Friction mediated by redox-active supramolecular connector molecules.

We report on a friction study at the nanometer scale using atomic force microscopy under electrochemical control. Friction arises from the interaction between two surfaces functionalized with cyclodextrin molecules. The interaction is mediated by connector molecules with (ferrocenylmethyl)ammonium end groups forming supramolecular complexes with the cyclodextrin molecules. With ferrocene connector molecules in solution, the friction increases by a factor of up to 12 compared to control experiments without connector molecules. The electrochemical oxidation of ferrocene to ferrocenium causes a decrease in friction owing to the lower stability of ferrocenium-cyclodextrin complex. Upon switching between oxidative and reduction potentials, a change in friction by a factor of 1.2-1.8 is observed. Isothermal titration calorimetry reveals fast dissociation and rebinding kinetics and thus an equilibrium regime for the friction experiments.

Abnormalities in the tricarboxylic acid (TCA) cycle in the brains of schizophrenia patients.

Images of brain metabolism and measurements of activities of components of the electron transport chain support earlier studies that suggest that brain glucose oxidation is inherently abnormal in a significant proportion of persons with schizophrenia. Therefore, we measured the activities of enzymes of the tricarboxylic (TCA) cycle in dorsolateral-prefrontal-cortex from schizophrenia patients (N=13) and non-psychiatric disease controls (N=13): the pyruvate dehydrogenase complex (PDHC), citrate synthase (CS), aconitase, isocitrate dehydrogenase (ICDH), the alpha-ketoglutarate dehydrogenase complex (KGDHC), succinate thiokinase (STH), succinate dehydrogenase (SDH), fumarase and malate dehydrogenase (MDH). Activities of aconitase (18.4%, p<0.05), KGDHC (26%) and STH (28.2%, p<0.05), enzymes in the first half of the TCA cycle, were lower, but SDH (18.3%, p<0.05) and MDH (34%, p<0.005), enzymes in the second half, were higher than controls. PDHC, CS, ICDH and fumarase activities were unchanged. There were no significant correlations between enzymes of TCA cycle and cognitive function, age or choline acetyl transferase activity, except for aconitase activity which decreased slightly with age (r=0.55, p=003). The increased activities of dehydrogenases in the second half of the TCA cycle may reflect a compensatory response to reduced activities of enzymes in the first half. Such alterations in the components of TCA cycle are adequate to alter the rate of brain metabolism. These results are consistent with the imaging studies of hypometabolism in schizophrenia. They suggest that deficiencies in mitochondrial enzymes can be associated with mental disease that takes the form of schizophrenia.

Intensive nutritional supplements can improve outcomes in stroke rehabilitation.

OBJECTIVE: Poor nutrition is a common complication of strokes severe enough to require inpatient rehabilitation. We therefore tested whether intensive nutritional supplements given to undernourished patients from the time of their admission to a specialized stroke rehabilitation service would improve patient outcomes. METHODS: Randomized, prospective, double-blind, single center study comparing intensive nutritional supplementation to routine nutritional supplementation in 116 undernourished patients admitted to a stroke service. The analysis included the 90% of patients who were not lost to follow-up due to acute or subacute hospitalization (n = 102; 51 in each group). The nutritional supplements are commercially available and Food and Drug Administration approved. The primary outcome variable was change in total score on the Functional Independence Measure (FIM). The secondary outcome measurements included the FIM motor and cognitive subscores, length of stay (taken from day of admission), 2-minute and 6-minute timed walk tests measured at admission and on discharge, and discharge disposition (home/not home). RESULTS: Patients receiving intensive nutritional supplementation improved more than those on standard nutritional supplements on measures of motor function (total FIM, FIM motor subscore, 2-minute and 6-minute timed walk tests, all significant at p < 0.002). They did not, however, improve on measures of cognition (FIM cognition score). A higher proportion of patients who received the intensive nutritional supplementation went home compared to those on standard supplementation (p = 0.05). CONCLUSION: Intensive nutritional supplementation, using readily available commercial preparations, improves motor recovery in previously undernourished patients receiving intensive in-patient rehabilitation after stroke.

