochemical properties of the leucine-rich repeat motif, we have studied a synthetic leucine-rich repeat peptide (LRP32) representing one of the repeats found in Drosophila chaoptin. We have shown that: (i) LRP32 is soluble in aqueous solution but will bind quantitatively to phospholipid vesicles; (ii) LRP32 has a partial beta structure in aqueous solution and is predominantly a beta structure in the presence of phospholipid; (iii) LRP32 integrates into lipid bilayers to form 60-A intramembrane particles as seen using freeze-fracture electron microscopy (these putative oligomeric structures appear to contain a central aqueous core as indicated by their ability to generate conductances in planar lipid bilayers); and (iv) LRP32-lipid complexes generate 2H NMR spectra characteristic of integral membrane proteins. This study is consistent with LRP32 forming an amphipathic beta sheet. We propose that protein segments containing tandem arrays of leucine-rich repeats also may form amphipathic beta sheets.

Amino Acid Sequence↗
PubMed1990

Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes.

Defensins are cationic, cysteine-rich peptides (Mr = 3500-4000) found in the cytoplasmic granules of neutrophils and macrophages. These peptides possess broad antimicrobial activity in vitro against bacteria, fungi, tumor cells, and enveloped viruses, and they are believed to contribute to the "oxygen-independent" antimicrobial defenses of neutrophils and macrophages. Pathophysiologic studies in vitro have pointed to the plasma membrane as a possible target for the cytotoxic action of defensins. We report here that defensins form voltage-dependent, weakly anion-selective channels in planar lipid bilayer membranes, and we suggest that this channel-forming ability contributes to their antimicrobial properties observed in vitro.

Anti-Bacterial Agents↗
PubMed1990

Oral S-adenosylmethionine in depression: a randomized, double-blind, placebo-controlled trial.

Methylation has been implicated in the etiology of psychiatric illness. Parenteral S-adenosylmethionine, a methyl group donor, has been shown to be an effective antidepressant. The authors studied the antidepressant effect of oral S-adenosylmethionine in a randomized, double-blind, placebo-controlled trial for 15 inpatients with major depression. The results suggest that oral S-adenosylmethionine is a safe, effective antidepressant with few side effects and a rapid onset of action. S-Adenosylmethionine induced mania in a patient with no history of mania. S-Adenosylmethionine may be useful for patients who cannot tolerate tricyclic anti-depressants. These findings support a role for methylation in the pathophysiology of depression.

Administration, Oral↗
PubMed1987

Effect of interrupted sleep patterns and partial sleep deprivation on DST and mood in psychiatric house officers.

To gain further insight into clinical associations seen in depression, the authors investigated the effect of interrupted night-time sleep on the HPA axis and mood in 20 psychiatric house officers taking overnight call. Specific interest was in whether multiple awakenings could induce a positive DST. No statistically significant association emerged between number of nocturnal awakenings, number of hours of sleep deprivation or temporal occurrence of sleep deprivation and cortisol, DST or mood. The results suggest that cortisol and DST changes are not likely to be causally linked to, or epiphenomenon of disrupted sleep. The implications of these findings for major depression are discussed.

Adult↗
PubMed1983

Mode of action of yeast killer toxins: channel formation in lipid bilayer membranes.

The toxic action of yeast killer proteins seems to involve selective functional damage to the plasma membrane of the sensitive cell. Physiological effects include leakage of K+ (refs 1, 2), inhibition of active transport of amino acids and acidification of the cell interior. These effects are strikingly similar to the effects of certain bacterial colicins which have been demonstrated previously to form channels in membranes. Proposed mechanisms of action have usually postulated a limited permeability change induced by the toxin in the plasma membrane. We report here that a killer toxin from the yeast Pichia kluyveri forms ion-permeable channels in phospholipid bilayer membranes, and we propose that the in vitro electrophysiological properties of these channels account for the morbid effects observed in intoxicated cells. A preliminary account of this work has appeared elsewhere.

Electric Conductivity↗
PubMed1982

Induction of ion-permeable channels by the venom of the fanged bloodworm Glycera dibranchiata.

Venom from the poison glands of the polychaete annelid Glycera convoluta has been reported to dramatically increase the frequency of miniature end-plate potentials at the frog and crayfish neuromuscular junctions, without causing detectable ultrastructural changes. We report here that addition of venom from the related annelid Glycera dibranchiata to one side of a lipid bilayer results in the formation of ion-permeable channels in the membrane. The channel forming activity was found in the void volume of a Sephadex G-25 column (mol. wt. greater than 5000). The conductance of a single channel is about 350 pmho in 0.1 M NaCl and is ohmic. The channels exhibit moderate (but not ideal) cation selectivity in NaCl or KCl gradients. Other selectivity measurements suggest that Ca2+ and Mg2+ are also permeable. The channels show a slight voltage sensitivity. The steady state conductance at--70 mV (side opposite venom) is about 5 times the conductance at + 70 mV. We suggest that these channels in the venom may evoke transmitter release at neuromuscular junctions either by (1) depolarizing the pre-synaptic terminal and thus opening voltage-dependent Ca2+ channels, or (2) directly allowing Ca2+ to enter the terminal. Black widow spider venom is known to produce similar effects on neuromuscular junctions and lipid bilayers. The single channel conductances and ionic selectivities of the channels found in the venoms of Glycera and Latrodectus are strikingly similar. Taken together, these results suggest that channel formation can explain the electrophysiologic effects of these two different venoms.

Animals↗