Effect of a fermented milk combining Lactobacillus acidophilus Cl1285 and Lactobacillus casei in the prevention of antibiotic-associated diarrhea: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Antibiotic-associated diarrhea is an important problem in hospitalized patients. The use of probiotics is gaining interest in the scientific community as a potential measure to prevent this complication. The main objective of the present study was to assess the efficacy and safety of a fermented milk combining Lactobacillus acidophilus and Lactobacillus casei that is widely available in Canada, in the prevention of antibiotic-associated diarrhea. METHODS: In this double-blind, randomized study, hospitalized patients were randomly assigned to receive either a lactobacilli-fermented milk or a placebo on a daily basis. RESULTS: Among 89 randomized patients, antibiotic-associated diarrhea occurred in seven of 44 patients (15.9%) in the lactobacilli group and in 16 of 45 patients (35.6%) in the placebo group (OR 0.34, 95% CI 0.125 to 0.944; P=0.05). The median hospitalization duration was eight days in the lactobacilli group, compared with 10 days in the placebo group (P=0.09). Overall, the lactobacilli-fermented milk was well tolerated. CONCLUSION: The daily administration of a lactobacilli-fermented milk was safe and effective in the prevention of antibiotic-associated diarrhea in hospitalized patients.

Pharmacokinetics and anesthetic activity of eugenol in male Sprague-Dawley rats.

Eugenol, the principle chemical constituent of clove oil, has recently been evaluated for its anesthetic and analgesic properties in fish and amphibians. The objective of this study was to determine the pharmacokinetic (PK) and anesthetic activity of eugenol in rats. Male Sprague-Dawley rats received single i.v. doses of eugenol (0, 5, 10, 20, 40 and 60 mg/kg) and anesthetic level was evaluated with the withdrawal reflex. For the 20 mg/kg dose level, blood and urinary samples were collected over 1 h for the PK assessment. Plasma and blood concentrations of eugenol, as well as metabolite identification in urine, were determined using a novel dansyl chloride derivatization method with liquid chromatography mass spectrometry (LC/MS/MS). PK parameters were calculated using noncompartmental methods. Eugenol-induced loss of consciousness in a dose-dependent manner, with mean (+/-SEM) recovery in reflex time of 167 +/- 42 sec observed at the highest dose level. Mean systemic clearance (Cl) in plasma and blood were 157 and 204 mL/min/kg, respectively. Glucuronide and sulfate conjugates were identified in urine. Overall, eugenol produced a reversible, dose-dependent anesthesia in male Sprague-Dawley rats.

The intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates to the nucleus in a notch-like manner.

The beta-amyloid precursor protein (APP) is a ubiquitous receptor-like molecule without a known function. However, the recent recognition that APP and Notch undergo highly similar proteolytic processing has suggested a potential signaling function for APP. After ligand binding, Notch is cleaved by the ADAM-17 metalloprotease followed by an intramembrane cleavage mediated by gamma-secretase. The gamma-secretase cut releases the Notch intracellular domain (NICD), which enters the nucleus and modulates transcription. Because APP is processed similarly by ADAM-17 and gamma-secretase, we reasoned that the APP intracellular domain (AICD) has a role analogous to the NICD. We therefore generated a plasmid encoding the AICD sequence and studied the subcellular localization of the expressed protein (C60). Our results demonstrate that the cytoplasmic domain of APP is a highly labile fragment that is stabilized by forming complexes with Fe65 and can then enter the nucleus in neurons and non-neural cells. These findings strongly support the hypothesis that APP signals in the nucleus in a manner analogous to the function of Notch.

No evidence for genetic association or linkage of the cathepsin D (CTSD) exon 2 polymorphism and Alzheimer disease.

Two recent case-control studies have suggested a strong association of a missense polymorphism in exon 2 of the cathepsin D gene (CTSD) and Alzheimer disease (AD). However, these findings were not confirmed in another independent study. We analyzed this polymorphism in two large and independent AD study populations and did not detect an association between CTSD and AD. The first sample was family-based and included 436 subjects from 134 sibships discordant for AD that were analyzed using the sibship disequilibrium test (SDT, p = 0.68) and the sib transmission/disequilibrium test (Sib-TDT, p = 0.81). The second sample of 200 AD cases and 182 cognitively normal controls also failed to show significant differences in the allele or genotype distribution in cases versus controls (chi2, p = 0.91 and p = 0.88, respectively). In addition, two-point linkage analyses in an enlarged family sample (n = 670) did not show evidence for linkage of the chromosomal region around CTSD. Thus, our analyses on more than 800 subjects suggest that if an association between the CTSD exon 2 polymorphism and AD exists, it is likely to be smaller than previously reported.