Deficits in a tricarboxylic acid cycle enzyme in brains from patients with Parkinson's disease.

Parkinson's disease (PD) is associated with mitochondrial dysfunction, specifically a deficiency of complex I of the electron transport chain. Most, although not all, studies indicate that this deficiency is limited to brain regions with neurodegeneration. The current studies tested for deficiencies in other mitochondrial components in PD brain in a neuropathologically unaffected region where the abnormality cannot be attributed to secondary effects of neurodegeneration. The activity of a key (and arguably rate-limiting) tricarboxylic acid cycle enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), was measured in the cerebellum of patients with PD. Activity in 19 PD brains was 50.5% of that in 18 controls matched for age, sex, post-mortem interval, and method of preservation (P<0.0019). The protein subunits of KGDHC were present in normal amounts in PD brains, indicating a relatively discrete abnormality in the enzyme. The activities of another mitochondrial enzyme, glutamate dehydrogenase (GDH), were normal in PD brains. These results demonstrate that specific reductions in KGDHC occur even in pathologically unaffected areas in PD, where the decline is unlikely to be a non-specific result of neurodegeneration. Reductions in the activity of this enzyme, if widespread in the brain, may predispose vulnerable regions to further damage.

Effect of inclusion of defatted grape seed meal in the diet on digestion and performance of growing rabbits.

The digestion and nutritive value of defatted grape seed meal (DGSM) was investigated. A basal diet was formulated to meet requirements of growing rabbits. Another diet was formulated by substituting 15.2% of the basal diet with DGSM. Two hundred eight weaned 30-d-old rabbits were fed these diets, and fattening performance was recorded. Eighty animals were used to study the effect of DGSM inclusion on cecal fermentation traits and intestinal disaccharidase activity at two ages (5 and 35 d after weaning). Fecal apparent digestibility of nutrients was measured in 18 rabbits. A third diet was formulated to contain DGSM (61.3%) as the sole source of fiber and a supplement consisting of wheat flour, casein, lard, and a mixture of vitamins and minerals to avoid nutrient imbalances. This semipurified diet was used to determine cecal digestion traits, disaccharidase activity in the small intestine, fecal apparent digestibility of nutrients, and rate of passage in 70-d-old rabbits. Digestible energy and NDF and CP digestibilities of DGSM calculated by difference were 5.51 +/- 0.89 MJ/kg DM, 24.5 +/- 5.76%, and 46.8 +/- 14.9%, respectively. Inclusion of 15% of DGSM in the basal diet increased ADFI in finishing rabbits (from 9 to 15%; P < 0.05), so that DE intake increased although dietary DE concentration decreased. As a consequence, ADG increased by 3.3% in the whole fattening period (P = 0.046). The increase in ADFI was parallel to an 8% decrease in the weight of cecal contents (P = 0.059), and it was in agreement with the relatively short cecal mean retention time of DGSM (7.61 h) in the semipurified diet. Inclusion of 15% of DGSM in the basal diet did not affect (P > or = 0.20) mortality (10.1%) or cecal concentrations of VFA, NH3 N, or cecal pH either at 5 d (71.9 mM, 17.7 mM, and 5.75, respectively) or at 35 d after weaning (74.6 mM, 10.1 mM, and 5.66, respectively) but improved the sucrase activity in the ileum by 36% (P = 0.031). Digestibility of NDF of DGSM in the semipurified diet was 8.57%, which agrees with the low acidity and weight of cecal contents of animals fed this diet (6.26 and 3.63% BW, respectively). From these results, we conclude that DGSM has a relatively high DE concentration and its inclusion at moderate levels (15%) in the diet exerts a positive effect on ADFI, DE intake, and ADG with no impairment of cecal fermentation and mortality.

N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

Brain metabolism and brain disease: is metabolic deficiency the proximate cause of Alzheimer dementia?

