About five out of 1,000 patients admitted to hospital develop bacterial sepsis leading to shock, the mortality rate for which is high despite antibiotic therapy. The infection results in hypotension and poor tissue perfusion, and eventually leads to the failure of several organ systems. Bacterial endotoxin is thought to be the direct cause of shock in Gram-negative sepsis, because it can cause shock in animals, and antibodies against endotoxin prevent Gram-negative shock in animals and in humans. But, the symptoms of septic shock are the result of the actions of host cytokines induced by the endotoxin. The cytokine interleukin-1 has been implicated as an important mediator of septic shock because it can induce tachycardia and hypotension and act synergistically with tumour necrosis factor to cause tissue damage and death. We now report that a specific interleukin-1 receptor antagonist reduces the lethality of endotoxin-induced shock in rabbits, indicating that interleukin-1 does indeed play an important part in endotoxin shock.

We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin. B. stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47C but not at 55C and above. Variants that were kanamycin resistant at 63°C were obtained by selection of spontaneous mutants, by passage ofa shuttle plasmid through theEscherichia colimutD5 mutator strain and introduction into B. stearothermophilus by transformation, and by growing the thermophile in a chemostat. The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80. Mutants that are even more heat stable were derived from the frt variant by selecting for kanamycin resistance at 70C, and these carry the additional change of threonine to lysine at position 130. This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays.The ability of some microorganisms to grow at extreme temperatures (1) implies that their enzymes are stable and active at these temperatures. This is largely borne out when enzymes from thermophilic sources are studied in vitro; such enzymes are indeed more thermostable than the equivalents isolated from phylogenetically related mesophilic organisms (2). Correlations between an increase in the proportion of hydrophobic residues and the degree of thermostability have been observed (3-5). Internal electrostatic interactions (6) and disulfide linkages (7) have also been proposed as features that stabilize proteins. However, the role of individual amino acid residues in enhancing the resistance to thermal denaturation of an enzyme from a thermophile is not known.We wish to understand the contributions of individual amino acids to the overall stability of a protein's structure. Comparisons between enzymes from mesophiles and thermophiles are complicated because these proteins, although homologous, usually differ in more than one residue. More precise inferences can be based on comparisons of temperature-sensitive mutations which encode, as a result of single amino acid changes, proteins that retain activity but are less resistant to heat denaturation than the wild-type counterpart. Thus, for example, many temperature-sensitive mutations of phage T4 lysozyme have been studied with the aim of correlating the changes in stability with changes in the protein structure (8). However, an x-ray crystallographic study of one of these mutant enzymes (9) revealed that a variant in which a histidine residue repla...

Synapse loss and synaptic dysfunction are pathological processes already involved in the early stages of Alzheimer's disease (AD). Synapses consist principally of neuronal membranes, and the neuronal and synaptic losses observed in AD have been linked to the degeneration and altered composition and structure of these membranes. Consequently, synapse loss and membrane-related pathology provide viable targets for intervention in AD. The specific nutrient combination Fortasyn Connect (FC) is designed to ameliorate synapse loss and synaptic dysfunction in AD by addressing distinct nutritional needs believed to be present in these patients. This nutrient combination comprises uridine, docosahexaenoic acid, eicosapentaenoic acid, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium, and is present in Souvenaid, a medical food intended for use in early AD. It has been hypothesized that FC counteracts synaptic loss and reduces membrane-related pathology in AD by providing nutritional precursors and cofactors that act together to support neuronal membrane formation and function. Preclinical studies formed the basis of this hypothesis which is being validated in a broad clinical study program investigating the potential of this nutrient combination in AD. Memory dysfunction is one key early manifestation in AD and is associated with synapse loss. The clinical studies to date show that the FC-containing medical food improves memory function and preserves functional brain network organization in mild AD compared with controls, supporting the hypothesis that this intervention counteracts synaptic dysfunction. This review provides a comprehensive overview of basic scientific studies that led to the creation of FC and of its effects in various preclinical models.

No abstract

BackgroundFolate, vitamin B-12, and vitamin B-6 are essential nutritional components in one-carbon metabolism and are required for methylation capacity. The availability of these vitamins may therefore modify methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) by PE-N-methyltransferase (PEMT) in the liver. It has been suggested that PC synthesis by PEMT plays an important role in the transport of polyunsaturated fatty acids (PUFAs) like docosahexaenoic acid (DHA) from the liver to plasma and possibly other tissues. We hypothesized that if B-vitamin supplementation enhances PEMT activity, then supplementation could also increase the concentration of plasma levels of PUFAs such as DHA. To test this hypothesis, we determined the effect of varying the combined dietary intake of these three B-vitamins on plasma DHA concentration in rats.MethodsIn a first experiment, plasma DHA and plasma homocysteine concentrations were measured in rats that had consumed a B-vitamin-poor diet for 4 weeks after which they were either continued on the B-vitamin-poor diet or switched to a B-vitamin-enriched diet for another 4 weeks. In a second experiment, plasma DHA and plasma homocysteine concentrations were measured in rats after feeding them one of four diets with varying levels of B-vitamins for 4 weeks. The diets provided 0% (poor), 100% (normal), 400% (enriched), and 1600% (high) of the laboratory rodent requirements for each of the three B-vitamins.ResultsPlasma DHA concentration was higher in rats fed the B-vitamin-enriched diet than in rats that were continued on the B-vitamin-poor diet (P = 0.005; experiment A). Varying dietary B-vitamin intake from deficient to supra-physiologic resulted in a non-linear dose-dependent trend for increasing plasma DHA (P = 0.027; experiment B). Plasma DHA was lowest in rats consuming the B-vitamin-poor diet (P > 0.05 vs. normal, P < 0.05 vs. enriched and high) and highest in rats consuming the B-vitamin-high diet (P < 0.05 vs. poor and normal, P > 0.05 vs. enriched). B-vitamin deficiency significantly increased plasma total homocysteine but increasing intake above normal did not significantly reduce it. Nevertheless, in both experiments plasma DHA was inversely correlated with plasma total homocysteine.ConclusionThese data demonstrate that dietary folate, vitamin B-12, and vitamin B-6 intake can influence plasma concentration of DHA.