Evidence against association of the FE65 gene (APBB1) intron 13 polymorphism in Alzheimer's patients.

A genetic polymorphism in intron 13 of the FE65 gene (APBB1) was reported to be associated with Alzheimer's disease (AD). Our analyses of this polymorphism, both in a family-based or a case-control sample, fail to support the association between the FE65 intron 13 polymorphism and AD. We performed the sibship disequilibrium test (SDT, P=0.77) and the sib transmission/disequilibrium test (Sib-TDT, P=0.56) in a family-based study which included 526 subjects from 158 sibships. In addition, we compared the genotype and allele frequencies of this biallelic polymorphism in 311 AD patients to those of a control group consisting of 260 subjects and found no significant difference (chi(2), P=0.847 and P=0.586, respectively). Furthermore, our two-point linkage analysis in a family-based sample was in agreement with a genome wide scan for linkage to AD and showed no evidence for linkage to the short arm of chromosome 11 where the FE65 gene is located. We conclude that the association of the FE65 intron 13 polymorphism with AD, if any, is smaller than previously reported.

Candidate genes showing no evidence for association or linkage with Alzheimer's disease using family-based methodologies.

Alzheimer's disease (AD) is a genetically complex and heterogeneous disorder. To date, a large number of candidate genes have been associated with the disease, however none of these findings has been consistently replicated in independent datasets. In this study we report the results of family-based analyses for polymorphisms of five such candidates on chromosomes 2 (interleukin-1beta, IL-1B), 3 (butyrylcholinesterase, BCHE), 11 (cathepsin D, CTSD; Fe65, APBB1) and 12 (lipoprotein receptor-related protein-1, LRP1) that were all suggested to be associated with AD in recent case-control studies. To minimize the possibility of spurious findings due to population admixture, we used a family-based design applying the sibship disequilibrium test (SDT) as well as two-point parametric linkage analyses on families from the National Institute of Mental Health (NIMH) Genetics Initiative. Contrary to the initial reports, none of the polymorphisms that were analyzed showed evidence for association or linkage with AD in our families. Our results suggest that the previously reported associations from case-control studies are either (a) false positive results, e.g. due to type I error or population admixture, (b) smaller than initially proposed, or (c) due to linkage disequilibrium with an as yet unidentified polymorphism nearby.

Progress toward valid transgenic mouse models for Alzheimer's disease.

A transgenic mouse model for Alzheimer's disease (AD) should mimic the age-dependent accumulation of beta-amyloid plaques, neurofibrillary tangles, neuronal cell death as well as display memory loss and behavioral deficits. Age-dependent accumulation of A beta deposits in mouse brain has been achieved in mice overexpressing mutant alleles of the amyloid precursor protein (APP). In contrast, mice bearing mutant alleles of the presenilin genes show increased production of the A beta42 peptide, but do not form amyloid deposits unless mutant alleles of APP are also overproduced. Furthermore, the onset of A beta deposition is greatly accelerated, paralleling the involvement of presenilins in early onset AD. Studies of APP and presenilin transgenic mice have shown 1) the absence of a requirement for a maturation step in dense core plaque formation, 2) evidence that beta-amyloid deposition is directed by regional factors, and 3) behavioral deficits are observed before A beta deposition. Crosses of APP transgenic mice with mice modified for known AD risk factors and "humanizing" the mouse may be necessary for complete replication of AD.

hFE65L influences amyloid precursor protein maturation and secretion.

The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective alpha-secretase-derived secreted APP (APPs alpha) and the Alzheimer's disease-associated beta-amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the C-terminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin-coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a three-fold increase in the amount of APPs alpha recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPs alpha secreted is due neither to a simple increase in the steady-state levels of APP nor to activation of the protein kinase C-regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway.

Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.

We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2.

The gene defects responsible for familial Alzheimer's disease.