The potential of impairments in oxidative/energy metabolism to cause diseases of the brain had been proposed even before the major pathways of oxidative/energy metabolism were described. Deficiencies associated with disease are known in all the pathways of oxidative/energy metabolism and are associated with some of the most common disorders of the nervous system, including Alzheimer's disease (AD) and Parkinson's disease. A common mechanism in these conditions appears to be a downward mitochondrial spiral, involving abnormalities in energy metabolism, calcium metabolism, and free radicals (reactive oxygen and nitrogen species). In AD, the spiral appears to interact with abnormalities in the metabolism of the Alzheimer amyloid precursor protein (APP) and its Abeta fragment. Several lines of evidence indicate that the mitochondrial spiral may be a proximate cause of the clinical disabilities in AD. Decreases in cerebral metabolic rate (CMR) characteristically occur in AD and in other dementias. Inducing decreases in CMR leads to clinical disabilities characteristically associated with AD and with analogous problems in experimental animals. Treatments directed toward normalizing CMR appear to help at least some patients. Further studies of this possibility and of treatments designed to ameliorate the mitochondrial spiral may prove useful for treating AD and perhaps some other dementing disorders.

Correlation of the clinical severity of Alzheimer's disease with an aberration in mitochondrial DNA (mtDNA).

Controversy exists about which of the well-established neurobiological abnormalities in Alzheimer's disease (AD) relate directly to the clinical disabilities. Because of an interest in the mitochondrial lesion in AD, we tested the correlation between clinical disability (measured by the Clinical Dementia Rating [CDR] scale) and an anomaly in mitochondrial DNA (mtDNA) in AD brain. Simultaneous polymerase chain reaction (PCR) amplification of the CO1 gene in mtDNA and CO1 pseudogenes in nuclear DNA (nDNA) were performed in samples from AD and non-AD brain, and the ratios of mtDNA/nDNA amplicons calculated. This approach utilizes PCR amplification of endogenous nDNA as a normalization standard for the amplification of mtDNA. We examined total DNA from the brains of Caucasian residents of a Jewish nursing home (86 AD and 26 non-AD "controls"). These patients had been closely followed clinically until death and then autopsied. In this sample, the degree of cognitive impairment in the AD patients correlated with the reduction in the amplification of the mtDNA gene (p = 0.23; p = 0.034), but not with the density of neuritic plaques (p = 0.109). These results agree with the suggestion that the well-documented impairment in brain-energy metabolism in AD may be a direct cause of the clinical disability.

A sensitive fluorometric assay for tissue transglutaminase.

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).

Selective loss of KGDHC-enriched neurons in Alzheimer temporal cortex: does mitochondrial variation contribute to selective vulnerability?

THE RESEARCH OBJECTIVE: of this study was to test whether variation in mitochondrial composition is associated with "selective vulnerability" in Alzheimer brain. The term "selective vulnerability" refers to the loss of relatively vulnerable brain cells and the sparing of relatively resistant brain cells in disorders in which a genetic defect or environmental agent acts on both types of cells. The mechanisms underlying selective vulnerability are largely unknown, but mitochondria may be involved; the composition of mitochondria varies among different types of neurons, and mitochondria have an important role in cell death. Alzheimer's Disease (AD) is one of a number of neurodegenerative disorders in which both selective vulnerability and abnormalities of mitochondria occur. METHODS: We examined by immunohistochemistry the cellular distribution of a mitochondrial constituent (the alpha-ketoglutarate dehydrogenase complex, KGDHC) known to be deficient in AD, in relation to the known selective vulnerability of neurons in areas 21 and 22 of the temporal lobe in this neurodegenerative disorder. RESULTS: In normal human brain, cortical layers III and V contain neurons intensely immunoreactive for KGDHC, compared to other cells in these areas. The KGDHC-enriched cells are lost in AD (p < 0.001). In layer III, the loss of KGDHC-enriched cells is proportional to total loss of neurons, as determined by immunoreactivity to neuron specific enolase (NSE). In layer V, a higher proportion of the KGDHC-enriched neurons are lost than of other (NSE positive) neurons (p < 0.001). SIGNIFICANCE: Variations in mitochondrial composition may be one of the factors determining which cells die first when different types of cells are exposed to the same stress.

Lysine-rich histone (H1) is a lysyl substrate of tissue transglutaminase: possible involvement of transglutaminase in the formation of nuclear aggregates in (CAG)(n)/Q(n) expansion diseases.

Histone H1, which contains about 27% lysine, is an excellent lysyl donor substrate of Ca(2+)-activated guinea pig liver tissue transglutaminase as judged by rapid fluorescence enhancement in the presence of the glutaminyl-donor substrate 1-N-(carbobenzoxy-L-glutaminylglycyl)-5-N-(5'N'N'-dimethylamino naphth alenesulfonyl) diamidopentane. Sodium dodecyl sulfate gel electrophoresis of a 30-min reaction mixture revealed the presence of fluorescent high-M(r) aggregates, which are also formed when histone H1 is incubated solely with activated tissue transglutaminase. Aggregate formation is even more pronounced when histone H1 is incubated with activated tissue transglutaminase and dimethylcasein (glutaminyl donor only). The findings suggest not only that histone H1 is an especially good lysyl substrate of tissue transglutaminase, but that it is also a glutaminyl substrate. Histone H1 is a good lysyl substrate of transglutaminase purified from Streptoverticillium mobaraense, suggesting that the ability of histone H1 to act as a transglutaminase lysyl substrate is widespread. In agreement with previous studies, it was found that human beta-endorphin is a moderately good substrate of tissue transglutaminase. At least 8 neurodegenerative diseases, including Huntington's disease, are caused by (CAG)(n) expansions in the genome and by an expansion of the corresponding polyglutamine domain within the expressed, mutated protein. Polyglutamine domains are excellent substrates of liver and brain transglutaminases. A hallmark of many of the (CAG)(n)/polyglutamine expansion diseases is the presence of polyglutamine-containing aggregates within the cytosol and nuclei of affected neurons. Transglutaminase activity occurs in both of these compartments in human brain. In future studies, it will be important to determine whether transglutaminases play a role in (1) cross-linking of histone H1 to glutaminyl donors (including polyglutamine domains) in nuclear chromatin, (2) the formation of nuclear aggregates in (CAG)(n)/polyglutamine expansion diseases, (3) DNA laddering and cell death in neurodegenerative diseases and (4) depletion of neuropeptides in vulnerable regions of Huntington's disease brain.

Mitochondrial damage in Alzheimer's disease varies with apolipoprotein E genotype.

Brain metabolism and the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a mitochondrial enzyme, are diminished in brains from patients with Alzheimer's disease (AD). In 109 subjects, the Clinical Dementia Rating (CDR) score was highly correlated with brain KGDHC activity. In AD patients who carried the epsilon 4 allele of the apolipoprotein E gene (ApoE4), the CDR score correlated better with KGDHC activity than with the densities of neuritic plaques or neuritic tangles. In contrast, in patients without ApoE4, the CDR score correlated significantly better with tangles and plaques than with KGDHC activity. The results suggest that mitochondrial/oxidative damage may be more important for the cognitive dysfunction in AD patients who carry ApoE4 than in those who do not.

Zn2+ inhibits alpha-ketoglutarate-stimulated mitochondrial respiration and the isolated alpha-ketoglutarate dehydrogenase complex.