Four different genes have now been found to contain AD-associated mutations or polymorphisms. While the pathogenic mutations in the early-onset FAD genes, APP, PS1, and PS2 directly cause AD with nearly 100% penetrance, in a larger subset of AD cases with onset over 60 years (maximally for onset at 61-65 years), inheritance of the APOE4 allele confers increased risk for AD but is not sufficient to cause the disease. Together, these four genes appear to account for approximately 50% of FAD cases. We are actively screening the genome for additional FAD loci by genotyping markers in over 400 FAD nuclear pedigrees and affected sib-pairs (83% late-onset and 17% early-onset). We have recently discovered genetic linkage to a novel FAD locus on chromosome 12 as well as another putative locus on chromosome 3 (unpublished findings). Positional cloning strategies are currently under way to identify these potentially novel FAD genes. A common event which is associated with all of the known FAD genes is the excessive accumulation of the A beta peptide and deposition of beta-amyloid in the brain. Thus, a common pathogenic pathway for AD neuropathogenesis appears to center around the cellular trafficking, maturation, and processing of APP, and the subsequent generation, aggregation, and deposition of A beta (or more specifically, A beta 1-42). APP and presenilin gene mutations most likely act as either gain-of-function or dominant negative gene defects which may ultimately lead to the transport of APP into intracellular compartments that promote the enhanced production of A beta or A beta 1-42. AD patients who carry an APOE4 allele experience increased amyloid burden in their brains compared to APOE4-negative AD cases. Thus, the presence of APOE4 would also appear to lead to abnormal generation, aggregation, or clearance of A beta in the brain A beta, perhaps by working in concert with its neuronal receptor, LRP. While the exact mechanisms by which the known FAD gene changes lead to the onset of AD remain unclear, the available data indicate that novel therapies aimed at curbing the generation, aggregation, and deposition of A beta would appear to carry the greatest potential for the effective treatment of this formidable disease.

Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes.

To identify proteins that regulate microtubule assembly in Saccharomyces cerevisiae, we screened for multicopy suppressors of a conditional mutation in alpha-tubulin. Cells expressing the recessive allele tub1-729 as their sole alpha-tubulin gene grow normally at permissive temperature. However, at 15 degrees C the cells lose viability and arrest primarily with large buds and quantitatively diminished microtubule structures. Transformation of mutant cells with genomic libraries repeatedly identified three different suppressors: the two wild-type alpha-tubulin genes, TUB1 and TUB3; and BUB3. BUB3 is a checkpoint gene that permits entry into mitosis depending upon the assembly state of microtubules. Excess BUB3 rescues both the loss of viability and microtubule defects but not the benomyl supersensitivity associated with tub1-729. The suppression is specific for the mutation ALA422VAL in TUB1, and does not affect several other mutations in TUB1 that produce the 'no microtubule' phenotype. Overexpression of BUB1, which interacts genetically with BUB3 and which is involved in the same checkpoint pathway, also rescues the cold sensitivity of tub1-729, but another checkpoint gene, MAD2, does not. Overexpression of BUB3 in wild-type cells has no detectable growth or microtubule defect, but disruption of the BUB3 gene produces slow growth and benomyl supersensitivity. Our results suggest that BUB1 and BUB3 overexpression modulate an event required for mitotic spindle function which is rate limiting for tub1-729 cells at the restrictive temperature.

Identification of a novel Brugia pahangi beta-tubulin gene (beta 2) and a 22-nucleotide spliced leader sequence on beta 1-tubulin mRNA.

We have examined the expression of beta-tubulin genes in the parasitic nematode, Brugia pahangi. A genomic library was constructed and screened by hybridization with a Haemonchus contortus beta-tubulin cDNA fragment which recognizes several B. pahangi beta-tubulin sequences, including sequences which correspond to the previously characterized beta 1-tubulin gene. The B. pahangi beta 2-tubulin gene was isolated by selecting clones which hybridize to the H. contortus beta-tubulin gene but which do not hybridize to the beta 1-tubulin gene. A partial sequence of the beta 2-tubulin gene confirms that it codes for a distinct beta-tubulin. Southern hybridization analyses show that the beta 2-tubulin sequence exists as a single copy gene within the B. pahangi genome. Expression of the beta 2-tubulin gene is developmentally regulated and the message is found predominantly in adult male worms, whereas the beta 1-tubulin gene is expressed in microfilariae and approximately equal levels of the transcript are found in male and female adult worms. During mRNA maturation the beta 1-tubulin mRNA of microfilariae and adult worms acquires a trans-spliced leader identical to the SL1 of Caenorhabditis elegans.

Characterization of a beta-tubulin gene and a beta-tubulin gene products of Brugia pahangi.

A genomic clone containing a beta-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the beta-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mNRA of 1.8 kb which codes for a protein of 448 amino acids. The predicted beta-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken beta 2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct beta-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi beta-tubulin was used to identify beta-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilariae and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved beta-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the beta-tubulin isoforms of B. pahangi.