Intracellular free Zn(2+) is elevated in a variety of pathological conditions, including ischemia-reperfusion injury and Alzheimer's disease. Impairment of mitochondrial respiration is also associated with these pathological conditions. To test whether elevated Zn(2+) and impaired respiration might be linked, respiration of isolated rat liver mitochondria was measured after addition of Zn(2+). Zn(2+) inhibition (K(i)(app) = approximately 1 micrometer) was observed for respiration stimulated by alpha-ketoglutarate at concentrations well within the range of intracellular Zn(2+) reported for cultured hepatocytes. The bc(1) complex is inhibited by Zn(2+) (Link, T. A., and von Jagow, G. (1995) J. Biol. Chem. 270, 25001-25006). However, respiration stimulated by succinate (K(i)(app) = approximately 6 micrometer) was less sensitive to Zn(2+), indicating the existence of a mitochondrial target for Zn(2+) upstream from bc(1) complex. Purified pig heart alpha-ketoglutarate dehydrogenase complex was strongly inhibited by Zn(2+) (K(i)(app) = 0.37 +/- 0.05 micrometer). Glutamate dehydrogenase was more resistant (K(i)(app) = 6 micrometer), malate dehydrogenase was unaffected, and succinate dehydrogenase was stimulated by Zn(2+). Zn(2+) inhibition of alpha-ketoglutarate dehydrogenase complex required enzyme cycling and was reversed by EDTA. Reversibility was inversely related to the duration of exposure and the concentration of Zn(2+). Physiological free Zn(2+) may modulate hepatic mitochondrial respiration by reversible inhibition of the alpha-ketoglutarate dehydrogenase complex. In contrast, extreme or chronic elevation of intracellular Zn(2+) could contribute to persistent reductions in mitochondrial respiration that have been observed in Zn(2+)-rich diseased tissues.

Inherent abnormalities in energy metabolism in Alzheimer disease. Interaction with cerebrovascular compromise.

Alzheimer disease (AD) is a form of the dementia syndrome. AD appears to have a variety of fundamental etiologies that lead to the neuropathological manifestations which define the disease. Patients who are at high risk to develop AD typically show impairments of cerebral metabolic rate in vivo even before they show any evidence of the clinical disease on neuropsychological, electrophysiological, and neuroimaging examinations. Therefore, impairment in energy metabolism in AD can not be attributed to loss of brain substance or to electrophysiological abnormalities. Among the characteristic abnormalities in the AD brain are deficiencies in several enzyme complexes which participate in the mitochondrial oxidation of substrates to yield energy. There include the pyruvate dehydrogenase complex (PDHC), the alpha-ketoglutarate dehydrogenase complex (KGDHC), and Complex IV of the electron transport chain (COX). The deficiency of KGDHC may be due to a mixture of causes including damage by free radicals and perhaps to genetic variation in the DLST gene encoding the core protein of this complex. Inherent impairment of glucose oxidation by the AD brain may reasonably be expected to interact synergistically with an impaired supply of oxygen and glucose to the AD brain, in causing brain damage. These considerations lead to the hypothesis that cerebrovascular compromise and inherent abnormalities in the brain's ability to oxidize substrates can interact to favor the development of AD, in individuals who are genetically predisposed to develop neuritic plaques.

Characterization of ferrochelatase (hemH) mutations in Haemophilus influenzae.

Haemophilus influenzae lacks most of the biosynthetic enzymes for hemin synthesis. However, the organism has retained ferrochelatase activity, which we identified to be encoded by a hemH-homologous gene. In this report we characterize the growth physiology conferred by hemH mutations under infection and laboratory conditions.

Randomized, double-blind, placebo-controlled, multicenter study to evaluate the safety and tolerability of metrifonate in patients with probable Alzheimer disease. The Metrifonate Study Group.

A randomized, double-blind, placebo-controlled, parallel-group study was undertaken to evaluate the safety and tolerability of a once-daily oral administration of metrifonate in patients with probable mild to moderate Alzheimer disease. Metrifonate was given as a loading dose of 125-225 mg based on weight (2.5 mg/kg) for 2 weeks, followed by a maintenance dose of 50-90 mg based on weight (1.0 mg/kg) for 4 weeks. Twenty-nine patients received metrifonate, and 10 patients received placebo. Metrifonate produced a mean erythrocyte acetylcholinesterase inhibition at the end of treatment of 86.3%. The proportion of patients who experienced at least one adverse event was comparable between the metrifonate (76%) and placebo (80%) groups. Selected adverse events in disfavor of metrifonate (defined as those for which the incidence in the metrifonate and placebo groups differed by at least 10%) were diarrhea, nausea, leg cramps, and accidental injury. Adverse events were predominantly mild in intensity and transient. No severe adverse events were experienced by any patient. The most notable hemodynamic change observed during metrifonate treatment was a clinically insignificant mean decrease in the heart rate (by electrocardiogram) of approximately 9 beats/min, compared with an approximate 3-beats/min decrease for the placebo group. No muscle weakness was observed in this study. No clinically relevant laboratory abnormalities, such as liver toxicity, or changes in exercise tolerance or pulmonary function tests were found with metrifonate treatment. This metrifonate dose provided a high level of acetylcholinesterase inhibition, which was associated in these patients with a favorable safety and tolerability profile. Indeed, the magnitude of the peripheral acetylcholinesterase inhibition is the highest tolerable inhibition level yet observed.

Neuronal subclass-selective loss of pyruvate dehydrogenase immunoreactivity following canine cardiac arrest and resuscitation.

Chronic impairment of aerobic energy metabolism accompanies global cerebral ischemia and reperfusion and likely contributes to delayed neuronal cell death. Reperfusion-dependent inhibition of pyruvate dehydrogenase complex (PDHC) enzyme activity has been described and proposed to be at least partially responsible for this metabolic abnormality. This study tested the hypothesis that global cerebral ischemia and reperfusion results in the loss of pyruvate dehydrogenase immunoreactivity and that such loss is associated with selective neuronal vulnerability to transient ischemia. Following 10 min canine cardiac arrest, resuscitation, and 2 or 24 h of restoration of spontaneous circulation, brains were either perfusion fixed for immunohistochemical analyses or biopsy samples were removed for Western immunoblot analyses of PDHC immunoreactivity. A significant decrease in immunoreactivity was observed in frontal cortex homogenates from both 2 and 24 h reperfused animals compared to samples from nonischemic control animals. These results were supported by confocal microscopic immunohistochemical determinations of pyruvate dehydrogenase immunoreactivity in the neuronal cell bodies located within different layers of the frontal cortex. Loss of immunoreactivity was greatest for pyramidal neurons located in layer V compared to neurons in layers IIIc/IV, which correlates with a greater vulnerability of layer V neurons to delayed death caused by transient global cerebral ischemia.

The alpha-ketoglutarate dehydrogenase complex in neurodegeneration.

Altered energy metabolism is characteristic of many neurodegenerative disorders. Reductions in the key mitochondrial enzyme complex, the alpha-ketoglutarate dehydrogenase complex (KGDHC), occur in a number of neurodegenerative disorders including Alzheimer's Disease (AD). The reductions in KGDHC activity may be responsible for the decreases in brain metabolism, which occur in these disorders. KGDHC can be inactivated by several mechanisms, including the actions of free radicals (Reactive Oxygen Species, ROS). Other studies have associated specific forms of one of the genes encoding KGDHC (namely the DLST gene) with AD, Parkinson's disease, as well as other neurodegenerative diseases. Reductions in KGDHC activity can be plausibly linked to several aspects of brain dysfunction and neuropathology in a number of neurodegenerative diseases. Further studies are needed to assess mechanisms underlying the sensitivity of KGDHC to oxidative stress and the relation of KGDHC deficiency to selective vulnerability in neurodegenerative diseases.

Genetic rearrangements of the regions adjacent to genes encoding heat-labile enterotoxins (eltAB) of enterotoxigenic Escherichia coli strains.

One of the most common bacterially mediated diarrheal infections is caused by enterotoxigenic Escherichia coli (ETEC) strains. ETEC-derived plasmids are responsible for the distribution of the genes encoding the main toxins, namely, the heat-labile and heat-stable enterotoxins. The origins and transfer modes (intra- or interplasmid) of the toxin-encoding genes have not been characterized in detail. In this study, we investigated the DNA regions located near the heat-labile enterotoxin-encoding genes (eltAB) of several clinical isolates. It was found that the eltAB region is flanked by conserved 236- and 280-bp regions, followed by highly variable DNA sequences which consist mainly of partial insertion sequence (IS) elements. Furthermore, we demonstrated that rearrangements of the eltAB region of one particular isolate, which harbors an IS91R sequence next to eltAB, could be produced by a recA-independent but IS91 sequence-dependent mechanism. Possible mechanisms of dissemination of IS element-associated enterotoxin-encoding genes are discussed.